[Show abstract][Hide abstract] ABSTRACT: Breast cancer is one of the leading causes of death worldwide, and therefore, new and improved approaches for the treatment of breast cancer are desperately needed. CtIP (RBBP8) is a multifunctional protein that is involved in various cellular functions, including transcription, DNA replication, DNA repair and the G1 and G2 cell cycle checkpoints. CtIP plays an important role in homologous recombination repair by interacting with tumor suppressor protein BRCA1. Here, we analyzed the expression profile of CtIP by data mining using published microarray data sets. We found that CtIP expression is frequently decreased in breast cancer patients, and the patient group with low-expressing CtIP mRNA is associated with a significantly lower survival rate. The knockdown of CtIP in breast cancer MCF7 cells reduced Rad51 foci numbers and enhanced f H2AX foci formation after f-irradiation, suggesting that deficiency of CtIP decreases homologous recombination repair and delays DNA double strand break repair.To explore the effect of CtIP on PARP inhibitor therapy for breast cancer, CtIP-depleted MCF7 cells were treated with PARP inhibitor olaparib (AZD2281) or veliparib (ABT-888). As in BRCA mutated cells, PARP inhibitors showed cytotoxicity to CtIP-depleted cells by preventing cells from repairing DNA damage, leading to decreased cell viability. Further, a xenograft tumor model in mice with MCF7 cells demonstrated significantly increased sensitivity towards PARP inhibition under CtIP deficiency. In summary, this study shows that low level of CtIP expression is associated with poor prognosis in breast cancer, and provides a rationale for establishing CtIP expression as a biomarker of PARP inhibitor response, and consequently offers novel therapeutic options for a significant subset of patients.
[Show abstract][Hide abstract] ABSTRACT: Contents
79 The Chicken Leads the Way in Avian Genomics.
Prepared by J. Smith.
80 The Chicken Genome: Current Status of Genome Assembly and Annotations.
Prepared by D.W. Burt, L. Eöry, A.L. Archibald, B.L. Aken, P. Flicek, K. Howe, W. Chow, M. Dunn, J.M.D. Wood, R. Nag, and W.C. Warren.
83 The Avian RNAseq Consortium: A Community Effort to Annotate the Chicken Genome.
Prepared by J. Smith, D.W. Burt, and the Avian RNAseq Consortium.
89 Noncoding RNAs in the Chicken Genome.
Prepared by J. Hertel, M. Fasold, A. Nitsche, I. Erb, P. Prieto, D. Kedra, C. Notredame, T.E. Steeves, P.P. Gardner, and P.F. Stadler.
91 Genome Sequencing in Birds and Evolutionary Inferences from Avian Genome Sequences.
Prepared by H. Ellegren.
94 The Use of Avian BAC Libraries and Clones.
Prepared by M.N. Romanov and D.K. Griffin.
96 Comparative Genomics.
Prepared by D.M. Larkin, M. Farré, and J. Damas.
100 Avian Cytogenetics Goes Functional.
Prepared by D.K. Griffin, M.N. Romanov, R. O’Connor, K.E. Fowler, and D.M. Larkin.
105 Hypermethylated Chromosome Regions in Chicken and Other Birds.
Prepared by M. Schmid, C. Steinlein, A.-S. Schneider, I. Nanda, and T. Haaf.
109 An Overview of Avian Evolution.
Prepared by S.B. Hedges.
114 An Update on Chicken Sex Determination and Gonadal Sex Differentiation.
Prepared by C.A. Smith.
119 Avian Epigenetics.
Prepared by H. Zhou.
122 Structural Variation and Copy Number Variation in Poultry.
Prepared by R.P.M.A. Crooijmans and M.A.M. Groenen.
124 SNPs and InDels – The Most Abundant Sources of Genetic Variations.
Prepared by A.A. Gheyas, C. Boschiero, and D.W. Burt.
130 Genetic Diversity of Village Chickens.
Prepared by T.T. Desta, R.A. Lawal, and O. Hanotte.
133 Mendelian Traits.
Prepared by D. Wragg.
137 Treasure the Exceptions: Utilizing Chicken Mutant Lines and Advanced Genetic Technologies to Uncover Genes Involved in Developmental Processes.
Prepared by E.A. O’Hare and M.E. Delany.
141 Genomic Landscape of the Chicken DT40 Cell Line.
Prepared by A. Motegi and M. Takata.
145 RNA-seq: Primary Cells, Cell Lines and Heat Stress.
Prepared by C.J. Schmidt, E.M. Pritchett, L. Sun, R.V.N. Davis, A. Hubbard, K.E. Kniel, S.M. Markland, Q. Wang, C. Ashwell, M. Persia, M.F. Rothschild, and S.J. Lamont.
148 Host-Viral Genome Interactions in Marek’s Disease.
Prepared by M.C. McPherson, C.M. Robinson, and M.E. Delany.
154 Transcriptome Variation in Response to Marek’s Disease Virus Acute Infection.
Prepared by L. Preeyanon, C.T. Brown, and H.H. Cheng.
163 The National Avian Research Facility.
Prepared by A. Hart, R. Kuo, L. Eöry, P. Kaiser, and D.W. Burt.
Full-text · Article · Jul 2015 · Cytogenetic and Genome Research
[Show abstract][Hide abstract] ABSTRACT: The E2 ubiquitin conjugating enzyme Ubc13 and the E3 ubiquitin ligases Rad18 and Rnf8 promote homologous recombination (HR)-mediated double-strand break (DSB) repair by enhancing polymerization of the Rad51 recombinase at γ-ray-induced DSB sites. To analyze functional interactions between the three enzymes, we created RAD18−/−, RNF8−/−, RAD18−/−/RNF8−/− and UBC13−/−clones in chicken DT40 cells. To assess the capability of HR, we measured the cellular sensitivity to camptothecin (topoisomerase I poison) and olaparib (poly(ADP ribose)polymerase inhibitor) because these chemotherapeutic agents induce DSBs during DNA replication, which are repaired exclusively by HR. RAD18−/−, RNF8−/− and RAD18−/−/RNF8−/− clones showed very similar levels of hypersensitivity, indicating that Rad18 and Rnf8 operate in the same pathway in the promotion of HR. Although these three mutants show less prominent defects in the formation of Rad51 foci than UBC13−/−cells, they are more sensitive to camptothecin and olaparib than UBC13−/−cells. Thus, Rad18 and Rnf8 promote HR-dependent repair in a manner distinct from Ubc13. Remarkably, deletion of Ku70, a protein essential for nonhomologous end joining (NHEJ) significantly restored tolerance of RAD18−/− and RNF8−/− cells to camptothecin and olaparib without affecting Rad51 focus formation. Thus, in cellular tolerance to the chemotherapeutic agents, the two enzymes collaboratively promote DSB repair by HR by suppressing the toxic effect of NHEJ on HR rather than enhancing Rad51 focus formation. In contrast, following exposure to γ-rays, RAD18−/−, RNF8−/−, RAD18−/−/RNF8−/− and UBC13−/−cells showed close correlation between cellular survival and Rad51 focus formation at DSB sites. In summary, the current study reveals that Rad18 and Rnf8 facilitate HR by two distinct mechanisms: suppression of the toxic effect of NHEJ on HR during DNA replication and the promotion of Rad51 focus formation at radiotherapy-induced DSB sites.
[Show abstract][Hide abstract] ABSTRACT: Reverse genetics is gaining importance in the field of modern biological sciences. Gene disruption and the use of siRNAs are the favored techniques for current research. Many researchers, however, are aware that the data from siRNA experiments are frequently inconsistent and that epistatic analysis of multiple genes using siRNAs is barely feasible. In recognition of the drawbacks of the siRNA technique, many researchers, especially in the field of DNA repair, are now introducing multiple genetic disruption techniques using the chicken DT40 cell line into their research. Thus, recent publications increasingly include data utilizing DT40 cells. In this chapter, we describe the current standard methods of multiple genetic manipulation in DT40 cells. We place a particular emphasis on describing the basic concepts and theoretical background of the genetic manipulation of DT40 cells for researchers who are new to such techniques.
No preview · Article · Feb 2014 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: SUMO conjugation is a reversible posttranslational modification that regulates protein function. SENP1 is one of the six SUMO-specific proteases present in vertebrate cells and its altered expression is observed in several carcinomas. To characterize SENP1 role in genome integrity, we generated Senp1 knockout chicken DT40 cells. SENP1(-/-) cells show normal proliferation, but are sensitive to spindle poisons. This hypersensitivity correlates with increased sister chromatid separation, mitotic slippage, and apoptosis. To test whether the cohesion defect had a causal relationship with the observed mitotic events, we restored the cohesive status of sister chromatids by introducing the TOP2α(+/-) mutation, which leads to increased catenation, or by inhibiting Plk1 and Aurora B kinases that promote cohesin release from chromosomes during prolonged mitotic arrest. Although TOP2α is SUMOylated during mitosis, the TOP2α(+/-) mutation had no obvious effect. By contrast, inhibition of Plk1 or Aurora B rescued the hypersensitivity of SENP1(-/-) cells to colcemid. In conclusion, we identify SENP1 as a novel factor required for mitotic arrest and cohesion maintenance during prolonged mitotic arrest induced by spindle poisons.
Full-text · Article · Aug 2012 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: DPB11/TopBP1 is an essential evolutionarily conserved gene involved in initiation of DNA replication and checkpoint signaling. Here, we show that Saccharomyces cerevisiae Dpb11 forms nuclear foci that localize to sites of DNA damage in G1, S and G2 phase, a recruitment that is conserved for its homologue TopBP1 in Gallus gallus. Damage-induced Dpb11 foci are distinct from Sld3 replication initiation foci. Further, Dpb11 foci are dependent on the checkpoint proteins Mec3 (9-1-1 complex) and Rad24, and require the C-terminal domain of Dpb11. Dpb11 foci are independent of the checkpoint kinases Mec1 and Tel1, and of the checkpoint mediator Rad9. In a site-directed mutagenesis screen, we identify a separation-of-function mutant, dpb11-PF, that is sensitive to DSB-inducing agents yet remains proficient for DNA replication and the S-phase checkpoint at the permissive temperature. The dpb11-PF mutant displays altered rates of heteroallelic and direct-repeat recombination, sensitivity to DSB-inducing drugs as well as delayed kinetics of mating-type switching with a defect in the DNA synthesis step thus implicating Dpb11 in homologous recombination. We conclude that Dpb11/TopBP1 plays distinct roles in replication, checkpoint response and recombination processes, thereby contributing to chromosomal stability.
[Show abstract][Hide abstract] ABSTRACT: Replicative DNA polymerases are frequently stalled by DNA lesions. The resulting replication blockage is released by homologous recombination (HR) and translesion DNA synthesis (TLS). TLS employs specialized TLS polymerases to bypass DNA lesions. We provide striking in vivo evidence of the cooperation between DNA polymerase η, which is mutated in the variant form of the cancer predisposition disorder xeroderma pigmentosum (XP-V), and DNA polymerase ζ by generating POLη(-/-)/POLζ(-/-) cells from the chicken DT40 cell line. POLζ(-/-) cells are hypersensitive to a very wide range of DNA damaging agents, whereas XP-V cells exhibit moderate sensitivity to ultraviolet light (UV) only in the presence of caffeine treatment and exhibit no significant sensitivity to any other damaging agents. It is therefore widely believed that Polη plays a very specific role in cellular tolerance to UV-induced DNA damage. The evidence we present challenges this assumption. The phenotypic analysis of POLη(-/-)/POLζ(-/-) cells shows that, unexpectedly, the loss of Polη significantly rescued all mutant phenotypes of POLζ(-/-) cells and results in the restoration of the DNA damage tolerance by a backup pathway including HR. Taken together, Polη contributes to a much wide range of TLS events than had been predicted by the phenotype of XP-V cells.
[Show abstract][Hide abstract] ABSTRACT: Proteasome-dependent protein degradation is involved in a variety of biological processes, including cell-cycle regulation, apoptosis, and stress-responses. Growing evidence from translational research and clinical trials proved the effectiveness of proteasome inhibitors (PIs) in treating several types of hematological malignancies. Although various key molecules in ubiquitin-dependent cellular processes have been proposed as relevant targets of therapeutic proteasome inhibition, our current understanding is far from complete. Recent rapid progress in DNA repair research has unveiled a crucial role of the ubiquitin-proteasome pathway (UPP) in regulating DNA repair. These findings thus bring up the idea that DNA repair pathways could be effective targets of PIs in mediating their cytotoxicity and enhancing the effect of radiotherapy and some DNA-damaging chemotherapeutic agents, such as cisplatin and camptothecin. In this review, we present the current perspective on the UPP-dependent regulatory mechanisms of DNA repair and discuss their therapeutic potential in the application of PIs to a broad spectrum of human cancers.
[Show abstract][Hide abstract] ABSTRACT: Chronic stalling of DNA replication forks caused by DNA damage can lead to genomic instability. Cells have evolved lesion bypass pathways such as postreplication repair (PRR) to resolve these arrested forks. In yeast, one branch of PRR involves proliferating cell nuclear antigen (PCNA) polyubiquitination mediated by the Rad5-Ubc13-Mms2 complex that allows bypass of DNA lesion by a template-switching mechanism. Previously, we identified human SHPRH as a functional homologue of yeast Rad5 and revealed the existence of RAD5-like pathway in human cells. Here we report the identification of HLTF as a second RAD5 homologue in human cells. HLTF, like SHPRH, shares a unique domain architecture with Rad5 and promotes lysine 63-linked polyubiquitination of PCNA. Similar to yeast Rad5, HLTF is able to interact with UBC13 and PCNA, as well as SHPRH; and the reduction of either SHPRH or HLTF expression enhances spontaneous mutagenesis. Moreover, Hltf-deficient mouse embryonic fibroblasts show elevated chromosome breaks and fusions after methyl methane sulfonate treatment. Our results suggest that HLTF and SHPRH are functional homologues of yeast Rad5 that cooperatively mediate PCNA polyubiquitination and maintain genomic stability.
Full-text · Article · Sep 2008 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Gross chromosomal rearrangement (GCR) is a type of genomic instability associated with many cancers. In yeast, multiple pathways cooperate to suppress GCR. In a screen for genes that promote GCR, we identified MPH1, which encodes a 3'-5' DNA helicase. Overexpression of Mph1p in yeast results in decreased efficiency of homologous recombination (HR) as well as delayed Rad51p recruitment to double-strand breaks (DSBs), which suggests that Mph1p promotes GCR by partially suppressing HR. A function for Mph1p in suppression of HR is further supported by the observation that deletion of both mph1 and srs2 synergistically sensitize cells to methyl methanesulfonate-induced DNA damage. The GCR-promoting activity of Mph1p appears to depend on its interaction with replication protein A (RPA). Consistent with this observation, excess Mph1p stabilizes RPA at DSBs. Furthermore, spontaneous RPA foci at DSBs are destabilized by the mph1Delta mutation. Therefore, Mph1p promotes GCR formation by partially suppressing HR, likely through its interaction with RPA.
Full-text · Article · Jul 2008 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Gross chromosomal rearrangements (GCRs), including translocations, deletions, amplifications and aneuploidy are frequently observed in various types of human cancers. Despite their clear importance in carcinogenesis, the molecular mechanisms by which GCRs are generated and held in check are poorly understood. By using a GCR assay, which can measure the rate of accumulation of spontaneous GCRs in Saccharomyces cerevisiae, we have found that many proteins involved in DNA replication, DNA repair, DNA recombination, checkpoints, chromosome remodeling, and telomere maintenance, play crucial roles in GCR metabolism. We describe here the theoretical background and practical procedures of this GCR assay. We will explain the breakpoint structure and DNA damage that lead to GCR formation. We will also summarize the pathways that suppress and enhance GCR formation. Finally, we will briefly describe similar assays developed by others and discuss their potential in studying GCR metabolism.
[Show abstract][Hide abstract] ABSTRACT: Ionizing radiation-induced mutagenesis (IR-IM) underlies a basis for radiation associated carcinogenesis as well as resistance to radiation therapy. This process was examined in Saccharomyces cerevisiae using an array of isogenic DNA repair deficient mutants. Mutations inactivating homologous recombination (rad51, 52, 54) or nucleotide excision repair (rad1, rad10, rad4) caused elevated IR-IM whereas inactivation of TransLesion Synthesis (TLS: rad6) caused severely defective IR-IM. Of the mutations inactivating TLS polymerases, rev3 and rev1 caused equally severe defects in IR-IM whereas rad30 did not significantly affect the process. The effects of the rev3, rev1, and rad6 mutations on IR-IM were epistatic, suggesting the requirement of both polymerase zeta and Rev1p in IR-IM related TLS. Although PCNA K164 SUMOylation/ubiquitination is a proposed prerequisite for TLS, the IR-IM defect of a rev3 or a rad6 mutant was worse than and epistatic to the pol30K164R mutant, a mutant in which the PCNA had been mutated to abolish such modifications. These results suggested that IR-IM related TLS occurs in the absence of PCNA K164 modification. Further analysis of a mutant simultaneously defective in SUMOylation and mono-ubiquitination (rad18 siz1) revealed that these modifications redundantly affected TLS as well as NHEJ. A genetic model based on these observations is proposed.
[Show abstract][Hide abstract] ABSTRACT: Differential modifications of proliferating cell nuclear antigen (PCNA) determine DNA repair pathways at stalled replication forks. In yeast, PCNA monoubiquitination by the ubiquitin ligase (E3) yRad18 promotes translesion synthesis (TLS), whereas the lysine-63-linked polyubiquitination of PCNA by yRad5 (E3) promotes the error-free mode of bypass. The yRad5-dependent pathway is important to prevent genomic instability during replication, although its exact molecular mechanism is poorly understood. This mechanism has remained totally elusive in mammals because of the lack of apparent RAD5 homologues. We report that a putative tumor suppressor gene, SHPRH, is a human orthologue of yeast RAD5. SHPRH associates with PCNA, RAD18, and the ubiquitin-conjugating enzyme UBC13 (E2) and promotes methyl methanesulfonate (MMS)-induced PCNA polyubiquitination. The reduction of SHPRH by stable short hairpin RNA increases sensitivity to MMS and enhances genomic instability. Therefore, the yRad5/SHPRH-dependent pathway is a conserved and fundamental DNA repair mechanism that protects the genome from genotoxic stress.
Full-text · Article · Jan 2007 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Gross chromosomal rearrangements (GCRs) are frequently observed in many cancers. Previously, we showed that inactivation of
Rad5 or Rad18, ubiquitin ligases (E3) targeting for proliferating cell nuclear antigen (PCNA), increases the de novo telomere
addition type of GCR (S. Smith, J. Y. Hwang, S. Banerjee, A. Majeed, A. Gupta, and K. Myung, Proc. Natl. Acad. Sci. USA 101:9039-9044, 2004). GCR suppression by Rad5 and Rad18 appears to be exerted by the RAD5-dependent error-free mode of bypass DNA repair. In contrast, Siz1 SUMO ligase and another ubiquitin ligase, Bre1, which target
for PCNA and histone H2B, respectively, have GCR-supporting activities. Inactivation of homologous recombination (HR) proteins
or the helicase Srs2 reduces GCR rates elevated by the rad5 or rad18 mutation. GCRs are therefore likely to be produced through the restrained recruitment of an HR pathway to stalled DNA replication
forks. Since this HR pathway is compatible with Srs2, it is not a conventional form of recombinational pathway. Lastly, we
demonstrate that selection of proper DNA repair pathways to stalled DNA replication forks is controlled by the Mec1-dependent
checkpoint and is executed by cooperative functions of Siz1 and Srs2. We propose a mechanism for how defects in these proteins
could lead to diverse outcomes (proper repair or GCR formation) through different regulation of DNA repair machinery.
Preview · Article · Mar 2006 · Molecular and Cellular Biology