George Iliakis

Publications of George Iliakis

• Dependence of adaptive response and its bystander transmission on the genetic background of tested cells.

  Authors: Holger Klammer, Li-Hua Zhang, Munira Kadhim, George Iliakis

  International journal of radiation biology. 05/2012;

  Abstract Purpose: Radiation-induced adaptive response (AR) is a phenomenon of increased radioresistance mediated by a low priming dose of ionizing radiation (IR) applied prior to a higher challengingAbstract Purpose: Radiation-induced adaptive response (AR) is a phenomenon of increased radioresistance mediated by a low priming dose of ionizing radiation (IR) applied prior to a higher challenging dose. We have previously shown that in mouse-embryo fibroblasts (MEF) and human A549 cells, AR is associated with enhanced repair of DNA double-strand breaks (DSB) by the DNA-PK-dependent pathway of non-homologous end-joining (D-NHEJ). Importantly, AR was "transmitted" to non-irradiated bystander cells through transfer of medium from cells that had received a priming dose of IR. Here, we examine the influence of the genetic background in these responses. Materials and Methods: Two plasmid-based assays specifically designed to measure the efficiency of NHEJ and HRR (homologous recombination repair) were deployed. MEF and the primary human fibroblast cell lines HF12 and HF19 were exposed to 10mGy - 5Gy X-rays. Bystander effects were investigated using the medium-transfer technique. Results: In contrast to MEF, which induce robust AR to NHEJ, even as a bystander response, human fibroblasts fail to develop such phenomena. Conclusions: The development of AR is cell-type-specific. The same holds true for the development of AR as a bystander effect. Better understanding of the underlying mechanisms will help to understand the molecular basis of these differences in response.

• Conformational transitions of proteins engaged in DNA double strand break repair, analyzed by tryptophan fluorescence emission and FRET.

  Authors: Katia Stankova, Katia Ivanova, Emil Mladenov, Bustanur Rosidi, Aparna Sharma, Rayna Boteva, George Iliakis

  The Biochemical journal. 02/2012;

  We analyzed protein-DNA and protein-protein interactions relevant to the repair of DNA double strand breaks (DSBs) by non-homologous end-joining (NHEJ). Conformational transitions in mammalian DNAWe analyzed protein-DNA and protein-protein interactions relevant to the repair of DNA double strand breaks (DSBs) by non-homologous end-joining (NHEJ). Conformational transitions in mammalian DNA ligases III (LigIII) and IV (LigIV), as well as in poly(ADP)ribose polymerase- 1 (PARP-1), were analyzed upon binding to double stranded DNA by changes in tryptophan (Trp) emission and FRET from Trp to DNA-conjugated Alexa-Fluor-532. For LigIII, two nonequivalent high and low affinity DNA-binding sites are detected interacting sequentially with DNA. PARP-1 displays a single high affinity DNA-binding site and can displace bound DNA fragments from the low affinity site of DNA ligase III, consistent with its mediator role in LigIIIDNA interactions. For LigIV/XRCC4 (LX-complex), a single DNA-binding site is detected. Binding of Ku to DNA was accompanied by conformational changes in the protein and inter2 molecular FRET from dansyl chromophores of the labeled Ku to the Alexa chromophores of Alexa-Fluor-532-conjugated DNA. The average distance of 5.7 nm calculated from FRET data is consistent with a location of Ku at the very end of the DNA molecule. Binding of LX to Ku- DNA complexes is associated with conformational changes in Ku, further translocating the protein towards the DNA ends. The protein-protein and protein-DNA interactions detected and analyzed generate a framework for the characterization of molecular interactions fundamental to the function of NHEJ pathways in higher eukaryotes.

• Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells.

  Authors: Hiroshi Arakawa, Theresa Bednar, Minli Wang, Katja Paul, Emil Mladenov, Alena A Bencsik-Theilen, George Iliakis

  Nucleic acids research. 11/2011;

  In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replicationIn eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excision (BER) and single strand break repair (SSBR). Here, using conditional knockout strategies for LIG3 and concomitant inactivation of the LIG1 and LIG4 genes, we show that in DT40 cells LigIII efficiently supports semi-conservative DNA replication. Our observations demonstrate a high functional versatility for the evolutionary new LigIII in DNA replication and mitochondrial metabolism, and suggest the presence of an alternative pathway for Okazaki fragment ligation.

• The Pathways of Double-Strand Break Repair

  Authors: Emil Mladenov, George Iliakis

  09/2011;

  ISBN: 978-953-307-649-2

• Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair.

  Authors: Satyendra K Singh, Minli Wang, Christian Staudt, George Iliakis

  Nucleic acids research. 07/2011; 39(19):8416-29.

  In cells exposed to ionizing radiation (IR), double-strand breaks (DSBs) form within clustered-damage sites from lesions disrupting the DNA sugar-phosphate backbone. It is commonly assumed that theseIn cells exposed to ionizing radiation (IR), double-strand breaks (DSBs) form within clustered-damage sites from lesions disrupting the DNA sugar-phosphate backbone. It is commonly assumed that these DSBs form promptly and are immediately detected and processed by the cellular DNA damage response (DDR) apparatus. This assumption is questioned by the observation that after irradiation of naked DNA, a fraction of DSBs forms minutes to hours after exposure as a result of temperature dependent, chemical processing of labile sugar lesions. Excess DSBs also form when IR-exposed cells are processed at 50°C, but have been hitherto considered method-related artifact. Thus, it remains unknown whether DSBs actually develop in cells after IR exposure from chemically labile damage. Here, we show that irradiation of 'naked' or chromatin-organized mammalian DNA produces lesions, which evolve to DSBs and add to those promptly induced, after 8-24 h in vitro incubation at 37°C or 50°C. The conversion is more efficient in chromatin-associated DNA, completed within 1 h in cells and delayed in a reducing environment. We conclude that IR generates sugar lesions within clustered-damage sites contributing to DSB formation only after chemical processing, which occurs efficiently at 37°C. This subset of delayed DSBs may challenge DDR, may affect the perceived repair kinetics and requires further characterization.

• Induction and repair of DNA double strand breaks: the increasing spectrum of non-homologous end joining pathways.

  Authors: Emil Mladenov, George Iliakis

  Mutation research. 02/2011; 711(1-2):61-72.

  A defining characteristic of damage induced in the DNA by ionizing radiation (IR) is its clustered character that leads to the formation of complex lesions challenging the cellular repair mechanisms.A defining characteristic of damage induced in the DNA by ionizing radiation (IR) is its clustered character that leads to the formation of complex lesions challenging the cellular repair mechanisms. The most widely investigated such complex lesion is the DNA double strand break (DSB). DSBs undermine chromatin stability and challenge the repair machinery because an intact template strand is lacking to assist restoration of integrity and sequence in the DNA molecule. Therefore, cells have evolved a sophisticated machinery to detect DSBs and coordinate a response on the basis of inputs from various sources. A central function of cellular responses to DSBs is the coordination of DSB repair. Two conceptually different mechanisms can in principle remove DSBs from the genome of cells of higher eukaryotes. Homologous recombination repair (HRR) uses as template a homologous DNA molecule and is therefore error-free; it functions preferentially in the S and G2 phases. Non-homologous end joining (NHEJ), on the other hand, simply restores DNA integrity by joining the two ends, is error prone as sequence is only fortuitously preserved and active throughout the cell cycle. The basis of DSB repair pathway choice remains unknown, but cells of higher eukaryotes appear programmed to utilize preferentially NHEJ. Recent work suggests that when the canonical DNA-PK dependent pathway of NHEJ (D-NHEJ), becomes compromised an alternative NHEJ pathway and not HRR substitutes in a quasi-backup function (B-NHEJ). Here, we outline aspects of DSB induction by IR and review the mechanisms of their processing in cells of higher eukaryotes. We place particular emphasis on backup pathways of NHEJ and summarize their increasing significance in various cellular processes, as well as their potential contribution to carcinogenesis.

• Evidence of an adaptive response targeting DNA nonhomologous end joining and its transmission to bystander cells.

  Authors: Holger Klammer, Munira Kadhim, George Iliakis

  Cancer research. 11/2010; 70(21):8498-506.

  Adaptive response (AR) is a term describing resistance to ionizing radiation-induced killing or formation of aberrant chromosomes that is mediated by pre-exposure to low ionizing radiation doses. TheAdaptive response (AR) is a term describing resistance to ionizing radiation-induced killing or formation of aberrant chromosomes that is mediated by pre-exposure to low ionizing radiation doses. The mechanism of AR remains elusive. Because cell killing and chromosome aberration formation derive from erroneous processing of DNA double-strand breaks (DSB), AR may reflect a modulation of DSB processing by nonhomologous end joining (NHEJ) or homologous recombination repair. Here, we use plasmid end-joining assays to quantify modulations induced by low ionizing radiation doses to NHEJ, the dominant pathway of DSB repair in higher eukaryotes, and investigate propagation of this response through medium transfer to nonirradiated bystander cells. Mouse embryo fibroblasts were conditioned with 10 to 1000 mGy and NHEJ quantified at different times thereafter by challenging with reporter plasmids containing a DSB. We show robust increases in NHEJ efficiency in mouse embryo fibroblasts exposed to ionizing radiation >100 mGy, irrespective of reporter plasmid used. Human tumor cells also show AR of similar magnitude that is compromised by caffeine, an inhibitor of DNA damage signaling acting by inhibiting ATM, ATR, and DNA-PKcs. Growth medium from pre-irradiated cells induces a caffeine-sensitive AR in nonirradiated cells, similar in magnitude to that seen in irradiated cells. In bystander cells, γH2AX foci are specifically detected in late S-G(2) phase and are associated with Rad51 foci that signify the function of homologous recombination repair, possibly on DNA replication-mediated DSBs. The results point to enhanced NHEJ as a mechanism of AR and suggest that AR may be transmitted to bystander cells through factors generating replication-mediated DSBs.

• Widespread dependence of backup NHEJ on growth state: ramifications for the use of DNA-PK inhibitors.

  Authors: Satyendra K Singh, Wenqi Wu, Lihua Zhang, Holger Klammer, Minli Wang, George Iliakis

  International journal of radiation oncology, biology, physics. 10/2010; 79(2):540-8.

  The backup pathway of nonhomologous end joining (B-NHEJ) enables cells to process DNA double-strand breaks (DSBs) when the DNA-PK-dependent pathway of NHEJ (D-NHEJ) is compromised. Our previousThe backup pathway of nonhomologous end joining (B-NHEJ) enables cells to process DNA double-strand breaks (DSBs) when the DNA-PK-dependent pathway of NHEJ (D-NHEJ) is compromised. Our previous results show marked reduction in the activity of B-NHEJ when LIG4(-/-) mouse embryo fibroblasts (MEFs) cease to grow and enter a plateau phase. The dependence of B-NHEJ on growth state is substantially stronger than that of D-NHEJ and points to regulatory mechanisms or processing determinants that require elucidation. Because the different D-NHEJ mutants show phenotypes distinct in their details, it is necessary to characterize the dependence of their DSB repair capacity on growth state and to explore species-specific responses. DSB repair was measured in cells of different genetic background from various species using pulsed-field gel electrophoresis, or the formation of γ-H2AX foci, at different stages of growth. Using pulsed-field gel electrophoresis, we report a marked reduction of B-NHEJ during the plateau phase of growth in KU and XRCC4, mouse or Chinese hamster, mutants. Notably, this reduction is only marginal in DNA-PKcs-deficient cells. However, reduced B-NHEJ is also observed in repair proficient, plateau-phase cells after treatment with DNA-PK inhibitors. The reduction of B-NHEJ activity in the plateau phase of growth does not derive from the reduced expression of participating proteins, is detectable by γ-H2AX foci analysis, and leads to enhanced cell killing. These results further document the marked dependence on growth state of an essential DSB repair pathway and show the general nature of the effect. Molecular characterization of the mechanism underlying this response will help to optimize the administration of DNA repair inhibitors as adjuvants in radiation therapy.

• Initial characterization of a low-molecular-weight factor enhancing the checkpoint response.

  Authors: Xiaoxiang Fan, Nge Cheong, George Iliakis

  Radiation research. 10/2010; 174(4):424-35.

  In higher eukaryotes, DNA double-strand breaks (DSBs) induced by ionizing radiation activate checkpoints that delay progression through the cell cycle. Compared to delays in other phases of the cellIn higher eukaryotes, DNA double-strand breaks (DSBs) induced by ionizing radiation activate checkpoints that delay progression through the cell cycle. Compared to delays in other phases of the cell cycle, delays induced in G(2) are longer and frequently correlate with resistance to killing by radiation. Therefore, modulation of the G(2) checkpoint offers a means to modulate cellular radiosensitivity. Although compounds are known that reduce the G(2) checkpoint and act as radiosensitizers, compounds enhancing this checkpoint have not been reported. Here we summarize evidence for a factor with such properties. We show that a highly radioresistant rat embryo fibroblast (REF) cell line displays a strong G(2) checkpoint partly as a result of a factor excreted into the growth medium by nonirradiated cells. Various tests indicate that this G(2)-arrest modulating activity (GAMA) is a small molecule showing detectable retention only after passing through filters with a molecular weight cutoff limit of less than 1,000 Da. GAMA is heat stable and resistant to treatment with proteases or nucleases. Electroelution tests show that GAMA is uncharged at neutral pH, a result that is in agreement with the observed failure to bind S- or Q-Sepharose. Investigations on the mechanism of GAMA function indicate ligand-receptor interactions and allow the classification of cells as producers, responders or both. Compounds with properties such as those of GAMA bridge intercellular communication with the DNA damage response and may function as radioprotectors.

• New partners for Chk1.

  Authors: George Iliakis

  Cell cycle (Georgetown, Tex.). 06/2010; 9(11):2061-2.

• Extensive Repair of DNA Double-Strand Breaks in Cells Deficient in the DNA-PK-Dependent Pathway of NHEJ after Exclusion of Heat-Labile Sites.

  Authors: Satyendra K Singh, Weizhong Wu, Wenqi Wu, Minli Wang, George Iliakis

  Radiation research. 09/2009; 172(2):152-64.

  Abstract Double-strand breaks (DSBs) can be generated in the DNA of mammalian cells when heat-labile sites induced by ionizing radiation within a clustered DNA damage site are thermally transformedAbstract Double-strand breaks (DSBs) can be generated in the DNA of mammalian cells when heat-labile sites induced by ionizing radiation within a clustered DNA damage site are thermally transformed to single-strand breaks (SSBs) and combine with other SSBs or heat-labile sites in the opposite DNA strand. When this thermal transformation of heat-labile sites to SSBs occurs during DNA preparation using high-temperature lysis, the DSB yield is overestimated by including DSBs not present in the tested cell. Low-temperature lysis avoids this artifact but shows slower repair kinetics for prompt DSBs than high-temperature lysis. The apparent slowdown of DSB repair after low-temperature lysis is particularly pronounced in mutants defective in the DNA-PK-dependent pathway of NHEJ (D-NHEJ) and has led to the postulate that these cells may actually be entirely deficient in DSB repair. Since our work as well as that of others provides strong evidence for DSB repair in D-NHEJ-deficient cells by alternative repair pathways operating as a backup (B-NHEJ), we re-examine here DSB repair in several D-NHEJ-deficient mutants using low-temperature lysis. The results demonstrate extensive but slow repair of DSBs after low-temperature lysis indicative of a robust function of B-NHEJ. At the same time, the results demonstrate a pronounced sensitivity of heat-labile sites to lysis at 37 degrees C, which indicates that DSBs generated by transformation of heat-labile sites may be in part a reality that the irradiated cell faces at all times.

• Backup pathways of NHEJ in cells of higher eukaryotes: Cell cycle dependence.

  Authors: George Iliakis

  Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology. 08/2009;

  DNA double-strand breaks (DSBs) induced by ionizing radiation (IR) in cells of higher eukaryotes are predominantly repaired by a pathway of non-homologous end joining (NHEJ) utilizing Ku, DNA-PKcs,DNA double-strand breaks (DSBs) induced by ionizing radiation (IR) in cells of higher eukaryotes are predominantly repaired by a pathway of non-homologous end joining (NHEJ) utilizing Ku, DNA-PKcs, DNA ligase IV, XRCC4 and XLF/Cernunnos (D-NHEJ) as central components. Work carried out in our laboratory and elsewhere shows that when this pathway is chemically or genetically compromised, cells do not shunt DSBs to homologous recombination repair (HRR) but instead use another form of NHEJ operating as a backup (B-NHEJ). Here I review our efforts to characterize this repair pathway and discuss its dependence on the cell cycle as well as on the growth conditions. I present evidence that B-NHEJ utilizes ligase III, PARP-1 and histone H1. When B-NHEJ is examined throughout the cell cycle, significantly higher activity is observed in G2 phase that cannot be attributed to HRR. Furthermore, the activity of B-NHEJ is compromised when cells enter the plateau phase of growth. Together, these observations uncover a repair pathway with unexpected biochemical constitution and interesting cell cycle and growth factor regulation. They generate a framework for investigating the mechanistic basis of HRR contribution to DSB repair.

• Application of alkaline sucrose gradient centrifugation in the analysis of DNA replication after DNA damage.

  Authors: Sascha Raschke, Jun Guan, George Iliakis

  Methods in molecular biology (Clifton, N.J.). 02/2009; 521:329-42.

  Sucrose density gradient ultracentrifugation is a powerful technique for fractionating macromolecules like DNA, RNA, and proteins. For this purpose, a sample containing a mixture of different sizeSucrose density gradient ultracentrifugation is a powerful technique for fractionating macromolecules like DNA, RNA, and proteins. For this purpose, a sample containing a mixture of different size macromolecules is layered on the surface of a gradient whose density increases linearly from top to bottom. During centrifugation, different size macromolecules sediment through the gradient at different rates. The rate of sedimentation depends, in addition to centrifugal force, on the size, shape, and density of the macromolecules, as well as on the density and viscosity of the gradient. In this way, macromolecules are separated by size with larger ones sedimenting towards the bottom and lighter ones remaining close to the top of the gradient. The method has been particularly successful in the size fractionation of large DNA molecules and has been extensively used to measure induction and repair of DNA breaks after exposure to clastogenic factors. Here, we describe an adaptation of this method that can be used in the analysis of newly synthesized DNA formed during DNA replication. Through size analysis of nascent DNA in alkaline sucrose gradients, variations in replication activity can be measured after exposure of cells to DNA-damaging agents. The method is particularly useful as it allows distinction between DNA damage-mediated effects on chain elongation vs. replicon initiation, which is essential for an in-depth analysis of the intra-S-phase checkpoint. This ability makes the technique unique and justifies its somewhat labour-intensive nature.

• Enhanced use of backup pathways of NHEJ in G2 in Chinese hamster mutant cells with defects in the classical pathway of NHEJ.

  Authors: Wenqi Wu, Minli Wang, Tamara Mussfeldt, George Iliakis

  Radiation research. 11/2008; 170(4):512-20.

  In higher eukaryotes DNA double-strand breaks (DSBs) are repaired by homologous recombination repair (HRR) or nonhomologous end joining (NHEJ). In addition to the DNA-PK dependent pathway of NHEJIn higher eukaryotes DNA double-strand breaks (DSBs) are repaired by homologous recombination repair (HRR) or nonhomologous end joining (NHEJ). In addition to the DNA-PK dependent pathway of NHEJ (D-NHEJ), cells employ a backup pathway (B-NHEJ) using DNA ligase III and PARP1. We have reported previously that mouse embryo fibroblasts (MEFs) defective in D-NHEJ show enhanced repair of DSBs in G2 not reflecting a contribution of HRR. Here we extend these studies to Chinese hamster mutant cells with defects in the DNA-PKcs, Ku80 or XRCC4 components of D-NHEJ or in the XRCC2 and XRCC3 components of HRR. Using cell sorting to separate cells at defined times after irradiation, we measure repair of DSBs with pulsed-field gel electrophoresis in unperturbed G1- and G2-phase cells. Wild-type cells and mutants of XRCC2 and XRCC3 repair DSBs with similar efficiency in G1 and G2. Mutants of DNA-PKcs, Ku80 and XRCC4 show more pronounced repair in G2 than in G1. These and previously published results provide support for the notion that the increased efficacy of DSB repair in G2 reflects the enhanced function of B-NHEJ, which may be a general feature of rodent cells that also holds for human cells.

• {gamma}-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin.

  Authors: Andrea Kinner, Wenqi Wu, Christian Staudt, George Iliakis

  Nucleic acids research. 10/2008;

  DNA double-strand breaks (DSBs) are extremely dangerous lesions with severe consequences for cell survival and the maintenance of genomic stability. In higher eukaryotic cells, DSBs in chromatinDNA double-strand breaks (DSBs) are extremely dangerous lesions with severe consequences for cell survival and the maintenance of genomic stability. In higher eukaryotic cells, DSBs in chromatin promptly initiate the phosphorylation of the histone H2A variant, H2AX, at Serine 139 to generate gamma-H2AX. This phosphorylation event requires the activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs, ATM, and ATR, and serves as a landing pad for the accumulation and retention of the central components of the signaling cascade initiated by DNA damage. Regions in chromatin with gamma-H2AX are conveniently detected by immunofluorescence microscopy and serve as beacons of DSBs. This has allowed the development of an assay that has proved particularly useful in the molecular analysis of the processing of DSBs. Here, we first review the role of gamma-H2AX in DNA damage response in the context of chromatin and discuss subsequently the use of this modification as a surrogate marker for mechanistic studies of DSB induction and processing. We conclude with a critical analysis of the strengths and weaknesses of the approach and present some interesting applications of the resulting methodology.

• Histone H1 functions as a stimulatory factor in backup pathways of NHEJ.

  Authors: Bustanur Rosidi, Minli Wang, Wenqi Wu, Aparna Sharma, Huichen Wang, George Iliakis

  Nucleic acids research. 04/2008; 36(5):1610-23.

  DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by ionizing radiation (IR) are predominantly removed by two pathways of non-homologous end-joining (NHEJ) termed D-NHEJ andDNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by ionizing radiation (IR) are predominantly removed by two pathways of non-homologous end-joining (NHEJ) termed D-NHEJ and B-NHEJ. While D-NHEJ depends on the activities of the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4/XLF, B-NHEJ utilizes, at least partly, DNA ligase III/XRCC1 and PARP-1. Using in vitro end-joining assays and protein fractionation protocols similar to those previously applied for the characterization of DNA ligase III as an end-joining factor, we identify here histone H1 as an additional putative NHEJ factor. H1 strongly enhances DNA-end joining and shifts the product spectrum from circles to multimers. While H1 enhances the DNA-end-joining activities of both DNA Ligase IV and DNA Ligase III, the effect on ligase III is significantly stronger. Histone H1 also enhances the activity of PARP-1. Since histone H1 has been shown to counteract D-NHEJ, these observations and the known functions of the protein identify it as a putative alignment factor operating preferentially within B-NHEJ.

• DNA double strand break repair inhibition as a cause of heat radiosensitization: re-evaluation considering backup pathways of NHEJ.

  Authors: George Iliakis, Wenqi Wu, Minli Wang

  International journal of hyperthermia : the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group. 03/2008; 24(1):17-29.

  Heat shock is one of the most effective radiosensitizers known. As a result, combination of heat with ionizing radiation (IR) is considered a promising strategy in the management of human cancer. TheHeat shock is one of the most effective radiosensitizers known. As a result, combination of heat with ionizing radiation (IR) is considered a promising strategy in the management of human cancer. The mechanism of heat radiosensitization has been the subject of extensive work but a unifying mechanistic model is presently lacking. To understand the cause of excessive death in irradiated cells after heat exposure, it is necessary to characterize the lesion(s) underlying the effect and to determine which of the pathways processing this lesion are affected by heat. Since DNA double strand breaks (DSBs) are the main cause for IR-induced cell death, inhibition of DSB processing has long been considered a major candidate for heat radiosensitization. However, effective radiosensitization of mutants with defects in homologous recombination repair (HRR), or in DNA-PK dependent non-homologous end joining (D-NHEJ), the two primary pathways of DSB repair, has led to the formulation of models excluding DSBs as a cause for this phenomenon and attributing heat radiosensitization to inhibition of base damage processing. Since direct evidence for a major role of base damage in heat radiosensitization, or in IR-induced killing for that matter, is scarce and new insights in DSB repair allow alternative interpretations of existing data with repair mutants, we attempt here a re-evaluation of the role of DSBs and their repair in heat radiosensitization. First, we reanalyse data obtained with various DSB repair mutants on first principles and in the light of the recent recognition that alternative pathways of NHEJ, operating as backup (B-NHEJ), substantially contribute to DSB repair and thus probably also to heat radiosensitization. Second, we review aspects of combined action of heat and radiation, such as modulation in the cell-cycle-dependent variation in radiosensitivity to killing, as well as heat radiosensitization as a function of LET, and examine whether the observed effects are compatible with DSB repair inhibition. We conclude with a model reclaiming a central role for DSBs in heat radiosensitization.

• Repair of radiation induced DNA double strand breaks by backup NHEJ is enhanced in G2.

  Authors: Wenqi Wu, Minli Wang, Weizhong Wu, Satyendra K Singh, Tamara Mussfeldt, George Iliakis

  DNA repair. 03/2008; 7(2):329-38.

  In higher eukaryotes DNA double strand breaks (DSBs) are repaired by homologous recombination (HRR) or non-homologous end joining (NHEJ). In addition to the DNA-PK dependent pathway of NHEJ (D-NHEJ),In higher eukaryotes DNA double strand breaks (DSBs) are repaired by homologous recombination (HRR) or non-homologous end joining (NHEJ). In addition to the DNA-PK dependent pathway of NHEJ (D-NHEJ), cells employ a backup pathway (B-NHEJ) utilizing Ligase III and PARP-1. The cell cycle dependence and coordination of these pathways is being actively investigated. We examine DSB repair in unperturbed G1 and G2 phase cells using mouse embryo fibroblast (MEF) mutants defective in D-NHEJ and/or HRR. WT and Rad54(-/-) MEFs repair DSBs with similar efficiency in G1 and G2 phase. LIG4(-/-), DNA-PKcs(-/-), and Ku70(-/-) MEFs show more pronounced repair defects in G1 than in G2. LIG4(-/-)/Rad54(-/-) MEFs repair DSBs as efficiently as LIG4(-/-) MEFs suggesting that the increased repair efficiency in G2 relies on enhanced function of B-NHEJ rather than HRR. In vivo and in vitro plasmid end joining assays confirm an enhanced function of B-NHEJ in G2. The results show a new and potentially important cell cycle regulation of B-NHEJ and generate a framework to investigate the mechanistic basis of HRR contribution to DSB repair.

• Low levels of DNA ligases III and IV sufficient for effective NHEJ.

  Authors: Frank Windhofer, Wenqi Wu, George Iliakis

  Journal of cellular physiology. 12/2007; 213(2):475-83.

  Cells of higher eukaryotes rejoin double strand breaks (DSBs) in their DNA predominantly by a non-homologous DNA end joining (NHEJ) pathway that utilizes the products of DNA-PKcs, Ku, LIG4, XRCC4,Cells of higher eukaryotes rejoin double strand breaks (DSBs) in their DNA predominantly by a non-homologous DNA end joining (NHEJ) pathway that utilizes the products of DNA-PKcs, Ku, LIG4, XRCC4, XLF/Cernunnos, Artemis as well as DNA polymerase lambda (termed D-NHEJ). Mutants with defects in these proteins remove a large proportion of DSBs from their genome utilizing an alternative pathway of NHEJ that operates as a backup (B-NHEJ). While D-NHEJ relies exclusively on DNA ligase IV, recent work points to DNA ligase III as a component of B-NHEJ. Here, we use RNA interference (RNAi) to further investigate the activity requirements for DNA ligase III and IV in the pathways of NHEJ. We report that 70-80% knock down of LIG3 expression has no detectable effect on DSB rejoining, either in D-NHEJ proficient cells, or in cells where D-NHEJ has been chemically or genetically compromised. Surprisingly, also LIG4 knock down has no effect on repair proficient cells, but inhibits DSB rejoining in a radiosensitive cell line with a hypomorphic LIG4 mutation that severely compromises its activity. The results suggest that complete coverage for D-NHEJ or B-NHEJ is afforded by very low ligase levels and demonstrate residual end joining by DNA ligase IV in cells of patients with mutations in LIG4.

• Marked dependence on growth state of backup pathways of NHEJ.

  Authors: Frank Windhofer, Wenqi Wu, Minli Wang, Satyendra K Singh, Janapriya Saha, Bustanur Rosidi, George Iliakis

  International journal of radiation oncology, biology, physics. 09/2007; 68(5):1462-70.

  PURPOSE: Backup pathways of nonhomologous end joining (B-NHEJ) enable cells to repair DNA double-strand breaks (DSBs) when DNA-PK-dependent NHEJ (D-NHEJ) is compromised. Recent evidence implicatesPURPOSE: Backup pathways of nonhomologous end joining (B-NHEJ) enable cells to repair DNA double-strand breaks (DSBs) when DNA-PK-dependent NHEJ (D-NHEJ) is compromised. Recent evidence implicates growth signaling in the regulation of D-NHEJ. This study was intended to determine whether the ability to repair DSBs by B-NHEJ also depends on growth state. METHODS AND MATERIALS: LIG4(-/-) and wild type (WT) mouse embryo fibroblasts (MEFs) were used. Repair of DSBs was measured by pulsed-field agarose gel electrophoresis. G1 cells were selected by centrifugal elutriation. A plasmid assay was used to measure DNA end-joining activity in whole cell extracts. RESULTS: Wild-type MEFs efficiently repaired DSBs by D-NHEJ in either the exponential or plateau phase of growth. Because of their defect in ligase IV, which compromises D-NHEJ, LIG4(-/-) MEFs showed reduced repair capacity but were slowly able to rejoin a large proportion of DSBs via B-NHEJ. B-NHEJ was markedly reduced in the plateau phase of growth or at high radiation doses. Elutriated G1 cells from exponentially growing or plateau-phase LIG4(-/-) cultures showed a response similar to nonelutriated cells, ruling out that the effect simply reflects redistribution in the cell cycle. An in vitro assay, gauging the activity of B-NHEJ, showed a reduction in DNA end joining during the plateau phase that could be corrected by recombinant DNA ligase IIIalpha. CONCLUSIONS: Suppression of growth signaling markedly compromises DSB repair by B-NHEJ. This effect is associated with a reduction in DNA ligase III mediated DNA end joining.