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At Loose Ends: Resecting a Double-Strand Break
Abstract
Double-strand break (DSB) repair is critical for maintaining genomic integrity and requires the processing of the 5' DSB ends. Recent studies have shed light on the mechanism and regulation of DNA end processing during DSB repair by homologous recombination.
... Cells commit to a homology-directed mechanism of repair when extensive resection is performed by the action of multiple nucleases. Short and long-term resection is mediated by MRN/CtIP in conjunction with EXO1 or BLM and DNA2 ( Figure 1B) [19]. This resection reveals 3 ssDNA ends, which are quickly coated by the replication protein A (RPA) complex [19,20]. ...
... Short and long-term resection is mediated by MRN/CtIP in conjunction with EXO1 or BLM and DNA2 ( Figure 1B) [19]. This resection reveals 3 ssDNA ends, which are quickly coated by the replication protein A (RPA) complex [19,20]. Filaments coated by RPA ensure that the ssDNA overhangs are not degraded, and prevent secondary structures from forming [19]. ...
... This resection reveals 3 ssDNA ends, which are quickly coated by the replication protein A (RPA) complex [19,20]. Filaments coated by RPA ensure that the ssDNA overhangs are not degraded, and prevent secondary structures from forming [19]. RAD51 then displaces RPA to form the pre-synaptic filament, and this requires the activity of several so-called RAD51 mediator proteins ( Figure 1C) [21]. ...
The accurate repair of DNA is critical for genome stability and cancer prevention. DNA double-strand breaks are one of the most toxic lesions; however, they can be repaired using homologous recombination. Homologous recombination is a high-fidelity DNA repair pathway that uses a homologous template for repair. One central HR step is RAD51 nucleoprotein filament formation on the single-stranded DNA ends, which is a step required for the homology search and strand invasion steps of HR. RAD51 filament formation is tightly controlled by many positive and negative regulators, which are collectively termed the RAD51 mediators. The RAD51 mediators function to nucleate, elongate, stabilize, and disassemble RAD51 during repair. In model organisms, RAD51 paralogs are RAD51 mediator proteins that structurally resemble RAD51 and promote its HR activity. New functions for the RAD51 paralogs during replication and in RAD51 filament flexibility have recently been uncovered. Mutations in the human RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and SWSAP1) are found in a subset of breast and ovarian cancers. Despite their discovery three decades ago, few advances have been made in understanding the function of the human RAD51 paralogs. Here, we discuss the current perspective on the in vivo and in vitro function of the RAD51 paralogs, and their relationship with cancer in vertebrate models.
... Cells commit to a homology-directed mechanism of repair when extensive resection is performed by the action of multiple nucleases. Short-and long-term resection is mediated by MRN/CtIP in conjunction with EXO1 or BLM and DNA2 ( Figure 1B) [19]. This resection reveals 3' ssDNA ends which are quickly coated by replication protein A complex [19,20]. ...
... Short-and long-term resection is mediated by MRN/CtIP in conjunction with EXO1 or BLM and DNA2 ( Figure 1B) [19]. This resection reveals 3' ssDNA ends which are quickly coated by replication protein A complex [19,20]. RPA-coated filaments ensure that the ssDNA overhangs are not degraded and prevent secondary structures from forming [19]. ...
... This resection reveals 3' ssDNA ends which are quickly coated by replication protein A complex [19,20]. RPA-coated filaments ensure that the ssDNA overhangs are not degraded and prevent secondary structures from forming [19]. RAD51 then displaces RPA to form the pre-synaptic filament, and this requires the activity of several so-called "RAD51 mediator" proteins ( Figure 1C) [21]. ...
Accurate repair of DNA is critical for genome stability and cancer prevention. DNA double-strand breaks are one of the most toxic lesions and can be repaired using homologous recombination (HR). HR is a high-fidelity DNA repair pathway that uses a homologous template for repair. One central HR step is RAD51 nucleoprotein filament formation on the single-stranded DNA ends, a step required for the homology search and strand invasion steps of HR. RAD51 filament formation is tightly controlled by many positive and negative regulators, collectively termed the RAD51 mediators. The RAD51 mediators function to nucleate, elongate, stabilize, and disassemble RAD51 during repair. In model organisms, RAD51 paralogs are RAD51 mediator proteins that structurally resemble RAD51 and promote its HR activity. New functions for the RAD51 paralogs during replication and in RAD51 filament flexibility have recently been uncovered. Mutations in the human RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and SWSAP1) are found in a subset of breast and ovarian cancers. Despite their discovery three decades ago, few advances have been made in understanding the function of the human RAD51 paralogs. Here we discuss the current perspective on the RAD51 paralogs in vivo and in vitro function and their relationship with cancer in vertebrate models.
... In eukaryotes, two major DSB repair pathways are known: non-homologous end joining (NHEJ) and homologous recombination (HR). The former is an error-prone process, while the latter mechanism exhibits high fidelity [2]. It has been suggested that in eukaryotes, DSBs that occur in the G1 phase of the cell cycle are most likely to be repaired via NHEJ, while those occurring in the S/G2 phase are preferentially processed via HRR [2,3]. ...
... The former is an error-prone process, while the latter mechanism exhibits high fidelity [2]. It has been suggested that in eukaryotes, DSBs that occur in the G1 phase of the cell cycle are most likely to be repaired via NHEJ, while those occurring in the S/G2 phase are preferentially processed via HRR [2,3]. ...
... Bacteria encode multiple pathways for DSB repair, including RecBCD, the primary HRR pathway, SbcC-SbcD, and one backup system, RecFOR [4,5]. The HRR pathway can be divided into five general steps: (1) recognition of the break sites and formation of a repair center (RC), (2) end-processing of the broken ends, (3) loading of RecA onto single-strand DNA, homology search, and strand invasion, (4) branch migration and resolution, or dissolution of the recombination intermediates and replication restart, and (5) disassembly of the recombination apparatus and segregation of sister chromosomes [5]. The eukaryotic HRR machinery is comprised of a core protein complex containing Mre11-Rad50-Xrs2/ Mre11-Rad50-NBS1(MRX/N); the nucleases/helicases exodeoxyribonuclease 1 (Exo1/EXO1), Dna2/DNA2, and Sgs1/BLM; the recombinase Rad51/RAD51; and several other accessory and regulatory proteins [6]. ...
Background
ATPase/Helicases and nucleases play important roles in homologous recombination repair (HRR). Many of the mechanistic details relating to these enzymes and their function in this fundamental and complicated DNA repair process remain poorly understood in archaea. Here we employed Sulfolobus islandicus, a hyperthermophilic archaeon, as a model to investigate the in vivo functions of the ATPase/helicase HerA, the nuclease NurA, and their associated proteins Mre11 and Rad50.
Results
We revealed that each of the four genes in the same operon, mre11, rad50, herA, and nurA, are essential for cell viability by a mutant propagation assay. A genetic complementation assay with mutant proteins was combined with biochemical characterization demonstrating that the ATPase activity of HerA, the interaction between HerA and NurA, and the efficient 5′-3′ DNA end resection activity of the HerA-NurA complex are essential for cell viability. NurA and two other putative HRR proteins: a PIN (PilT N-terminal)-domain containing ATPase and the Holliday junction resolvase Hjc, were co-purified with a chromosomally encoded N-His-HerA in vivo. The interactions of HerA with the ATPase and Hjc were further confirmed by in vitro pull down.
Conclusion
Efficient 5′-3′ DNA end resection activity of the HerA-NurA complex contributes to necessity of HerA and NurA in Sulfolobus, which is crucial to yield a 3′-overhang in HRR. HerA may have additional binding partners in cells besides NurA.
Electronic supplementary material
The online version of this article (doi:10.1186/s12867-015-0030-z) contains supplementary material, which is available to authorized users.
... DNA replication proteins then act to amplify this region resulting in an accurate repair of the break. As this mechanism requires the homologous sequence such as a sister chromatid, it is only active during late S-G2 phases of the cell cycle (Bernstein and Rothstein 2009). NHEJ by comparison is able to act at all times due to its ability to function without a homologous template, NHEJ is known to compete for DSBs even when a homologous sequence is present (Shrivastav et al. 2008). ...
... HR processes differ due to the requirement for greater nuclease dependent processing of DSB ends to create sufficient overhangs for recognition of homologous sequences. A review by Bernstein and Rothstein (2009) highlights each step of HR in detail. The MRN complex performs initial resection of the DSB in conjunction with DNA endonuclease SAE2; further progressive processing can also be performed using EXO1 or DNA2 nucleases. ...
... This end processing (thought to be performed by MRE11 at the perception stage) creates compatible ends for rejoining but disregards sequence homology. This process contrasts to HR which utilises sister chromatids during S phase to act as a template for accurate repair (Bernstein and Rothstein 2009). ...
... DNA double-strand breaks (DSBs) are highly cytotoxic lesions that, if unrepaired or repaired incorrectly, can cause cell death, mutations, and chromosomal instability. DSBs are repaired by two major pathways-nonhomologous end joining (NHEJ) and homologous recombination (HR) [1][2][3]. NHEJ involves errorprone end-to-end ligation of the DSB end, and this pathway is most prevalent in the G1 phase of the cell cycle [1][2][3]. In contrast, HR allows precise repair of DSBs and occurs primarily in the S and G2 phase [1][2][3]. ...
... DSBs are repaired by two major pathways-nonhomologous end joining (NHEJ) and homologous recombination (HR) [1][2][3]. NHEJ involves errorprone end-to-end ligation of the DSB end, and this pathway is most prevalent in the G1 phase of the cell cycle [1][2][3]. In contrast, HR allows precise repair of DSBs and occurs primarily in the S and G2 phase [1][2][3]. ...
... NHEJ involves errorprone end-to-end ligation of the DSB end, and this pathway is most prevalent in the G1 phase of the cell cycle [1][2][3]. In contrast, HR allows precise repair of DSBs and occurs primarily in the S and G2 phase [1][2][3]. HR depends on the presence of a sister chromatid and requires extensive DNA-end resection to generate stretches of single-stranded DNA (ssDNA) that can invade the homologous DNA strand [1][2][3]. DNA-end resection occurs via a two-step process that can be divided into resection initiation and resection extension [1][2][3]. ...
Background:
Repair of DNA double-strand breaks (DSBs) by homologous recombination requires 5'-3' resection of the DSB ends. In vertebrates, DSB resection is initiated by the collaborative action of CtIP and the MRE11-RAD50-NBS1 (MRN) complex. However, how this process occurs within the context of chromatin is still not well understood.
Results:
Here we identify the human SRCAP chromatin remodeling complex as a factor that promotes CtIP-dependent DNA-end resection. We show that SRCAP, which is mutated in Floating-Harbor syndrome, confers resistance to DNA damage-inducing agents and is recruited to DSBs. Moreover, we demonstrate that SRCAP is required for DNA-end resection, and thereby for recruitment of RPA and RAD51 to DSBs, and for the ensuing homologous recombination. Finally, we reveal that SRCAP forms a complex with CtIP and promotes accumulation of CtIP at DSBs through a mechanism involving its ATPase activity.
Conclusions:
Our study implicates the human SRCAP chromatin remodeling complex as a novel regulator of DNA damage responses that orchestrates proper signaling and repair of DSBs in the context of chromatin.
... In contrast to NHEJ, HR is a complex, multi-step repair pathway that requires the sequential activity of a cohort of proteins and occurs primarily in the S and G2 phases of the cell cycle [2][3][4][5][6] . HR relies on the presence of a sister chromatid as a donor template, and is initiated by nuclease-mediated extensive 5′-3′ resection of DSB ends, resulting in long stretches of 3′ single-stranded DNA (ssDNA) that subsequently invades the homologous duplex DNA [12][13][14] . It is now well-established that DSBs are resected in a two-step manner [12][13][14] . ...
... HR relies on the presence of a sister chromatid as a donor template, and is initiated by nuclease-mediated extensive 5′-3′ resection of DSB ends, resulting in long stretches of 3′ single-stranded DNA (ssDNA) that subsequently invades the homologous duplex DNA [12][13][14] . It is now well-established that DSBs are resected in a two-step manner [12][13][14] . Initially, the evolutionarily-conserved MRE11-RAD50-NBS1/XRS2 (MRN/X) complex and its associated factor CtIP/Sae2 carry out limited resection near the break site to generate a short 3′ overhang [15][16][17][18][19] . ...
DNA double-strand breaks (DSBs) are mainly repaired by either homologous recombination (HR) or non-homologous end-joining (NHEJ). Here, we identify AUNIP/C1orf135, a largely uncharacterized protein, as a key determinant of DSB repair pathway choice. AUNIP physically interacts with CtIP and is required for efficient CtIP accumulation at DSBs. AUNIP possesses intrinsic DNA-binding ability with a strong preference for DNA substrates that mimic structures generated at stalled replication forks. This ability to bind DNA is necessary for the recruitment of AUNIP and its binding partner CtIP to DSBs, which in turn drives CtIP-dependent DNA-end resection and HR repair. Accordingly, loss of AUNIP or ablation of its ability to bind to DNA results in cell hypersensitivity toward a variety of DSB-inducing agents, particularly those that induce replication-associated DSBs. Our findings provide new insights into the molecular mechanism by which DSBs are recognized and channeled to the HR repair pathway.
... As outlined above, the defining feature of HR is the utilization of the sister chromatid to guide repair of a DSB during the S and G2 phases of the cell cycle [32]. During HR, DSBs are resected to give extensive 3′ single-strand DNA (ssDNA) overhangs on each side of the break [33,34]. ...
... As outlined above, the defining feature of HR is the utilization of the sister chromatid to guide repair of a DSB during the S and G2 phases of the cell cycle [32]. During HR, DSBs are resected to give extensive 3 single-strand DNA (ssDNA) overhangs on each side of the break [33,34]. ...
Genomic integrity is of outmost importance for the survival at the cellular and the organismal level and key to human health. To ensure the integrity of their DNA, cells have evolved maintenance programs collectively known as the DNA damage response. Particularly challenging for genome integrity are DNA double-strand breaks (DSB) and defects in their repair are often associated with human disease, including leukemia. Defective DSB repair may not only be disease-causing, but further contribute to poor treatment outcome and poor prognosis in leukemia. Here, we review current insight into altered DSB repair mechanisms identified in leukemia. While DSB repair is somewhat compromised in all leukemic subtypes, certain key players of DSB repair are particularly targeted: DNA-dependent protein kinase (DNA-PK) and Ku70/80 in the non-homologous end-joining pathway, as well as Rad51 and breast cancer 1/2 (BRCA1/2), key players in homologous recombination. Defects in leukemia-related DSB repair may not only arise from dysfunctional repair components, but also indirectly from mutations in key regulators of gene expression and/or chromatin structure, such as p53, the Kirsten ras oncogene (K-RAS), and isocitrate dehydrogenase 1 and 2 (IDH1/2). A detailed understanding of the basis for defective DNA damage response (DDR) mechanisms for each leukemia subtype may allow to further develop new treatment methods to improve treatment outcome and prognosis for patients.
... HR has been investigated extensively in bacteria and eukarya. It has been suggested that in eukaryotes, DSBs that occur in the G1 phase of the cell cycle are most likely to be repaired via NHEJ, while those occurring in the S/G2 phase are preferentially processed via HR [5,6]. The HR machinery is comprised of a core protein complex containing Mre11-Rad50-Nbs1 (human, MRN) or Mre11-Rad50-Xrs2 (Saccharomyces cerevisiae, MRX); this complex, in conjunction with Ctp1/CtIP (or Sae2), recognizes DSBs and removes few nucleotides to form early intermediates during HR [7][8][9][10][11][12][13]. EXO1/Exo1 nuclease, BLM/Sgs1 helicase, and DNA2/Dna2 nuclease bind to these intermediates and generate 3 0 -ssDNA overhangs [1,9,[11][12][13] that are then utilized in Rad51-dependent strand exchange [9,11,[14][15][16]. ...
... As shown in Fig 7C, the pattern observed was comparable to the one obtained for the single stranded ODN2 (Fig 7B). Moreover, comparing the effect of HerA on NurA in the absence (lanes [4][5][6] or in the presence (lanes 9-11) of ATP, once again, we observed a stronger stimulation when ATP was present. ...
The nuclease NurA and the ATPase HerA are present in all known thermophilic archaea and cooperate with the highly conserved MRE11/RAD50 proteins to facilitate efficient DNA double-strand break end processing during homologous recombinational repair. However, contradictory results have been reported on the exact activities and mutual dependence of these two enzymes. To understand the functional relationship between these two enzymes we deeply characterized Sulfolobus solfataricus NurA and HerA proteins. We found that NurA is endowed with exo- and endonuclease activities on various DNA substrates, including linear (single-stranded and double stranded) as well as circular molecules (single stranded and supercoiled double-stranded). All these activities are not strictly dependent on the presence of HerA, require divalent ions (preferably Mn2+), and are inhibited by the presence of ATP. The endo- and exonculease activities have distinct requirements: whereas the exonuclease activity on linear DNA fragments is stimulated by HerA and depends on the catalytic D58 residue, the endonuclease activity on circular double-stranded DNA is HerA-independent and is not affected by the D58A mutation. On the basis of our results we propose a mechanism of action of NurA/HerA complex during DNA end processing.
... The first step of HR involves extensive processing of the DSB such that the 5' ends of the DNA duplex that flank the DSB are resected to generate long, 3' single-stranded tails 127 . ...
... These lesions are primarily repaired via two distinct pathways: non-homologous end joining during the G1 cell phase and homologous recombination during later cell phases after DNA synthesis 23,24,26,122,123 . The hallmark of homologous recombination is the processing of the DNA ends to reveal 3' single stranded DNA overhangs (known as resection) that subsequently searches for a homologous region of the genome to use as a template for repair 127 . A major hurdle for repair in this process is the chromatin structure, which must be modified to allow for repair machinery to access the DNA. ...
DNA double-strand break (DSB) repair is essential for maintenance of genome stability. However, the compaction of the eukaryotic genome into chromatin creates an inherent barrier to any DNA-mediated event, such as during DNA repair. This demands that there be mechanisms to modify the chromatin structure and thus access DNA. Recent work has implicated a host of chromatin regulators in the DNA damage response and several functional roles have been defined. Yet the mechanisms that control their recruitment to DNA lesions, and their relationship with concurrent histone modifications, remain unclear. We find that efficient DSB recruitment of many yeast chromatin regulators is cell-cycle dependent. Furthering this, we find recruitment of the INO80, SWR-C, NuA4, SWI/SNF, and RSC enzymes is inhibited by the non-homologous end joining machinery, and that their recruitment is controlled by early steps of homologous recombination. Strikingly, we find no significant role for H2A.X phosphorylation (γH2AX) in the recruitment of chromatin regulators, but rather that their recruitment coincides with reduced levels of γH2AX. We go on to determine the chromatin remodeling enzyme Fun30 functions in histone dynamics surround a DSB, but does not significantly affect γH2AX dynamics. Additionally, we describe a conserved functional interaction among the chromatin remodeling enzyme, SWI/SNF, the NuA4 and Gcn5 histone acetyltransferases, and phosphorylation of histone H2A.X. Specifically, we find that the NuA4 and Gcn5 enzymes are both required for the robust recruitment of SWI/SNF to a DSB, which in turn promotes the phosphorylation of H2A.X.
... Besides t 6 A modification, KEOPS also plays a role in homologous recombination (HR) repair and telomere maintenance in budding yeast, both of which are critical for genome integrity and cell survival 13,14 . End resection of DNA double-strand breaks (DSBs) is an essential step in HR repair 15,16 . The Mre11-Rad50-Xrs2/Mre11-Rad50-NBS1(MRX/N) complex, in conjunction with Sae2/CtIP, initiates the resection, which is followed by two distinct pathways mediated by Exo1/EXO1 and Dna2/DNA2-Sgs1/BLM, respectively, generating a long 3ʹ-tail of single-stranded DNA (ssDNA) [17][18][19][20] . ...
Kinase, putative Endopeptidase, and Other Proteins of Small size (KEOPS) is a multisubunit protein complex conserved in eukaryotes and archaea. It is composed of Pcc1, Kae1, Bud32, Cgi121, and Gon7 in eukaryotes and is primarily involved in N 6-threonylcarbamoyl adenosine (t 6 A) modification of transfer RNAs (tRNAs). Recently, it was reported that KEOPS participates in homologous recombination (HR) repair in yeast. To characterize the KEOPS in archaea (aKEOPS), we conducted genetic and biochemical analyses of its encoding genes in the hyperthermophilic archaeon Saccharolobus islandicus. We show that aKEOPS also possesses five subunits, Pcc1, Kae1, Bud32, Cgi121, and Pcc1-like (or Gon7-like), just like eukaryotic KEOPS. Pcc1-like has physical interactions with Kae1 and Pcc1 and can mediate the monomerization of the dimeric sub-complex (Kae1-Pcc1-Pcc1-Kae1), suggesting that Pcc1-like is a functional homolog of the eukaryotic Gon7 subunit. Strikingly, none of the genes encoding aKEOPS subunits, including Pcc1 and Pcc1-like, can be deleted in the wild type and in a t 6 A modification complementary strain named TsaKI, implying that the aKEOPS complex is essential for an additional cellular process in this archaeon. Knock-down of the Cgi121 subunit leads to severe growth retardance in the wild type that is partially rescued in TsaKI. These results suggest that aKEOPS plays an essential role independent of the cellular t 6 A modification level. In addition, archaeal Cgi121 possesses dsDNA-binding activity that relies on its tRNA 3ʹ CCA tail binding module. Our study clarifies the subunit organization of archaeal KEOPS and suggests an origin of eukaryotic Gon7. The study also reveals a possible link between the function in t 6 A modification and the additional function, presumably HR.
... These findings suggest that the effects of sperm DNA damage on ICSI outcomes may be hidden when oocyte quality is diminished, and thus support that oocyte alterations have a larger influence on embryo development than sperm DNA breaks do. This is in agreement with previous research reporting that oocyte quality is one of the main causes of embryo aneuploidy and implantation failure [29,61,62]. Conversely, when oocytes from greater quality-because they come from donors-were involved, ICSI outcomes were better and it was thus easier to determine the influence of sperm DNA integrity on embryo development. ...
Abstract Based on the inconsistent literature published thus far involving infertile patients, whether intracytoplasmic sperm injection (ICSI) allows overcoming total fertilization failure due to sperm DNA fragmentation is still unclear. Related to this, female factors, which may have a significant impact on assisted reproduction outcomes, can mask male infertility. In this scenario, evaluating ICSI outcomes following cycles using healthy donor gametes could shed light on this realm, as it would avoid the influence of (un)known confounding factors present in infertile individuals. The present work, therefore, aimed to address whether single- and double-stranded sperm DNA fragmentation leads to impaired ICSI outcomes in double gamete donation cycles. The study also compared these double-gamete donation cycles to cycles in which only sperm were donated and oocytes were obtained from infertile patients. Two cohorts were included: (a) the Donor-Donor (DD) cohort, which included 27 semen donor samples used in 49 ICSI cycles with young healthy oocyte donors; and (b) the Donor-Infertile (DI) cohort, which involved 34 semen donor samples used in 57 ICSI cycles with oocytes from patients. Single- and double-stranded sperm DNA breaks were determined with alkaline and neutral Comet assays, respectively; ICSI was conducted following standard protocols and embryos were monitored through time-lapse microscopy. In the DD cohort, the percentage of sperm with high overall DNA damage correlated with fertilization rates (Rs = − 0.666; P
... However, oocytes from women of advanced maternal age have diminished repair functions compared to young women [122]. Single-stranded DNA breakage is more likely to be repaired than double-stranded DNA breakage [123,124]. Therefore, the final impact of sperm DNA fragmentation on the pregnancy outcome still relies on the balance of the oocyte repairing system and the extent of sperm DNA damage [90]. ...
Male factor infertility contribute approximately at 50% for the cause of infertility. The steady declination of semen quality in men for all over the world might be from various factors such as life style changes, environmental toxicity, dietary contribution and social problems. Assisted reproduction is the main treatment of choice for male infertility; However, in severe male factor infertility, the treatment outcomes could end up with recurrent implantation failure or recurrent pregnancy loss. Basic semen analysis still has limitation to explain the cause of failure for the part of male factors. The purposes of developing new sperm evaluation methods are to improve the diagnostic tools for identifying the sperm defects, appraise of fertility potential and provide suitable treatment for an infertile couple, explain the cause of treatment failure from male factor part and measure the efficacy of male contraception.
... Mechanistically, MMS22L interacts with TONSL and is required for efficient RAD51 foci formation (68), whereas EXD2 is an interactor of the MRE11-RAD50-NBS1 (MRN) complex necessary for DNA end resection upstream of RAD51 foci formation (10). Importantly, MRE11A, RAD50 and NBS1 are also known targets of miR-494-3p (69) and EXO1, which mediates long-range end resection following the initial resection facilitated by the MRN complex (70,71), is among the predicted targets of miR-494-3p. Taken together, these observations strongly suggest that miR-494-3p exerts a multi-target based inhibition during the initial stages of HR and they can readily explain the strong regulatory effect of transient mir-494-3p transfection on RAD51 foci formation, despite its only weak effects on RAD51D. ...
The Fanconi anemia (FA) and homologous recombination (HR) pathways, which partially overlap and include RAD51 and its paralogs, are key for the repair of different types of DNA damage, such as DNA interstrand crosslinks. Here, as a first step to broadly assess the impact of microRNA-mediated regulation, we examined microRNA expression profiles in five isogenic fibroblast cell pairs, either deficient in DNA repair due to germ-line mutations in FANCA, FANCB, FANCC, FANCI or BRIP1/FANCJ or proficient due to correction with retroviral vectors. In each pair, we observed lower abundance of specific microRNAs in the FA-deficient cells. From the list of microRNAs, we experimentally confirmed the effects of miR-141-3p and miR-369-3p targeting RAD51B and miR-15a-5p, miR-494-3p as well as miR-544a targeting RAD51D. However, by western blotting, only RAD51D protein was reduced by a mixture of its regulating microRNAs. Gene ontology analyses, and identification of additional FA/HR factors as targets of miR-15a-5p, miR-494-3p and miR-544a strongly suggested the widespread influence of these three microRNAs on HR. Interestingly, only miR-494-3p directly reduced RAD51 foci formation, while a mixture of miR-15a-5p, miR-494-3p and miR-544a strongly reduced HR activity in GFP repair assays. Summarized, by successfully employing this novel loss- and gain-of-function strategy, we have identified new microRNAs strongly inhibiting HR in mammalian cells. Understanding and modulating such miRNA regulation of DNA repair genes/pathways might help to overcome the reduced repair capacity of FA patients with bi-allelic hypomorphic mutations or help to engineer synthetic lethality strategies for patients with mutations in cancer-associated FA/HR genes.
... While NHEJ can occur throughout the cell cycle, HR is limited to S and G2 phases because of the presence of the homologous copy of the damaged DNA. The choice between these two pathways is dictated by a DNA mechanism known as DNA end resection, a tightly regulated machinery that ensures genomic stability [4,5]. During this process, DNA ends are resected through the joint action of helicases and nucleases that unwind the DNA duplex and generate 3 overhangs required in S and G2 phases for the subsequent repair steps of HR [6,7]. ...
Generation of the 3' overhang is a critical step during homologous recombination (HR) and replication fork rescue processes. This event is usually performed by a series of DNA nucleases and/or helicases. The nuclease NurA and the ATPase HerA, together with the highly conserved MRE11/RAD50 proteins, play an important role in generating 3' single-stranded DNA during archaeal HR. Little is known, however, about HerA-NurA function and activation of this fundamental and complicated DNA repair process. Herein, we analyze the functional relationship among NurA, HerA and the single-strand binding protein SSB from Saccharolubus solfataricus. We demonstrate that SSB clearly inhibits NurA endonuclease activity and its exonuclease activities also when in combination with HerA. Moreover, we show that SSB binding to DNA is greatly stimulated by the presence of either NurA or NurA/HerA. In addition, if on the one hand NurA binding is not influenced, on the other hand, HerA binding is reduced when SSB is present in the reaction. In accordance with what has been observed, we have shown that HerA helicase activity is not stimulated by SSB. These data suggest that, in archaea, the DNA end resection process is governed by the strictly combined action of NurA, HerA and SSB.
... DFS70/LEDGFp75 enhances DNA repair via recruiting replication protein A 32kDa subunit (RPA32) to DNA damage foci (Bernstein and Rothstein, 2009 via its high-affinity interaction with the HIV integrase (Debyser et al., 2015;Shun et al., 2008). HIV binds to the C-terminal IBD (Figure 1) of DFS70/LEDGFp75, this stabilizes HIV integrase and leads to DFS70/LEDGFp75-mediated shuffling of HIV into the nucleus, promoting the viral integration into transcriptionally active sites (Debyser et al., 2015;Maertens et al., 2003). ...
Lens epithelium derived growth factor (LEDGF), is known to be overexpressed in different solid cancers and cancer cell lines but is also involved in acquired immunodeficiency syndrome (AIDS) and diverse inflammatory diseases. Due to its chromatin-binding ability, it acts as a transcriptional coactivator, promoting anti-apoptotic pathways leading to increased tumor aggressiveness and chemotherapy resistance. In this thesis, the role of LEDGF in laryngeal cancer cells was investigated. Thus, a knock-out of LEDGF in HEp-2 cells was successfully generated by CRISPR/Cas9 technology. Moreover, LEDGF-deficient HEp-2 cells exhibited a decreased proliferation and migration, as well as an increased chemotherapy sensitivity towards topoisomerase II inhibitor etoposide. The DNA damage response was in more detail investigated, where LEDGF depleted cells showed a drastic reduction in the recruitment of downstream damage-response related proteins, more precisely replication protein A subunit of 32kDa (RPA32), and an increased amount of DNA fragmentation, highlighting the role of LEDGF in homology-directed DNA repair. In addition, a efficient method was established to allow genomic tagging of LEDGF at its C-terminus with EGFP which enables the monitoring of LEDGF in living cells. Therefore, an efficient Cas9-mediated DNA DSB induction was supplemented with a DNA template containing homology arms to the C-terminus as well as the EGFP sequence to allow homology-directed repair. Subsequently, EGFP-expressing cells were FACS sorted and single cells expanded. Almost all test clones showed a homozygous genotype for LEDGF-EGFP fusion which exhibited the typical nuclear pattern of LEDGF.
... HR is considered error-free because it uses a homologous template for repair to restore any missing nucleotides at the break site (12). After DSB formation, the DNA ends are resected and coated by the single-stranded DNA (ssDNA)-binding heterotrimer replication protein A (RPA), consisting of RFA1, RFA2 and RFA3 ( Figure 1A) (13). Subsequently, RPA becomes displaced by RAD51, which forms a nucleoprotein filament of RAD51 protomers around the ssDNA end (Figure 1B) (14). ...
Regulation of homologous recombination (HR) is central for cancer prevention. However, too little HR can increase cancer incidence, whereas too much HR can drive cancer resistance to therapy. Importantly, therapeutics targeting HR deficiency have demonstrated a profound efficacy in the clinic improving patient outcomes, particularly for breast and ovarian cancer. RAD51 is central to DNA damage repair in the HR pathway. As such, understanding the function and regulation of RAD51 is essential for cancer biology. This review will focus on the role of RAD51 in cancer and beyond and how modulation of its function can be exploited as a cancer therapeutic.
... Both the oocyte repair capacity and the type and/or complexity of SDF vary from one cell to another, thus differentially affecting the em-bryo′s implantation potential. While both SS-DBs and DS-DBs can be repaired at the same DNA strand by direct ligation of 5′-3′ free ends, thereby evading the production of structural chromosomal abnormalities (Obe et al., 2002;van Gent et al., 2001), DS-DNA breaks are more difficult to repair because there is no complementary strand to use as a template (Bernstein & Rothstein, 2009;Price & D′Andrea, 2013). Unrepaired DNA motifs may produce chromosomal rearrangements, which can generate high levels of genome instability. ...
We herein summarise the evidence concerning the impact of sperm DNA fragmentation in various clinical infertility scenarios and the advances on sperm DNA fragmentation tests. The collected evidence was used to formulate 41 recommendations. Of these, 13 recommendations concern technical aspects of sperm DNA fragmentation testing, including pre-analytical information, clinical thresholds and interpretation of results. The remaining 28 recommendations relate to indications for sperm DNA fragmentation testing and clinical management. Clinical scenarios like varicocele, unexplained infertility, idiopathic infertility, recurrent pregnancy loss, intrauterine insemination, in vitro fertilisation/intracytoplasmic sperm injection, fertility counselling for men with infertility risk factors and sperm cryopreservation have been contemplated. The bulk evidence supporting the recommendations has increased in recent years, but it is still of moderate to low quality. This practice guideline provides clinicians with advice on best practices in sperm DNA fragmentation testing. Also, recommendations are provided on possible management strategies to overcome infertility related to sperm DNA fragmentation, based on the best available evidence. Lastly, we identified gaps in knowledge and opportunities for research and elaborated a list of recommendations to stimulate further investigation concerning sperm DNA fragmentation testing and management of sperm DNA fragmentation-related infertility conditions.
... HR initiates DSB repair by resecting DSBs, leading to the formation of 39 single-strand overhangs, followed by polymerization of Rad51 on the single-strand DNA (31)(32)(33). The resulting Rad51 nucleoprotein filaments undergo homology search and pairing with the intact duplex DNA donor to form joint molecules (JMs) such as double Holliday junctions (dHJs) with the help of Rad54 (33)(34)(35). ...
Homologous recombination (HR) repairs DNA double-strand breaks using intact homologous sequences as template DNA. Broken DNA and intact homologous sequences form joint molecules (JMs), including Holliday junctions (HJs), as HR intermediates. HJs are resolved to form crossover and noncrossover products. A mismatch repair factor, MLH3 endonuclease produces the majority of crossovers during meiotic HR, but it remains elusive whether mismatch repair factors promote HR in non-meiotic cells. We disrupted genes encoding the MLH3 and PMS2 endonucleases in the human B cell line, TK6, generating null MLH3-/- and PMS2-/- mutant cells. We also inserted point mutations into the endonuclease motif of MLH3 and PMS2 genes, generating endonuclease death MLH3DN/DN and PMS2EK/EK cells. MLH3-/- and MLH3DN/DN cells showed a very similar phenotype, a 2.5 times decrease in the frequency of heteroallelic HR-dependent repair of a restriction-enzyme-induced double-strand breaks. PMS2-/- and PMS2EK/EK cells showed a phenotype very similar to that of the MLH3 mutants. These data indicate that MLH3 and PMS2 promote HR as an endonuclease. The MLH3DN/DN and PMS2EK/EK mutations had an additive effect on the heteroallelic HR. MLH3DN/DN/PMS2EK/EK cells showed normal kinetics of g-irradiation-induced Rad51 foci but a significant delay in the resolution of Rad51 foci and three times decrease in the number of cisplatin-induced sister chromatid exchange (SCE). The ectopic expression of the Gen1 HJ resolvase partially reversed the defective heteroallelic HR of MLH3DN/DN/PMS2EK/EK cells. Taken together, we propose that MLH3 and PMS2 promote HR as endonucleases, most likely by processing JMs in mammalian somatic cells.
... Several studies showed that the generation of RPA-coated ssDNAs is an intermediate step not only for HR repair but also for ATR/Chk1 activation. [67,68] We found that BRD7 depletion also inhibited HU-induced RPA2 focus formation, suggesting that BRD7 depletion impairs RPA2 recruitment in response to DNA damage (CPT or IR), as well as replication stress ( Figure 6C,D). Indeed, we also found that phosphorylation of BRD7 at Thr 515 by ATR was important for the activation of ATR-CHK1 signaling pathway but not for ATM-CHK2 pathway ( Figure S10A,E, Supporting Information). ...
Repair of DNA double‐strand breaks (DSBs) is essential for genome integrity, and is accompanied by transcriptional repression at the DSB regions. However, the mechanisms how DNA repair induces transcriptional inhibition remain elusive. Here, it is identified that BRD7 participates in DNA damage response (DDR) and is recruited to the damaged chromatin via ATM signaling. Mechanistically, BRD7 joins the polycomb repressive complex 2 (PRC2), the nucleosome remodeling and histone deacetylation (NuRD) complex at the damaged DNA and recruits E3 ubiquitin ligase RNF168 to the DSBs. Furthermore, ATM‐mediated BRD7 phosphorylation is required for recruitment of the PRC2 complex, NuRD complex, DSB sensor complex MRE11‐RAD50‐NBS1 (MRN), and RNF168 to the active transcription sites at DSBs, resulting in transcriptional repression and DNA repair. Moreover, BRD7 deficiency sensitizes cancer cells to PARP inhibition. Collectively, BRD7 is crucial for DNA repair and DDR‐mediated transcription repression, which may serve as a therapeutic target. The findings identify the missing link between DNA repair and transcription regulation that maintains genome integrity. Repair of DNA double‐strand breaks (DSBs) is essential for genome integrity, and is accompanied by transcriptional repression at the DSB regions. ATM‐mediated BRD7 phosphorylation at the Ser 263 site is required for recruitment of the PRC2 complex, NuRD complex, DSB sensor complex MRE11‐RAD50‐NBS1 (MRN) and RNF168 to the active transcription sites at DSBs, resulting in transcriptional repression and DNA repair.
... The other DSB repair pathways depend on DNA end resection, i.e. the generation of single stranded DNA (ssDNA). Alternative end joining (alt-EJ) utilizes PARP-1-mediated annealing at short homologous DNA sequences (microhomologies) 8,9 . Homology directed repair (HDR) and single strand annealing (SSA), however, both depend on extensive end resection, as replication factor A (RPA)-a crucial initiation factor for both pathways-can only bind to ssDNA. ...
Severe side effects often restrict clinical application of the widely used chemotherapeutic drug doxorubicin. In order to decrease required substance concentrations, new concepts for successful combination therapy are needed. Since doxorubicin causes DNA damage, combination with compounds that modulate DNA repair could be a promising strategy. Very recently, a role of nuclear actin for DNA damage repair has been proposed, making actin a potential target for cancer therapy in combination with DNA-damaging therapeutics. This is of special interest, since actin-binding compounds have not yet found their way into clinics. We find that low-dose combination treatment of doxorubicin with the actin polymerizer chondramide B (ChB) synergistically inhibits tumor growth in vivo. On the cellular level we demonstrate that actin binders inhibit distinctive double strand break (DSB) repair pathways. Actin manipulation impairs the recruitment of replication factor A (RPA) to the site of damage, a process crucial for homologous recombination. In addition, actin binders reduce autophosphorylation of DNA-dependent protein kinase (DNA-PK) during nonhomologous end joining. Our findings substantiate a direct involvement of actin in nuclear DSB repair pathways, and propose actin as a therapeutic target for combination therapy with DNA-damaging agents such as doxorubicin.
... DSB are the most lethal alteration that may happen in a zygote, since paternal and maternal pronucleus remain separated in early mammalian embryos and, therefore, no complementary chain would be available for DNA repair [73][74][75] and a few number of DSB are sufficient to delay cell cycle [76]. It is important to note that paternal double-strand breaks remain attached to the nuclear matrix and probably to other proteins such as TOP2B [20,46,77] and the nuclear matrix is inherited at male pronucleus until first mitotic division [49,78]. ...
Reproductive diseases have become a growing worldwide problem and male factor plays an important role in the reproductive diagnosis, prognosis and design of assisted reproductive treatments. Sperm cell holds the mission of carrying the paternal genetic complement to the oocyte in order to contribute to an euploid zygote with proper DNA integrity. Sperm DNA fragmentation had been used for decades as a male fertility test, however, its usefulness have arisen multiple debates, especially around Intracytoplasmic Sperm Injection (ICSI) treatments. In the recent years, it has been described that different types of sperm DNA breaks (single and double strand DNA breaks) cause different clinical reproductive effects. On one hand, single-strand DNA breaks are present extensively as a multiple break points in all regions of the genome, are related to oxidative stress and cause a lack of clinical pregnancy or an increase of the conception time. On the other hand, double-strand DNA breaks are mainly localized and attached to the sperm nuclear matrix as a very few break points, are possibly related to a lack of DNA repair in meiosis and cause a higher risk of miscarriage, low embryo quality and higher risk of implantation failure in ICSI cycles. The present work also reviews different studies that may contribute in the understanding of sperm chromatin as well as treatments to prevent sperm DNA damage.
... Les résolvasespermettent la résolution des structures formées sous forme de jonction de Hollyday. (Bernstein and Rothstein, 2009). Les noms en magenta correspondent aux noms des facteurs humains quand ils sont différents des noms des protéines de levure (noir) ...
Au cours du cycle cellulaire, de nombreux agents génotoxiques conduisent à la formation de cassures double brins (CDB) de d’ADN. Ces lésions extrêmement dangereuses doivent être réparées afin de ne pas altérer l’intégrité du génome et la survie cellulaire. Chez les mammifères, la plupart de ces CDB sont prise en charge par la voie de réparation NHEJ (pour Non Homologous End Joining, recombinaison non homologue en français). La première étape de cette voie, la reconnaissance des extrémités d’ADN, est assurée par l’hétérodimère Ku70-Ku80. Ce complexe protège les extrémités d’ADN des activités de résection et recrute les facteurs impliqués dans la poursuite de la voie de réparation (la DNA-PKcs, XLF et le complexe de ligation XRCC4-LigIV) ainsi que des facteurs accessoires (PAXX, WRN et APLF). De cette observation, une vision plus générale de la fonction de Ku a émergé, une fonction de coordination de la voie NHEJ. La manière dont Ku interagit avec tous les facteurs du NHEJ, pour la réparation de toutes les CDB (propres ou non, accidentelles ou programmées) reste une question ouverte. Notre équipe travaillait précédemment sur l’étude des facteurs XRCC4 et XLF, dont ils ont pu mettre en évidence l’association en filaments dans un cristal.Au cours de ma thèse, j’ai caractérisé les structures cristallines de complexes comprenant Ku70-Ku80, un ADN et des peptides portant un motif de liaison à Ku (KBM) issus des protéines APLF et XLF. Le motif KBM de APLF se fixe de manière très forte à Ku (avec une affinité de 33 nM) et interagit avec une surface conservée sur Ku80. Cette interaction se déroule loin du site de fixation de Ku à l’ADN, à la périphérie du complexe. La connaissance des détails moléculaires de cette interaction a permis de comprendre et de rationnaliser la présence de résidus très conservés à la surface de Ku80 et dans la séquence d’APLF.Le motif KBM présent chez XLF, présentent une affinité plus modérée (un Kd de quelques μM) malgré une similarité de séquence importante avec le KBM de APLF (une région basique suivi d’une région hydrophobe). La structure du complexe Ku70-Ku80-ADN et du KBM de XLF montre que ce peptide se fixe sur un site différent de celui d’APLF. En effet ce peptide induit un large changement de conformation de Ku80 et vient se loger dans le sillon ainsi découvert. Par la suite, j’ai caractérisé deux mutants du KBM de XLF (L297E et L297W) qui respectivement abroge et renforce l’interaction de ce motif KBM avec Ku.Ensemble ces résultats confortent notre idée que Ku joue le rôle de chef d’orchestre de la voie de réparation NHEJ chez l’humain.
... On the contrary, HR is a highfidelity mechanism since it relies on homologous DNA sequences generating error-free products (Niu et al 2010;Longhese et al 2010). It has been suggested that in eukaryotes, DSBs that take place in the G1 phase of the cell cycle are most probably repaired via NHEJ, while those occurring in the S/G2 phase are mainly processed via HR (Bernstein and Rothstein 2009;Chapman et al 2012). During HR a core protein complex formed by Mre11-Rad50-Nbs1/ Mre11-Rad50-Xrs2 (human MRN and Saccharomyces cerevisiae MRX, respectively) in conjunction with Ctp1/ CtIP (or Sae2), recognizes the lesion and removes a few nucleotides to form an early intermediate of HR (Lengsfeld et al 2007;Nicolette et al 2010;Mimitou and Symington 2008;Cejka et al 2010). ...
ATPase/Helicases and nucleases play important roles in DNA end-resection, a critical step during homologous recombination repair in all organisms. In hyperthermophilic archaea the exo-endonuclease NurA and the ATPase HerA cooperate with the highly conserved Mre11-Rad50 complex in 3′ single-stranded DNA (ssDNA) end processing to coordinate repair of double-stranded DNA breaks. Little is known, however, about the assembly mechanism and activation of the HerA-NurA complex. In this study we demonstrate that the NurA exonuclease activity is inhibited by the Sulfolobus solfataricus RecQ-like Hel112 helicase. Inhibition occurs both in the presence and in the absence of HerA, but is much stronger when NurA is in complex with HerA. In contrast, the endonuclease activity of NurA is not affected by the presence of Hel112. Taken together these results suggest that the functional interaction between NurA/HerA and Hel112 is important for DNA end-resection in archaeal homologous recombination.
... CDKs, RNF138, chromatin remodeling factors, and others (14,(54)(55)(56)(57). Initiation of DNA end resection by the MRN complex and CtIP removes Ku from DNA ends to generate ssDNA overhangs that not only inhibit NHEJ but also provide a platform to recruit proteins involved in HR repair (47). DNA end resection is a 2-step process in which the MRN complex and CtIP are involved in the initial step, and EXO1 and DNA2-BLM are involved in the second step (54,58). CDKs promote these 2 steps in the process of DNA resection by phosphorylating CtIP, NBS1, or EXO1, respectively (14,55). ...
DNA double-strand breaks (DSBs) are mainly repaired either by homologous recombination (HR) or by nonhomologous end-joining (NHEJ) pathways. Here, we showed that myeloid cell leukemia sequence 1 (Mcl-1) acts as a functional switch in selecting between HR and NHEJ pathways. Mcl-1 was cell cycle-regulated during HR, with its expression peaking in S/G2 phase. While endogenous Mcl-1 depletion reduced HR and enhanced NHEJ, Mcl-1 overexpression resulted in a net increase in HR over NHEJ. Mcl-1 directly interacted with the dimeric Ku protein complex via its Bcl-2 homology 1 and 3 (BH1 and BH3) domains, which are required for Mcl-1 to inhibit Ku-mediated NHEJ. Mcl-1 also promoted DNA resection mediated by the Mre11 complex and HR-dependent DSB repair. Using the Mcl-1 BH1 domain as a docking site, we identified a small molecule, MI-223, that directly bound to BH1 and blocked Mcl-1-stimulated HR DNA repair, leading to sensitization of cancer cells to hydroxyurea- or olaparib-induced DNA replication stress. Combined treatment with MI-223 and hydroxyurea or olaparib exhibited a strong synergy against lung cancer in vivo. This mechanism-driven combination of agents provides a highly attractive therapeutic strategy to improve lung cancer outcomes.
... The key components of c-NHEJ include the DNA end-binding heterodimer Ku70/80, the kinase DNA-PKcs, the nuclease Artemis, the DNA ligase IV, the scaffolding proteins XRCC4 and XLF, and the newly characterized PAXX 4 In contrast to NHEJ, HR operates with slower kinetics and is executed primarily in the late S and G2 phases of the cell cycle when sister chromatids are available as repair templates 2,3 . HR is initiated by the 5′ to 3′ nucleolytic resection of DSB ends, a process mediated by the MRE11-RAD50-NBS1/XRS2 (MRN/X) complex in conjunction with CtIP/Sae2 that carries out limited resection, and the 5′-3′ exonuclease EXO1 or the helicase-nuclease protein complex BLM/Sgs1-DNA2 that carries out extensive resection 6,7 . The resulting 3′ single-stranded DNA (ssDNA) overhangs are rapidly coated by replication protein A (RPA) to prevent the formation of secondary structures such as hairpins 8 . ...
BRCA2-deficient cells exhibit gross genomic instability, but the underlying mechanisms are not fully understood. Here we report that inactivation of BRCA2 but not RAD51 destabilizes RPA-coated single-stranded DNA (ssDNA) structures at resected DNA double-strand breaks (DSBs) and greatly enhances the frequency of nuclear fragmentation following cell exposure to DNA damage. Importantly, these BRCA2-associated deficits are fueled by the aberrant activation of classical (c)- and alternative (alt)- nonhomologous end-joining (NHEJ), and rely on the well-defined DNA damage signaling pathway involving the pro-c-NHEJ factor 53BP1 and its downstream effector RIF1. We further show that the 53BP1–RIF1 axis promotes toxic end-joining events via the retention of Artemis at DNA damage sites. Accordingly, loss of 53BP1, RIF1, or Artemis prolongs the stability of RPA-coated DSB intermediates in BRCA2-deficient cells and restores nuclear integrity. We propose that BRCA2 antagonizes 53BP1, RIF1, and Artemis-dependent c-NHEJ and alt-NHEJ to prevent gross genomic instability in a RAD51-independent manner.
... The choice between these repair pathways is dictated by end resection, a DNA processing mechanism that selectively degrades the 5 strand DNA from the ends to generate long 3 ssDNA overhangs required for HR in S and G2 phases of the cell cycle. By converting dsDNA ends into ssDNA structures, resection promotes HR and averts NHEJ (11,(15)(16)(17)(18). DSB resection also controls the checkpoint responses that coordinate DNA repair with other cellular processes such as cell cycle progression and gene expression (19)(20)(21)(22). Checkpoint responses are controlled by ATM and ATR protein kinases, both of which are activated by DSBs (23)(24)(25). ...
Nucleolytic resection of DNA double-strand breaks (DSBs) is essential for both checkpoint activation and homology-mediated repair; however, the precise mechanism of resection, especially the initiation step, remains incompletely understood. Resection of blocked ends with protein or chemical adducts is believed to be initiated by the MRN complex in conjunction with CtIP through internal cleavage of the 5' strand DNA. However, it is not clear whether resection of clean DSBs with free ends is also initiated by the same mechanism. Using the Xenopus nuclear extract system, here we show that the Dna2 nuclease directly initiates the resection of clean DSBs by cleaving the 5' strand DNA ∼10-20 nucleotides away from the ends. In the absence of Dna2, MRN together with CtIP mediate an alternative resection initiation pathway where the nuclease activity of MRN apparently directly cleaves the 5' strand DNA at more distal sites. MRN also facilitates resection initiation by promoting the recruitment of Dna2 and CtIP to the DNA substrate. The ssDNA-binding protein RPA promotes both Dna2- and CtIP-MRN-dependent resection initiation, but a RPA mutant can distinguish between these pathways. Our results strongly suggest that resection of blocked and clean DSBs is initiated via distinct mechanisms.
... Lack of PARP activity through genetic modification or pharmacological inhibitors increases SSB counts. Unrepaired SSBs are then converted into DSBs at fork replication [50]; as a result, cells are flooded with DSBs and succumb to apoptotic cell death [51][52][53]. In most cell lines with intact DSB DNA repair mechanisms, treatment with PARP inhibitors at doses that successfully inhibit PARP activity does not cause cell death, providing an exquisite approach to specifically targeting cancer cells, especially those harboring mutant BRCA1 and BRCA2 [43][44][45]54]. ...
Liver kinase B1 (LKB1) functions as a tumor suppressor encoded by STK11, a gene that mutated in Peutz-Jeghers syndrome and in sporadic cancers. Previous studies showed that LKB1 participates in IR- and ROS-induced DNA damage response (DDR). However, the impact of LKB1 mutations on targeted cancer therapy remains unknown. Herein, we demonstrated that LKB1 formed DNA damage-induced nuclear foci and co-localized with ataxia telangiectasia mutated kinase (ATM), ?-H2AX, and breast cancer susceptibility 1 (BRCA1). ATM mediated LKB1 phosphorylation at Thr 363 following the exposure of cells to ionizing radiation (IR). LKB1 interacted with BRCA1, a downstream effector in DDR that is recruited to sites of DNA damage and functions directly in homologous recombination (HR) DNA repair. LKB1 deficient cells exhibited delayed DNA repair due to insufficient HR. Notably, LKB1 deficiency sensitized cells to poly (ADP-ribose) polymerase (PARP) inhibitors. Thus, we have demonstrated a novel function of LKB1 in DNA damage response. Cancer cells lacking LKB1 are more susceptible to DNA damage-based therapy and, in particular, to drugs that further impair DNA repair, such as PARP inhibitors.
... DNA end resection is a pivotal step in HR repair to produce 3' overhangs that not only inhibit NHEJ but also provide a platform to recruit proteins involved in HR repair [11]. DNA end resection is completed through a two-step process in which the MRN/X complex and CtIP/Sae2 protein are involved in the initial step, and EXO1 and DNA2-BLM/Sgs1 are involved in the second step [79]. However, precisely how these resection factors are regulated in a coordinated manner is still unclear. ...
DNA double-strand breaks (DSBs), which arise following exposure to a number of endogenous and exogenous agents, can be repaired by either the homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways in eukaryotic cells. A vital step in HR repair is DNA end resection, which generates a long 3′ single-stranded DNA (ssDNA) tail that can invade the homologous DNA strand. The generation of 3′ ssDNA is not only essential for HR repair, but also promotes activation of the ataxia telangiectasia and Rad3-related protein (ATR). Multiple factors, including the MRN/X complex, C-terminal-binding protein interacting protein (CtIP)/Sae2, exonuclease 1 (EXO1), Bloom syndrome protein (BLM)/Sgs1, DNA2 nuclease/helicase, and several chromatin remodelers, cooperate to complete the process of end resection. Here we review the basic machinery involved in DNA end resection in eukaryotic cells.
... NHEJ repairs the break through direct re-ligation of the broken DNA ends with no or limited end processing and thus is error-prone. By comparison, HR repairs the break in an error-free manner, and is initiated by nucleolytic processing of the 5′ ends of a DSB through a process called DNA end resection [8][9][10][11][12][13][14][15][16]. Resection occurs in 5′→3′ direction to generate 3′ ssDNA overhangs, which are initially bound by ssDNA-binding protein replication protein A (RPA) and then replaced by Rad51 during HR. ...
DNA end resection is a key process in the cellular response to DNA double-strand break damage that is essential for genome
maintenance and cell survival. Resection involves selective processing of 5′ ends of broken DNA to generate ssDNA overhangs,
which in turn control both DNA repair and checkpoint signaling. DNA resection is the first step in homologous recombination-mediated
repair and a prerequisite for the activation of the ataxia telangiectasia mutated and Rad3-related (ATR)-dependent checkpoint
that coordinates repair with cell cycle progression and other cellular processes. Resection occurs in a cell cycle-dependent
manner and is regulated by multiple factors to ensure an optimal amount of ssDNA required for proper repair and genome stability.
Here, we review the latest findings on the molecular mechanisms and regulation of the DNA end resection process and their
implications for cancer formation and treatment.
... Together with other DNA repair factors, including BRCA1 and MRN complex, RBBP8/CtIP catalyses an initial 5 -3 DNA resection of the broken DNA ends generating 3 single-stranded DNA (ssDNA) overhangs, which are rapidly coated by the heteromeric ssDNA-binding RPA complex (27)(28)(29). The RPA-coated ssDNA then triggers assembly of the homologous recombination repair signalling machinery governed by Serine/threonine-protein kinase ATR, which leads to the phosphorylation of multiple target proteins, including the RPA2 subunit of RPA (30). In order to assess the role of HDGFRP2 in the initial steps of homologous recombination, we first monitored the phosphorylation of RPA2 at S4/S8, a site whose phosphorylation depends on ssDNA-induced activation of ATR (31). ...
We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage.
... NHEJ can take place throughout the cell cycle but is predominant during the G1 phase when HR is not efficient (Frank-Vaillant and Marcand 2002). NHEJ differs also from HR by its conceptual simplicity, since it consists of a straightforward religation of the DSB gap, while HR requires sequence homology between damaged DNA and the template DNA used to religate the gap (Bernstein and Rothstein 2009;Chilton and Que 2003;Dudas and Chovanec 2004). HR is mainly active during late S-G2 phases of cell cycle where sister chromatids mutually provide undamaged DNA templates to be used (Jackson 2002). ...
Agrobacterium species are soil borne Gram-negative bacteria exhibiting predominantly a saprophytic lifestyle. Only few species are capable of parasitic growth on plants, causing either hairy root or crown gall diseases. The core of the infection strategy of pathogenic Agrobacteria is a genetic transformation of the host cell, via stable integration into the host genome of a DNA fragment called T-DNA. This genetic transformation results in oncogenic reprogramming of the host to the benefit of the pathogen. This unique ability of inter-kingdom DNA transfer was largely used as a tool for genetic engineering. Thus, the artificial host range of Agrobacterium is continuously expanding and includes plant and non-plant organisms. In the meanwhile, the increasing availability of genomic tools encouraged genome wide surveys of T-DNA tagged libraries, and the pattern of T-DNA integration in eukaryotic genomes was studied. Therefore, data was made available for a better understanding of T-DNA integration mechanisms and potential biases. This review focuses on the intra-nuclear mechanisms necessary for proper targeting and stable expression of Agrobacterium oncogenic T-DNA in the host cell. More specifically, the role of genome features and the putative involvement of host's transcriptional machinery in addressing the T-DNA to regions suitable for gene expression will be discussed. Also, the mechanisms underlying T-DNA integration into specific genome compartments will be reviewed, and a theoretical model for T-DNA intra-nuclear targeting will be presented.
... ). In cancer cells however, these repair mechanisms are faulty, leading to random fusion of chromosomes once breakage occurs, generating CIN(Phillips and McKinnon 2007;Shrivastav, De Haro et al. 2008;Bernstein and Rothstein 2009). Flow cytometry information generated from the Plk4 MEFs indicates steadily increasing ploidy with passage number in the heterozygous cells. ...
... HR is a highly accurate process that uses a homologous DNA template to repair breaks during the S or G2 phases of the eukaryotic cell cycle. In this repair pathway, the first proteins recruited to a DSB is the MRX/MRN complex, consisting of Mre11-Rad50-Xrs2 (MRX) in yeast and MRE11-Rad50-NBS1 (MRN) in humans (Bernstein and Rothstein, 2009). These complexes perform the initial processing of damaged DNA ends by producing 3' ssDNA at each end of the break. ...
... The MRN complex comprised of Mre11, Rad50, and Nbs1 binds to the ends of DSBs and mediates end resection required for HRR (29). BRCA1 has been shown to promote MRN-mediated end resection through interaction with the BRCA1-associated protein CtIP (24,30). ...
Unlabelled:
PARP inhibitors exploit synthetic lethality to target epithelial ovarian cancer (EOC) with hereditary BRCA mutations and defects in homologous recombination repair (HRR). However, such an approach is limited to a small subset of EOC patients and compromised by restored HRR due to secondary mutations in BRCA genes. Here, it was demonstrated that triapine, a small-molecule inhibitor of ribonucleotide reductase, enhances the sensitivity of BRCA wild-type EOC cells to the PARP inhibitor olaparib and the topoisomerase II inhibitor etoposide. Triapine abolishes olaparib-induced BRCA1 and Rad51 foci, and disrupts the BRCA1 interaction with the Mre11-Rad50-Nbs1 (MRN) complex in BRCA1 wild-type EOC cells. It has been shown that phosphorylation of CtIP (RBBP8) is required for the interaction with BRCA1 and with MRN to promote DNA double-strand break (DSB) resection during S and G(2) phases of the cell cycle. Mechanistic studies within reveal that triapine inhibits cyclin-dependent kinase (CDK) activity and blocks olaparib-induced CtIP phosphorylation through Chk1 activation. Furthermore, triapine abrogates etoposide-induced CtIP phosphorylation and DSB resection as evidenced by marked attenuation of RPA32 phosphorylation. Concurrently, triapine obliterates etoposide-induced BRCA1 foci and sensitizes BRCA1 wild-type EOC cells to etoposide. Using a GFP-based HRR assay, it was determined that triapine suppresses HRR activity induced by an I-SceI-generated DSB. These results suggest that triapine augments the sensitivity of BRCA wild-type EOC cells to drug-induced DSBs by disrupting CtIP-mediated HRR.
Implications:
These findings provide a strong rationale for combining triapine with PARP or topoisomerase inhibitors to target HRR-proficient EOC cells.
... HR is often initiated by DNA double-strand breaks (DSBs), either in a programmed manner involving the meiosis-specific Spo11 protein complex (8) or in an unscheduled manner, mounted in response to the exposure of cells to DSB causative agents, such as ionizing radiation (5,6). For HR to occur, the 5' ends of the DSBs must first be resected to yield 3' single-stranded DNA (ssDNA) tails (9)(10)(11)(12). The RAD51 or DMC1 recombinase then polymerizes on these ssDNA tails, in an ATP-dependent manner, to form a highly ordered helical polymer known as the presynaptic filament (5,6). ...
The Hop2-Mnd1 complex functions with the DMC1 recombinase in meiotic recombination. Hop2-Mnd1 stabilizes the DMC1-single-stranded DNA (ssDNA) filament and promotes the capture of the double-stranded DNA partner by the recombinase filament to assemble the synaptic complex. Herein, we define the action mechanism of Hop2-Mnd1 in DMC1-mediated recombination. Small angle X-ray scattering analysis and electron microscopy reveal that the heterodimeric Hop2-Mnd1 is a V-shaped molecule. We show that the protein complex harbors three distinct DNA binding sites, and determine their functional relevance. Specifically, the N-terminal double-stranded DNA binding functions of Hop2 and Mnd1 co-operate to mediate synaptic complex assembly, whereas ssDNA binding by the Hop2 C-terminus helps stabilize the DMC1-ssDNA filament. A model of the Hop2-Mnd1-DMC1-ssDNA ensemble is proposed to explain how it mediates homologous DNA pairing in meiotic recombination.
RAD51 is integral in homologous recombination DNA damage repair and has garnered much interest as both a biomarker and potential therapeutic target in oncology. Multiple in vitro and in vivo studies have demonstrated its role as a predictive marker, particularly in the context of platinum-based therapies and poly ADP-ribose polymerase (PARP) inhibitors. In this review, we highlight the development of RAD51 inhibitors, with a focus on novel molecules and ongoing clinical trials. Despite many efforts to develop effective and tolerable direct RAD51 inhibitors, identification of these agents remains challenging. Clinically, however, there may be a role of pharmacological indirect RAD51 inhibition.
Exonucleases are often found associated with polymerase or helicase domains in the same enzyme or can function as autonomous entities to maintain genome stability. Here, we uncovered Chaetomium thermophilum RecQ family proteins that also have exonuclease activity in addition to their main helicase function. The novel exonuclease activity is separate from the helical core domain and coexists with the latter two enzymatic activities on the same polypeptide. The CtRecQ¹²¹⁻³⁶⁶ exonuclease region performs independently as an exonuclease. We describe its catalytic mechanism and biological characteristics. We demonstrate unequivocally that CtRecQ¹²¹⁻³⁶⁶ exclusively displays exonuclease activity and that this activity has a 3′–5′ polarity that can both hydrolyze ssDNA and cleave dsDNA substrates. The hydrolytic activity of majority exonuclease is driven by bimetal ions, and this appears to be the case for the CtRecQ¹²¹⁻³⁶⁶ exonuclease as well. Additionally, the maximum activity of CtRecQ¹²¹⁻³⁶⁶ was observed at pH 8.0–9.0, low salt with Mg²⁺. The two helices in the structure, a6 and a7, play significant roles in the execution by anticipating their shape and changing essential amino acids.
In humans, hematopoietic stem cells (HSCs) adopt unique responsive pathways counteracting with the DNA-damaging assaults to weigh the balance between the maintenance of normal stem cell poor for whole-life blood regeneration and the transformation to leukemia stem cells (LSCs) for leukemia initiation. LSCs also take actions of combating with the attack launched by externally therapeutic drugs that can kill most leukemic cells, to avoid extermination and promote disease relapse. Therefore, the collection of knowledge about all these underlined mechanisms would present a preponderance for later studies. In this chapter, the universal DNA damage response (DDR) mechanisms were firstly introduced, and then DDR of HSCs were presented focusing on the DNA double-strand breaks in the quiescent state of HSCs, which poses a big advantage in promoting its transformation into preleukemic HSCs. Lastly, the DDR of LSCs were summarized based on the major outcomes triggered by different pathways in specific leukemia, upon which some aspects for future investigations were envisioned under our currently limited scope of knowledge.
Chromosomal translocations are now well understood to not only constitute signature molecular markers for certain human cancers but often also to be causative in the genesis of that tumor. Despite the obvious importance of such events, the molecular mechanism of chromosomal translocations in human cells remains poorly understood. Part of the explanation for this dearth of knowledge is due to the complexity of the reaction and the need to archaeologically work backwards from the final product (a translocation) to the original unrearranged chromosomes to infer mechanism. Although not definitive, these studies have indicated that the aberrant usage of endogenous DNA repair pathways likely lies at the heart of the problem. An equally obfuscating aspect of this field, however, has also originated from the unfortunate species-specific differences that appear to exist in the relevant model systems that have been utilized to investigate this process. Specifically, yeast and murine systems (which are often used by basic science investigators) rely on different DNA repair pathways to promote chromosomal translocations than human somatic cells. In this chapter, we will review some of the basic concepts of chromosomal translocations and the DNA repair systems thought to be responsible for their genesis with an emphasis on underscoring the differences between other species and human cells. In addition, we will focus on a specific subset of translocations that involve the very end of a chromosome (a telomere). A better understanding of the relationship between DNA repair pathways and chromosomal translocations is guaranteed to lead to improved therapeutic treatments for cancer.
The bone marrow (BM) is the primary organ in adults in which blood cell formation occurs. Hematopoiesis is orchestrated by a rare population of pluripotent stem cells, known as hematopoietic stem cells (HSCs). The maintenance of genomic stability is essential for cell survival and for preventing malignant transformation. DNA damage checkpoints and DNA repair mechanisms cooperate to restore genomic stability following genotoxic insult. Cytochrome C is one of the most important proapoptotic factors released in this process that interacts with apoptotic protease-activating factor 1 and deoxyadenosine triphosphate in the cytosol, collectively forming the apoptosome. Studies to date clearly indicate that mesenchymal stromal cells (MSCs) are radioresistant. Long-term proliferation of primary human MSCs in vitro following exposure to high-dose irradiation is comparable to that of known radioresistant cell types, such as the A549 lung cancer cell line. MSCs are radioresistant progenitors that reside in hypoxic niches in vivo, particularly in the BM.
Gene conversions occur when genomic double strand DNA breaks (DSBs) trigger unidirectional transfer of genetic material from a homologous template sequence. Exogenous or mutated sequence can be introduced through this homology directed repair (HDR). We leveraged gene conversion to develop a method for genomic editing of existing transgenic insertions in Drosophila melanogaster The Homology Assisted CRISPR Knock-in (HACK) method utilizes the CRISPR/Cas9 system to induce DSBs in a GAL4 transgene, which is repaired by a single genomic transgenic construct containing GAL4 homologous sequences flanking a T2A-QF2 cassette. With two crosses, this technique converts existing GAL4 lines, including enhancer traps, into functional QF2 expressing lines. We used HACK to convert the most commonly used GAL4 lines (labelling tissues such as neurons, fat, glia, muscle, and haemocytes) to QF2 lines. We also identified regions of the genome that exhibited differential efficiencies of HDR. The HACK technique is robust and readily adaptable for targeting and replacement of other genomic sequences, and could be a useful approach to re-purpose existing transgenes as new genetic reagents become available.
Chronic infection with hepatitis B virus (HBV) occurs in 6% of the world's population. These carriers of the virus are at high risk for the life-threatening complications of cirrhosis and liver cancer. Although effective vaccines are available to prevent HBV infection, they are of no use to individuals who are already infected with the virus. Therefore, complications of chronic HBV infection are likely to remain a significant public health problem for some time. Stability of the HBV covalently closed circular DNA (cccDNA), together with resistance of this replication intermediate to licensed therapies, are the main reasons for the limited success of currently available treatment interventions. Gene therapy approaches, particularly using exogenous RNA interference (RNAi) activators and derivatives of DNA sequence-specific proteins, have shown potential as HBV therapeutics. Synthetic and expressed RNAi activators have been used successfully to inhibit HBV replication in cell culture and murine models of the infection. A candidate drug comprising a short interfering RNA conjugate is currently in phase II of clinical trial. Although efficacy against the virus is impressive, it remains to be established whether gene silencing will be curative of persistent HBV infection. In one of the first studies using sequence-specific DNA binding proteins, zinc finger proteins (ZFPs) were shown to inhibit transcription from duck HBV cccDNA. Subsequent investigations showed that coupling FokI endonuclease elements to ZFPs or transcription activator-like effectors achieved specific cleavage of HBV DNA, with resultant disabling of viral gene expression. This was an important development in HBV gene therapy because it demonstrated that inactivation of the problematic viral cccDNA replication intermediate is feasible.
In this review we focus on new insights that challenge our understanding of homologous recombination (HR) and Rad51 regulation. Recent advances using high-resolution microscopy and single molecule techniques have broadened our knowledge of Rad51 filament formation and strand invasion at double-strand break (DSB) sites and at replication forks, which are one of most physiologically relevant forms of HR from yeast to humans. Rad51 filament formation and strand invasion is regulated by many mediator proteins such as the Rad51 paralogues and the Shu complex, consisting of a Shu2/SWS1 family member and additional Rad51 paralogues. Importantly, a novel RAD51 paralogue was discovered in Caenorhabditis elegans, and its in vitro characterization has demonstrated a new function for the worm RAD51 paralogues during HR. Conservation of the human RAD51 paralogues function during HR and repair of replicative damage demonstrate how the RAD51 mediators play a critical role in human health and genomic integrity. Together, these new findings provide a framework for understanding RAD51 and its mediators in DNA repair during multiple cellular contexts.
High expression levels of SLFN11 correlate with the sensitivity of human cancer cells to DNA-damaging agents. However, little is known about the underlying mechanism. Here, we show that SLFN11 interacts directly with RPA1 and is recruited to sites of DNA damage in an RPA1-dependent manner. Furthermore, we establish that SLFN11 inhibits checkpoint maintenance and homologous recombination repair by promoting the destabilization of the RPA-ssDNA complex, thereby sensitizing cancer cell lines expressing high endogenous levels of SLFN11 to DNA-damaging agents. Finally, we demonstrate that the RPA1-binding ability of SLFN11 is required for its function in the DNA damage response. Our findings not only provide novel insight into the molecular mechanisms underlying the drug sensitivity of cancer cell lines expressing SLFN11 at high levels, but also suggest that SLFN11 expression can serve as a biomarker to predict responses to DNA-damaging therapeutic agents.
DNA double-strand breaks (DSBs) are repaired mainly by non-homologous end joining or homologous recombination (HR). Cell cycle stage and DNA end resection are believed to regulate the commitment to HR repair. Here we identify RNF138 as a ubiquitin E3 ligase that regulates the HR pathway. RNF138 is recruited to DNA damage sites through zinc fingers that have a strong preference for DNA with 5'- or 3'-single-stranded overhangs. RNF138 stimulates DNA end resection and promotes ATR-dependent signalling and DSB repair by HR, thereby contributing to cell survival on exposure to DSB-inducing agents. Finally, we establish that RNF138-dependent Ku removal from DNA breaks is one mechanism whereby RNF138 can promote HR. These results establish RNF138 as an important regulator of DSB repair pathway choice.
Our genome is constantly challenged by sources that cause DNA damage. To repair DNA damage and maintain genomic stability eukaryotes have evolved a complex network of pathways termed the DNA damage response (DDR). The DDR consists of signal transduction pathways that sense DNA damage and mediate tightly coordinated reactions to halt the cell cycle and repair DNA with a collection of different enzymes. In this manner, the DDR protects the genome by preventing the accumulation of mutations and DNA aberrations that promote cellular transformation and cancer development. Loss of function mutations in DDR genes and genomic instability occur frequently in many tumor types and underlie numerous cancer-prone hereditary syndromes such as Fanconi Anemia (FA).
My thesis research applies candidate-based and unbiased experimental approaches to investigate the role of several tumor suppressor genes (TSGs) in the DDR. My dissertation will first describe a novel function for the breast and ovarian cancer tumor suppressor and FA-associated gene FANCJ in the DDR to ultraviolet (UV) irradiation. In response to UV irradiation FANCJ supports checkpoint induction, the arrest of DNA synthesis, and suppresses UV induced point mutations. Suggesting that FANCJ could suppress UV induced cancers, in sequenced melanomas from multiple databases I found somatic mutations in FANCJ previously associated with breast/ovarian cancer and FA syndrome.
The second part of my dissertation will describe an RNA interference screen to identify genes modulating cellular sensitivity to the chemotherapeutic drug cisplatin. The hereditary breast/ovarian cancer tumor suppressor BRCA2 is essential for DNA repair, thus BRCA2 mutant ovarian cancer cells are initially sensitive to cisplatin chemotherapy that induces DNA damage. However, drug resistance develops and remains a major problem in the clinic. My screen identified the chromatin remodeling factor CHD4 as a potent modulator of cisplatin sensitivity and predictor of response to chemotherapy in BRCA2 mutant cancers. Taken together, my investigations highlight the important contribution of the DDR and the role they play in tumorigenesis and predicting therapeutic response.
Chronic infection with hepatitis B virus (HBV) occurs in approximately 5 % of the world's human population and persistence of the virus is associated with serious complications of cirrhosis and liver cancer. Currently available treatments are modestly effective and advancing novel therapeutic strategies is a medical priority. Stability of the viral cccDNA replication intermediate is a major factor that has impeded the development of therapies that are capable of eliminating chronic infection. Recent advances that employ gene therapy strategies offer useful advantages over current therapeutics. Silencing of HBV gene expression by harnessing the RNA interference pathway has been shown to be highly effective in cell culture and in vivo. However, a potential limitation of this approach is that the post-transcriptional mechanism of gene silencing does not disable cccDNA. Early results using designer transcription activator-like effector nucleases (TALENs) and repressor TALEs (rTALEs) have shown potential as a mode of inactivating cccDNA. In this article, we review the recent advances that have been made in HBV gene therapy, with a particular emphasis on the potential anti-HBV therapeutic utility of designed sequence-specific DNA binding proteins and their derivatives.
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The Hop2–Mnd1 complex functions with the DMC1 recombinase in meiotic recombination. Hop2–Mnd1 stabilizes the DMC1-single-stranded DNA (ssDNA) filament and promotes the capture of the double-stranded DNA partner by the recombinase filament to assemble the synaptic complex. Herein, we define the action mechanism of Hop2–Mnd1 in DMC1-mediated recombination. Small angle X-ray scattering analysis and electron microscopy reveal that the heterodimeric Hop2–Mnd1 is a V-shaped molecule. We show that the protein complex harbors three distinct DNA binding sites, and deter-mine their functional relevance. Specifically, the N-terminal double-stranded DNA binding functions of Hop2 and Mnd1 co-operate to mediate synaptic complex assembly, whereas ssDNA binding by the Hop2 C-terminus helps stabilize the DMC1-ssDNA filament. A model of the Hop2-Mnd1-DMC1-ssDNA ensemble is proposed to explain how it mediates homologous DNA pairing in meiotic recombination.
A number of studies have investigated the association between NBS1 Glu185Gln (rs1805794, E185Q) polymorphism and cancer risk, but the results remained controversial. Previous meta-analysis found a borderline significant impact of this polymorphism on cancer risk; however, the result might be relatively unreliable due to absence of numerous newly published studies. Thus, we conducted an updated meta-analysis. A systematic search was performed in PubMed and Embase databases until April 9, 2013. The odds ratios were pooled by the fixed-effects/random-effects model in STATA 12.0 software. As a result, a total of 48 case-control studies with 17,159 cases and 22,002 controls were included. No significant association was detected between the Glu185Gln polymorphism and overall cancer risk. As to subgroup analysis by cancer site, the results showed that this polymorphism could increase the risk for leukemia and nasopharyngeal cancer. Notably, the Glu185Gln polymorphism was found to be related to increased risk for urinary system cancer, but decreased risk for digestive system cancer. No significant associations were obtained for other subgroup analyses such as ethnicity, sample size and smoking status. In conclusion, current evidence did not suggest that the NBS1 Glu185Gln polymorphism was associated with overall cancer risk, but this polymorphism might contribute to the risk for some specific cancer sites due to potential different mechanisms. More well-designed studies are imperative to identify the exact function of this polymorphism in carcinogenesis.
The error-free repair of double-stranded DNA breaks by homologous recombination requires processing of broken ends. These processed ends are substrates for assembly of DNA strand exchange proteins that mediate DNA strand invasion. Here, we establish that human BLM helicase, a member of the RecQ family, stimulates the nucleolytic activity of human exonuclease 1 (hExo1), a 5′→3′ double-stranded DNA exonuclease. The stimulation is specific because other RecQ homologs fail to stimulate hExo1. Stimulation of DNA resection by hExo1 is independent of BLM helicase activity and is, instead, mediated by an interaction between the 2 proteins. Finally, we show that DNA ends resected by hExo1 and BLM are used by human Rad51, but not its yeast or bacterial counterparts, to promote homologous DNA pairing. This in vitro system recapitulates initial steps of homologous recombination and provides biochemical evidence for a role of BLM and Exo1 in the initiation of recombinational DNA repair.
• Bloom syndrome
• Rad51
• recombination
• RecQ
• DNA pairing
Mutations in human homologues of the bacterial RecQ helicase cause diseases leading to cancer predisposition and/or shortened lifespan (Werner, Bloom, and Rothmund-Thomson syndromes). The budding yeast Saccharomyces cerevisiae has one RecQ helicase, Sgs1, which functions with Top3 and Rmi1 in DNA repair. Here, we report separation-of-function alleles of SGS1 that suppress the slow growth of top3Delta and rmi1Delta cells similar to an SGS1 deletion, but are resistant to DNA damage similar to wild-type SGS1. In one allele, the second acidic region is deleted, and in the other, only a single aspartic acid residue 664 is deleted. sgs1-D664Delta, unlike sgs1Delta, neither disrupts DNA recombination nor has synthetic growth defects when combined with DNA repair mutants. However, during S phase, it accumulates replication-associated X-shaped structures at damaged replication forks. Furthermore, fluorescent microscopy reveals that the sgs1-D664Delta allele exhibits increased spontaneous RPA foci, suggesting that the persistent X-structures may contain single-stranded DNA. Taken together, these results suggest that the Sgs1 function in repair of DNA replication intermediates can be uncoupled from its role in homologous recombinational repair.
DNA ends exposed after introduction of double-strand breaks (DSBs) undergo 5'-3' nucleolytic degradation to generate single-stranded DNA, the substrate for binding by the Rad51 protein to initiate homologous recombination. This process is poorly understood in eukaryotes, but several factors have been implicated, including the Mre11 complex (Mre11-Rad50-Xrs2/NBS1), Sae2/CtIP/Ctp1 and Exo1. Here we demonstrate that yeast Exo1 nuclease and Sgs1 helicase function in alternative pathways for DSB processing. Novel, partially resected intermediates accumulate in a double mutant lacking Exo1 and Sgs1, which are poor substrates for homologous recombination. The early processing step that generates partly resected intermediates is dependent on Sae2. When Sae2 is absent, in addition to Exo1 and Sgs1, unprocessed DSBs accumulate and homology-dependent repair fails. These results suggest a two-step mechanism for DSB processing during homologous recombination. First, the Mre11 complex and Sae2 remove a small oligonucleotide(s) from the DNA ends to form an early intermediate. Second, Exo1 and/or Sgs1 rapidly process this intermediate to generate extensive tracts of single-stranded DNA that serve as substrate for Rad51.
Cells of all living organisms have evolved complex mechanisms to maintain genome stability. There is increasing evidence that spontaneous genomic instability occurs primarily during DNA replication. RecQ DNA helicases function during DNA replication and are essential for the maintenance of genome stability. In human cells, there exist five RecQ DNA helicases, and mutations of three of these helicases, encoded by the BLM, WRN and RECQL4 genes, give rise to the cancer predisposition disorders, Bloom syndrome (BS), Werner syndrome (WS) and Rothmund-Thomson syndrome (RTS), respectively. Individuals suffering from WS and RTS also show premature aging phenotypes. Although the two remaining helicases, RECQL1 and RECQL5, have not yet been associated with heritable human diseases, a single nucleotide polymorphism of RECQL1 is associated with reduced survival of pancreatic cancer, and RecQl5 knockout mice show a predisposition to cancer. Here, we review the functions of eukaryotic RecQ helicases, focusing primarily on BLM in the maintenance of genome stability through various pathways of nucleic acid metabolism and with special reference to DNA replication.
DNA double-strand breaks (DSBs) are repaired by two principal mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR). HR is the most accurate DSB repair mechanism but is generally restricted to the S and G2 phases of the cell cycle, when DNA has been replicated and a sister chromatid is available as a repair template. By contrast, NHEJ operates throughout the cell cycle but assumes most importance in G1 (refs 4, 6). The choice between repair pathways is governed by cyclin-dependent protein kinases (CDKs), with a major site of control being at the level of DSB resection, an event that is necessary for HR but not NHEJ, and which takes place most effectively in S and G2 (refs 2, 5). Here we establish that cell-cycle control of DSB resection in Saccharomyces cerevisiae results from the phosphorylation by CDK of an evolutionarily conserved motif in the Sae2 protein. We show that mutating Ser 267 of Sae2 to a non-phosphorylatable residue causes phenotypes comparable to those of a sae2Delta null mutant, including hypersensitivity to camptothecin, defective sporulation, reduced hairpin-induced recombination, severely impaired DNA-end processing and faulty assembly and disassembly of HR factors. Furthermore, a Sae2 mutation that mimics constitutive Ser 267 phosphorylation complements these phenotypes and overcomes the necessity of CDK activity for DSB resection. The Sae2 mutations also cause cell-cycle-stage specific hypersensitivity to DNA damage and affect the balance between HR and NHEJ. These findings therefore provide a mechanistic basis for cell-cycle control of DSB repair and highlight the importance of regulating DSB resection.
We have identified two novel intermediates of homothallic switching of the yeast mating type gene, from MATa to MAT alpha. Following HO endonuclease cleavage, 5' to 3' exonucleolytic digestion is observed distal to the HO cut, creating a 3'-ended single-stranded tail. This recision is more extensive in a rad52 strain unable to switch. Surprisingly, the proximal side of the HO cut is protected from degradation; this stabilization depends on the presence of the silent copy donor sequences. A second intermediate was identified by a quantitative application of the polymerase chain reaction (PCR). The Y alpha-MAT distal covalent fragment of the switched product appears 30 min prior to the appearance of the MAT proximal Y alpha junction. No covalent joining of MAT distal to HML distal sequences is detected. We suggested that the MAT DNA distal to the HO cut invades the intact donor and is extended by DNA synthesis. This step is prevented in a rad52 strain. These intermediates are consistent with a model for MAT switching in which only the distal side of the HO cut is initially active in strand invasion and transfer of information from the donor.
Repair of double-strand breaks by gene conversions between homologous sequences located on different Saccharomyces cerevisiae chromosomes or plasmids requires RAD51. When repair occurs between inverted repeats of the same plasmid, both RAD51-dependent and RAD51-independent repairs are found. Completion of RAD51-independent plasmid repair events requires RAD52, RAD50, RAD59, TID1 (RDH54), and SRS2 and appears to involve break-induced replication coupled to single-strand annealing. Surprisingly, RAD51-independent recombination requires much less homology (30 bp) for strand invasion than does RAD51-dependent repair (approximately 100 bp); in fact, the presence of Rad51p impairs recombination with short homology. The differences
between the RAD51- and RAD50/RAD59-dependent pathways account for the distinct ways that two different recombination processes maintain yeast telomeres in the
absence of telomerase.
A single double-strand break (DSB) induced by HO endonuclease triggers both repair by homologous recombination and activation of the Mec1-dependent DNA damage checkpoint in budding yeast. Here we report that DNA damage checkpoint activation by a DSB requires the cyclin-dependent kinase CDK1 (Cdc28) in budding yeast. CDK1 is also required for DSB-induced homologous recombination at any cell cycle stage. Inhibition of homologous recombination by using an analogue-sensitive CDK1 protein results in a compensatory increase in non-homologous end joining. CDK1 is required for efficient 5' to 3' resection of DSB ends and for the recruitment of both the single-stranded DNA-binding complex, RPA, and the Rad51 recombination protein. In contrast, Mre11 protein, part of the MRX complex, accumulates at unresected DSB ends. CDK1 is not required when the DNA damage checkpoint is initiated by lesions that are processed by nucleotide excision repair. Maintenance of the DSB-induced checkpoint requires continuing CDK1 activity that ensures continuing end resection. CDK1 is also important for a later step in homologous recombination, after strand invasion and before the initiation of new DNA synthesis.
When eukaryotic chromosomes undergo double strand breaks (DSBs), several evolutionarily conserved proteins, among which the MRX complex, are recruited to the break site, leading to checkpoint activation and DNA repair. The function of the Saccharomyces cerevisiae Sae2 protein, which is known to work together with the MRX complex in meiotic DSB processing and in specific mitotic DSB repair events, is only beginning to be elucidated. Here we provide new insights into the role of Sae2 in mitotic DSB repair. We show that repair by single strand annealing of a single DSB, which is generated by the HO endonuclease between direct repeats, is defective both in the absence of Sae2 and in the presence of the hypomorphic rad50s allele altering the Rad50 subunit of MRX. Moreover, SAE2 overexpression partially suppresses the rad50s single strand annealing repair defects, suggesting that the latter might arise from defective MRX-Sae2 interactions. Finally, SAE2 deletion slows down resection of an HO-induced DSB and impairs DSB end bridging. Thus, Sae2 participates in DSB single strand annealing repair by ensuring both resection and intrachromosomal association of the broken ends.
To ensure proper replication and segregation of the genome, eukaryotic cells have evolved surveillance systems that monitor and react to impaired replication fork progression. In budding yeast, the intra-S phase checkpoint responds to stalled replication forks by downregulating late-firing origins, preventing spindle elongation and allowing efficient resumption of DNA synthesis after recovery from stress. Mutations in this pathway lead to high levels of genomic instability, particularly in the presence of DNA damage. Here we demonstrate by chromatin immunoprecipitation that when yeast replication forks stall due to hydroxyurea (HU) treatment, DNA polymerases alpha and epsilon are stabilized for 40-60 min. This requires the activities of Sgs1, a member of the RecQ family of DNA helicases, and the ATM-related kinase Mec1, but not Rad53 activation. A model is proposed whereby Sgs1 helicase resolves aberrantly paired structures at stalled forks to maintain single-stranded DNA that allows RP-A and Mec1 to promote DNA polymerase association.
We showed previously that rad50 and mre11 genes of thermophilic archaea are organized in an operon‐like structure with a third gene (nurA) encoding a 5′ to 3′ exonuclease. Here, we show that the rad50, mre11 and nurA genes from the hyperthermo philic archaeon Sulfolobus acidocaldarius are co‐transcribed with a fourth gene encoding a DNA helicase. This enzyme (HerA) is the prototype of a new class of DNA
helicases able to utilize either 3′ or 5′ single‐stranded DNA extensions for loading and subsequent DNA duplex unwinding.
To our knowledge, DNA helicases capable of translocating along the DNA in both directions have not been identified previously.
Sequence analysis of HerA shows that it is a member of the TrwB, FtsK and VirB4/VirD4 families of the PilT class NTPases.
HerA homologs are found in all thermophilic archaeal species and, in all cases except one, the rad50, mre11, nurA and herA genes are grouped together. These results suggest that the archaeal Rad50–Mre11 complex might act in association with a 5′
to 3′ exonuclease (NurA) and a bipolar DNA helicase (HerA) indicating a probable involvement in the initiation step of homologous
recombination.
DNA double strand breaks (DSBs) can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HR). HR requires nucleolytic degradation of 5' DNA ends to generate tracts of single-stranded DNA (ssDNA), which are also important for the activation of DNA damage checkpoints. Here we describe a quantitative analysis of DSB processing in the budding yeast Saccharomyces cerevisiae. We show that resection of an HO endonuclease-induced DSB is less extensive than previously estimated and provide evidence for significant instability of the 3' ssDNA tails. We show that both DSB resection and checkpoint activation are dose-dependent, especially during the G1 phase of the cell cycle. During G1, processing near the break is inhibited by competition with NHEJ, but extensive resection is regulated by an NHEJ-independent mechanism. DSB processing and checkpoint activation are more efficient in G2/M than in G1 phase, but are most efficient at breaks encountered by DNA replication forks during S phase. Our findings identify unexpected complexity of DSB processing and its regulation, and provide a framework for further mechanistic insights.
We have identified two novel intermediates of homothallic switching of the yeast mating type gene, from MATa to MAT alpha. Following HO endonuclease cleavage, 5′ to 3′ exonucleolytic digestion is observed distal to the HO cut, creating a 3′‐ended single‐stranded tail. This recision is more extensive in a rad52 strain unable to switch. Surprisingly, the proximal side of the HO cut is protected from degradation; this stabilization depends on the presence of the silent copy donor sequences. A second intermediate was identified by a quantitative application of the polymerase chain reaction (PCR). The Y alpha‐MAT distal covalent fragment of the switched product appears 30 min prior to the appearance of the MAT proximal Y alpha junction. No covalent joining of MAT distal to HML distal sequences is detected. We suggested that the MAT DNA distal to the HO cut invades the intact donor and is extended by DNA synthesis. This step is prevented in a rad52 strain. These intermediates are consistent with a model for MAT switching in which only the distal side of the HO cut is initially active in strand invasion and transfer of information from the donor.
The Mre11/Rad50 complex has been implicated in the early steps of DNA double-strand break (DSB) repair through homologous recombination in several organisms. However, the enzymatic properties of this complex are incompatible with the generation of 3' single-stranded DNA for recombinase loading and strand exchange. In thermophilic archaea, the Mre11 and Rad50 genes cluster in an operon with genes encoding a helicase, HerA, and a 5' to 3' exonuclease, NurA, suggesting a common function. Here we show that purified Mre11 and Rad50 from Pyrococcus furiosus act cooperatively with HerA and NurA to resect the 5' strand at a DNA end under physiological conditions in vitro. The 3' single-stranded DNA generated by these enzymes can be utilized by the archaeal RecA homolog RadA to catalyze strand exchange. This work elucidates how the conserved Mre11/Rad50 complex promotes DNA end resection in archaea and may serve as a model for DSB processing in eukaryotes.
A key cellular response to DNA double-strand breaks (DSBs) is 5'-to-3' DSB resection by nucleases to generate regions of ssDNA that then trigger cell cycle checkpoint signaling and DSB repair by homologous recombination (HR). Here, we reveal that in the absence of exonuclease Exo1 activity, deletion or mutation of the Saccharomyces cerevisiae RecQ-family helicase, Sgs1, causes pronounced hypersensitivity to DSB-inducing agents. Moreover, we establish that this reflects severely compromised DSB resection, deficient DNA damage signaling, and strongly impaired HR-mediated repair. Furthermore, we show that the mammalian Sgs1 ortholog, BLM--whose deficiency causes cancer predisposition and infertility in people--also functions in parallel with Exo1 to promote DSB resection, DSB signaling and resistance to DSB-generating agents. Collectively, these data establish evolutionarily conserved roles for the BLM and Sgs1 helicases in DSB processing, signaling, and repair.
Formation of single-strand DNA (ssDNA) tails at a double-strand break (DSB) is a key step in homologous recombination and DNA-damage signaling. The enzyme(s) producing ssDNA at DSBs in eukaryotes remain unknown. We monitored 5'-strand resection at inducible DSB ends in yeast and identified proteins required for two stages of resection: initiation and long-range 5'-strand resection. We show that the Mre11-Rad50-Xrs2 complex (MRX) initiates 5' degradation, whereas Sgs1 and Dna2 degrade 5' strands exposing long 3' strands. Deletion of SGS1 or DNA2 reduces resection and DSB repair by single-strand annealing between distant repeats while the remaining long-range resection activity depends on the exonuclease Exo1. In exo1Deltasgs1Delta double mutants, the MRX complex together with Sae2 nuclease generate, in a stepwise manner, only few hundred nucleotides of ssDNA at the break, resulting in inefficient gene conversion and G2/M damage checkpoint arrest. These results provide important insights into the early steps of DSB repair in eukaryotes.
Extensive work on the maturation of lagging strands during the replication of simian virus 40 DNA suggests that the initiator RNA primers of Okazaki fragments are removed by the combined action of two nucleases, RNase HI and Fen1, before the Okazaki fragments join. Despite the well established in vitro roles of these two enzymes, genetic analyses in yeast revealed that null mutants of RNase HI and/or Fen1 are not lethal, suggesting that an additional enzymatic activity may be required for the removal of RNA. One such enzyme is the Saccharomyces cerevisiae Dna2 helicase/endonuclease, which is essential for cell viability and is well suited to removing RNA primers of Okazaki fragments. In addition, Dna2 interacts genetically and physically with several proteins involved in the elongation or maturation of Okazaki fragments. Here we show that the endonucleases Dna2 and Fen1 act sequentially to facilitate the complete removal of the primer RNA. The sequential action of these enzymes is governed by a single-stranded DNA-binding protein, replication protein-A (RPA). Our results demonstrate that the processing of Okazaki fragments in eukaryotes differs significantly from, and is more complicated than, that occurring in prokaryotes. We propose a novel biochemical mechanism for the maturation of eukaryotic Okazaki fragments.
The stability of DNA ends generated by the HO endonuclease in yeast is surprisingly high with a half-life of more than an hour. This transient stability is unaffected by mutations that abolish nonhomologous end joining (NHEJ). The unprocessed ends interact with Yku70p and Yku80p, two proteins required for NHEJ, but not significantly with Rad52p, a protein involved in homologous recombination (HR). Repair of a double-strand break by NHEJ is unaffected by the possibility of HR, although the use of HR is increased in NHEJ-defective cells. Partial in vitro 5' strand processing suppresses NHEJ but not HR. These results show that NHEJ precedes HR temporally, and that the availability of substrate dictates the particular pathway used. We propose that transient stability of DNA ends is a foundation for the permanent stability of telomeres.
RecQ helicases, together with topoisomerase III and Rmi1 family proteins, form an evolutionarily conserved complex that is essential for the maintenance of genome integrity. This complex, which we term RTR, is capable of, or has been implicated in, the processing of a diverse array of DNA structures, and we propose here that it functions in a coordinated fashion as a DNA structure-specific 'dissolvasome'. Little is known about how the RTR complex might be regulated or targeted to various DNA structures in vivo. Recent findings indicate that the components of the RTR complex might activate the cell cycle checkpoint machinery as well as be a target of checkpoint kinases, suggesting that these events are crucial to ensure faithful DNA replication and chromosome segregation.
Mre11/Rad50 complexes in all organisms function in the repair of DNA double-strand breaks. In budding yeast, genetic evidence suggests that the Sae2 protein is essential for the processing of hairpin DNA intermediates and meiotic double-strand breaks by Mre11/Rad50 complexes, but the biochemical basis of this functional relationship is not known. Here we demonstrate that recombinant Sae2 binds DNA and exhibits endonuclease activity on single-stranded DNA independently of Mre11/Rad50 complexes, but hairpin DNA structures are cleaved cooperatively in the presence of Mre11/Rad50 or Mre11/Rad50/Xrs2. Hairpin structures are not processed at the tip by Sae2 but rather at single-stranded DNA regions adjacent to the hairpin. Truncation and missense mutants of Sae2 inactivate this endonuclease activity in vitro and fail to complement Deltasae2 strains in vivo for meiosis and recombination involving hairpin intermediates, suggesting that the catalytic activities of Sae2 are important for its biological functions.
Double-strand breaks (DSBs) are potentially lethal DNA lesions that can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). We show that DSBs induced by ionizing radiation (IR) are efficiently processed for HR and bound by Rfa1 during G1, while endonuclease-induced breaks are recognized by Rfa1 only after the cell enters S phase. This difference is dependent on the DNA end-binding Yku70/Yku80 complex. Cell-cycle regulation is also observed in the DNA damage checkpoint response. Specifically, the 9-1-1 complex is required in G1 cells to recruit the Ddc2 checkpoint protein to damaged DNA, while, upon entry into S phase, the cyclin-dependent kinase Cdc28 and the 9-1-1 complex both serve to recruit Ddc2 to foci. Together, these results demonstrate that the DNA repair machinery distinguishes between different types of damage in G1, which translates into different modes of checkpoint activation in G1 and S/G2 cells.
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