The controlling role of ATM in homologous recombinational repair of DNA damage

The human genetic disorder ataxia telangiectasia (A-T), caused by mutation in the ATM gene, is characterized by chromosomal instability, radiosensitivity and defective cell cycle checkpoint activation. DNA double-strand breaks (dsbs) persist in A-T cells after irradiation, but the underlying defect is unclear. To investigate ATM's interactions with dsb repair pathways, we disrupted ATM along with other genes involved in the principal, complementary dsb repair pathways of homologous recombination (HR) or non-homologous end-joining (NHEJ) in chicken DT40 cells. ATM(-/-) cells show altered kinetics of radiation-induced Rad51 and Rad54 focus formation. Ku70-deficient (NHEJ(-)) ATM(-/-) chicken DT40 cells show radiosensitivity and high radiation-induced chromosomal aberration frequencies, while Rad54-defective (HR(-)) ATM(-/-) cells show only slightly elevated aberration levels after irradiation, placing ATM and HR on the same pathway. These results reveal that ATM defects impair HR-mediated dsb repair and may link cell cycle checkpoints to HR activation.     

Genetic dissection of vertebrate 53BP1: a major role in non-homologous end joining of DNA double strand breaks

53BP1 (p53 binding protein) is a BRCT domain-containing protein that is rapidly recruited to DNA double strand breaks (DSBs). To investigate the role of 53BP1 in the DNA damage response, we generated 53BP1(-/-) cells from the chicken DT40 cell line. As in mammalian cells, mutation of 53BP1 increased cellular sensitivity to ionizing radiation. Although depletion of 53BP1 resulted in checkpoint defects in mammalian cells, DT40 53BP1(-/-) cells had normal intra S phase and G2/M checkpoints. G1 specific radiosensitivity and a higher sensitivity to topoisomerase II suggested defective non-homologous end joining (NHEJ) defects in DT40 53BP1(-/-) cells. Genetic analyses confirm this suggestion as we have demonstrated an epistatic relationship between 53BP1 and the NHEJ genes, Ku70 and Artemis, but not with Rad54, a gene essential for repair of DSBs by homologous recombination. We conclude that the major role of 53BP1 in supporting survival of DT40 cells that have suffered DNA DSBs is in facilitating repair by NHEJ.     

Evaluation of recombination repair pathways in thermal radiosensitization

Thermal radiosensitization has been shown to cause inhibition of repair of sublethal and potentially lethal damage and DNA DSBs. In this study we assessed thermal radiosensitization in mutants deficient in homologous recombinational (HR) repair and nonhomologous end joining repair (NHEJ). Using cells of the mouse wild-type embryo fibroblast cell line MEF and its Ku80(-/-) derivative that is deficient in NHEJ, we showed that thermal radiosensitization is the same in both cell lines. Further studies with cells of the wild-type CHO-AA8 cell line and its derivative IRS(ISF), which is deficient in HR, also showed comparable thermal radiosensitization in both cell lines. Further experiments using cells of chicken DT40 cell lines also showed comparable thermal radiosensitization between the wild-type HR mutant Rad54, the NHEJ mutant Ku70, and the double mutant Rad 54-Ku70. These results indicate that the HR and NHEJ pathways may not be targets for thermal radiosensitization.     

Genetic evidence for involvement of two distinct nonhomologous end-joining pathways in repair of topoisomerase II-mediated DNA damage

In vertebrate cells, DNA double-strand breaks are efficiently repaired by homologous recombination or nonhomologous end-joining (NHEJ). The latter pathway relies on Ku (the Ku70/Ku86 heterodimer), DNA-PKcs, Artemis, Xrcc4, and DNA ligase IV (Lig4). Here, we show that a human pre-B cell line nullizygous for Lig4 exhibits hypersensitivity to topoisomerase II (Top2) inhibitors, demonstrating a crucial role for the NHEJ pathway in repair of Top2-induced DNA damage in vertebrates. We also show that in the chicken DT40 cell line, all NHEJ mutants (i.e., Ku70-, Lig4-, and DNA-PKcs-null cells) are equally hypersensitive to the Top2 inhibitor ICRF-193, indicating that the drug-induced damage is repaired by NHEJ involving DNA-PKcs. Intriguingly, however, DNA-PKcs-null cells display considerably less severe phenotype than other NHEJ mutants in terms of hypersensitivity to VP-16, a Top2 poison that stabilizes cleavable complexes. The results indicate that two distinct NHEJ pathways, involving or not involving DNA-PKcs, are important for the repair of VP-16-induced DNA damage, providing additional evidence for the biological relevance of DNA-PKcs-independent NHEJ. Our results provide significant insights into the mechanisms of repair of Top2-mediated DNA damage, with implications for chemotherapy involving Top2 inhibitors.     

Down-regulation of PARP-1, but not of Ku80 or DNA-PKcs', results in higher gene targeting efficiency

The viability of non-homologous end-joining (NHEJ)-defective mice suggests that homologous recombination (HR) might take over its role in DNA repair. To test this hypothesis, we examined gene targeting frequencies (TF) in DNA-PK(cs), Ku80 and poly(ADP-ribose) polymerase (PARP-1) nullizygous cells. We observed a 3-fold TF increase in PARP-1 knockout embryonic stem (ES) cells, which is consistent with the predicted role of PARP-1 as a switch between HR and NHEJ. To a lesser extent, such effect could be reproduced upon chemical inhibition of PARP-1. However, TF was not enhanced in Ku80- or DNA-PK(cs)-defective cells. Our study also suggests an unexpected involvement of DNA-PK(cs) in HR.     

Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells.

Eukaryotic cells repair DNA double-strand breaks (DSBs) by at least two pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). Rad54 participates in the first recombinational repair pathway while Ku proteins are involved in NHEJ. To investigate the distinctive as well as redundant roles of these two repair pathways, we analyzed the mutants RAD54(-/-), KU70(-/-) and RAD54(-/-)/KU70(-/-), generated from the chicken B-cell line DT40. We found that the NHEJ pathway plays a dominant role in repairing gamma-radiation-induced DSBs during G1-early S phase while recombinational repair is preferentially used in late S-G2 phase. RAD54(-/-)/KU70(-/-) cells were profoundly more sensitive to gamma-rays than either single mutant, indicating that the two repair pathways are complementary. Spontaneous chromosomal aberrations and cell death were observed in both RAD54(-/-) and RAD54(-/-)/KU70(-/-) cells, with RAD54(-/-)/KU70(-/-) cells exhibiting significantly higher levels of chromosomal aberrations than RAD54(-/-) cells. These observations provide the first genetic evidence that both repair pathways play a role in maintaining chromosomal DNA during the cell cycle.     

PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways

Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer.     

Alternative pathways for the repair of RAG-induced DNA breaks

RAG1 and RAG2 cleave DNA to generate blunt signal ends and hairpin coding ends at antigen receptor loci in lymphoid cells. During V(D)J recombination, repair of these RAG-generated double-strand breaks (DSBs) by the nonhomologous end-joining (NHEJ) pathway contributes substantially to the antigen receptor diversity necessary for immune system function, although recent evidence also supports the ability of RAG-generated breaks to undergo homology-directed repair (HDR). We have determined that RAG-generated chromosomal breaks can be repaired by pathways other than NHEJ in mouse embryonic stem (ES) cells, although repair by these pathways occurs at a significantly lower frequency than NHEJ. HDR frequency was estimated to be >or=40-fold lower than NHEJ frequency for both coding end and signal end reporters. Repair by single-strand annealing was estimated to occur at a comparable or lower frequency than HDR. As expected, V(D)J recombination was substantially impaired in cells deficient for the NHEJ components Ku70, XRCC4, and DNA-PKcs. Concomitant with decreased NHEJ, RAG-induced HDR was increased in each of the mutants, including cells lacking DNA-PKcs, which has been implicated in hairpin opening. HDR was increased to the largest extent in Ku70-/- cells, implicating the Ku70/80 DNA end-binding protein in regulating pathway choice. Thus, RAG-generated DSBs are typically repaired by the NHEJ pathway in ES cells, but in the absence of NHEJ components, a substantial fraction of breaks can be efficiently channeled into alternative pathways in these cells.     

Loss of nonhomologous end joining confers camptothecin resistance in DT40 cells. Implications for the repair of topoisomerase I-mediated DNA damage

DNA topoisomerase I (Top1) generates transient DNA single-strand breaks via the formation of cleavage complexes in which the enzyme is linked to the 3'-phosphate of the cleavage strand. The anticancer drug camptothecin (CPT) poisons Top1 by trapping cleavage complexes, thereby inducing Top1-linked single-strand breaks. Such DNA lesions are converted into DNA double-strand breaks (DSBs) upon collision with replication forks, implying that DSB repair pathways could be involved in the processing/repair of Top1-mediated DNA damage. Here we report that Top1-mediated DNA damage is repaired primarily by homologous recombination, a major pathway of DSB repair. Unexpectedly, however, we found that nonhomologous end joining (NHEJ), another DSB repair pathway, has no positive role in the relevant repair; notably, DT40 cell mutants lacking either of the NHEJ factors (namely, Ku70, DNA-dependent protein kinase catalytic subunit, and DNA ligase IV) were resistant to killing by CPT. In addition, we showed that the absence of NHEJ alleviates the requirement of homologous recombination in the repair of CPT-induced DNA damage. Our results indicate that NHEJ can be a cytotoxic pathway in the presence of CPT, shedding new light on the molecular mechanisms for the formation and repair of Top1-mediated DNA damage in vertebrates. Thus, our data have significant implications for cancer chemotherapy involving Top1 inhibitors.     

Characteristics of DNA-binding proteins determine the biological sensitivity to high-linear energy transfer radiation

Non-homologous end-joining (NHEJ) and homologous recombination repair (HRR), contribute to repair ionizing radiation (IR)-induced DNA double-strand breaks (DSBs). Mre11 binding to DNA is the first step for activating HRR and Ku binding to DNA is the first step for initiating NHEJ. High-linear energy transfer (LET) IR (such as high energy charged particles) killing more cells at the same dose as compared with low-LET IR (such as X or γ rays) is due to inefficient NHEJ. However, these phenomena have not been demonstrated at the animal level and the mechanism by which high-LET IR does not affect the efficiency of HRR remains unclear. In this study, we showed that although wild-type and HRR-deficient mice or DT40 cells are more sensitive to high-LET IR than to low-LET IR, NHEJ deficient mice or DT40 cells are equally sensitive to high- and low-LET IR. We also showed that Mre11 and Ku respond differently to shorter DNA fragments in vitro and to the DNA from high-LET irradiated cells in vivo. These findings provide strong evidence that the different DNA DSB binding properties of Mre11 and Ku determine the different efficiencies of HRR and NHEJ to repair high-LET radiation induced DSBs.     

PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA

Poly-ADP ribose polymerase 1 (PARP-1) is activated by DNA damage and has been implicated in the repair of single-strand breaks (SSBs). Involvement of PARP-1 in other DNA damage responses remains controversial. In this study, we show that PARP-1 is required for replication fork slowing on damaged DNA. Fork progression in PARP-1^−/− DT40 cells is not slowed down even in the presence of DNA damage induced by the topoisomerase I inhibitor camptothecin (CPT). Mammalian cells treated with a PARP inhibitor or PARP-1–specific small interfering RNAs show similar results. The expression of human PARP-1 restores fork slowing in PARP-1^−/− DT40 cells. PARP-1 affects SSB repair, homologous recombination (HR), and nonhomologous end joining; therefore, we analyzed the effect of CPT on DT40 clones deficient in these pathways. We find that fork slowing is correlated with the proficiency of HR-mediated repair. Our data support the presence of a novel checkpoint pathway in which the initiation of HR but not DNA damage delays the fork progression.     

Competition between PARP-1 and Ku70 control the decision between high-fidelity and mutagenic DNA repair

Affinity maturation of antibodies requires a unique process of targeted mutation that allows changes to accumulate in the antibody genes while the rest of the genome is protected from off-target mutations that can be oncogenic. This targeting requires that the same deamination event be repaired either by a mutagenic or a high-fidelity pathway depending on the genomic location. We have previously shown that the BRCT domain of the DNA-damage sensor PARP-1 is required for mutagenic repair occurring in the context of IgH and IgL diversification in the chicken B cell line DT40. Here we show that immunoprecipitation of the BRCT domain of PARP-1 pulls down Ku70 and the DNA-PK complex although the BRCT domain of PARP-1 does not bind DNA, suggesting that this interaction is not DNA dependent. Through sequencing the IgL variable region in PARP-1(-/-) cells that also lack Ku70 or Lig4, we show that Ku70 or Lig4 deficiency restores GCV to PARP-1(-/-) cells and conclude that the mechanism by which PARP-1 is promoting mutagenic repair is by inhibiting high-fidelity repair which would otherwise be mediated by Ku70 and Lig4.     

The embryonic lethality in DNA ligase IV-deficient mice is rescued by deletion of Ku: implications for unifying the heterogeneous phenotypes of NHEJ mutants

There are two general pathways by which multicellular eukaryotes repair double-strand DNA breaks (DSB): homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). All mammalian mutants in the NHEJ pathway demonstrate a lack of B and T lymphocytes and ionizing radiation sensitivity. Among these NHEJ mutants, the DNA-PK(cs) and Artemis mutants are the least severe, having no obvious phenotype other than the general defects described above. Ku mutants have an intermediate severity with accelerated senescence. The XRCC4 and DNA ligase IV mutants are the most severe, resulting in embryonic lethality. Here we show that the lethality of DNA ligase IV-deficiency in the mouse can be rescued when Ku86 is also absent. To explain the fact that simultaneous gene mutations in the NHEJ pathway can lead to viability when a single mutant is not viable, we propose a nuclease/ligase model. In this model, disrupted NHEJ is more severe if the Artemis:DNA-PK(cs) nuclease is present in the absence of a ligase, and Ku mutants are of intermediate severity, because the nuclease is less efficient. This model is also consistent with the order of severity in organismal phenotypes; consistent with chromosomal breakage observations reported here; and consistent with the NHEJ mutation identified in radiation sensitive human SCID patients.     

Roles of vertebrate Smc5 in sister chromatid cohesion and homologous recombinational repair

The structural maintenance of chromosomes (Smc) family members Smc5 and Smc6 are both essential in budding and fission yeasts. Yeast smc5/6 mutants are hypersensitive to DNA damage, and Smc5/6 is recruited to HO-induced double-strand breaks (DSBs), facilitating intersister chromatid recombinational repair. To determine the role of the vertebrate Smc5/6 complex during the normal cell cycle, we generated an Smc5-deficient chicken DT40 cell line using gene targeting. Surprisingly, Smc5(-) cells were viable, although they proliferated more slowly than controls and showed mitotic abnormalities. Smc5-deficient cells were sensitive to methyl methanesulfonate and ionizing radiation (IR) and showed increased chromosome aberration levels upon irradiation. Formation and resolution of Rad51 and gamma-H2AX foci after irradiation were altered in Smc5 mutants, suggesting defects in homologous recombinational (HR) repair of DNA damage. Ku70(-/-) Smc5(-) cells were more sensitive to IR than either single mutant, with Rad54(-/-) Smc5(-) cells being no more sensitive than Rad54(-/-) cells, consistent with an HR function for the vertebrate Smc5/6 complex. Although gene targeting occurred at wild-type levels, recombinational repair of induced double-strand breaks was reduced in Smc5(-) cells. Smc5 loss increased sister chromatid exchanges and sister chromatid separation distances in mitotic chromosomes. We conclude that Smc5/6 regulates recombinational repair by ensuring appropriate sister chromatid cohesion.     

The Ku-dependent non-homologous end-joining but not other repair pathway is inhibited by high linear energy transfer ionizing radiation

Ionizing radiation (IR) induced DNA double strand breaks (DSBs) are repaired by both non-homologous end-joining (NHEJ) and homologous recombination repair (HRR) in mammalian cells. The NHEJ repair includes a Ku-dependent main pathway and a PARP-1-dependent complementary pathway. Compared with low linear energy transfer (LET) IR (X or gamma ray) at the same doses, high LET IR (high-charge particles) induces more cell death because of ineffective DNA repair. However, it remains unclear whether high LET IR inhibits all repair or specifically one repair pathway. By combining the assays of clonogenic survival, G2M checkpoint and gammaH2AX in the cell lines with deficiencies in different repair genes, we show here that high LET IR inhibits only the Ku-dependent main NHEJ pathway and does not inhibit either the HRR pathway or the PARP-1-dependent complementary NHEJ pathway. In addition, by developing an assay to detect small fragments of DSB (<400 bp) and by detecting the binding abilities of purified Ku and PARP to different sized dsDNA, we present a possible link for explaining the phenotypes. When compared with low LET IR at the same dose, high LET IR might induce similar yields of DNA DSBs in total but it might induce more small fragments of DNA DSBs (<40 base pairs) that prevent Ku binding efficiently to two ends of one DSB fragment at the same time, thus delaying Ku-dependent repair.     

Enhancement of radiation induced cell death in chicken B lymphocytes by withaferin A

Withaferin A (WA), a plant withanolide, has shown significant radiosensitizing effect in vitro and in vivo. Inhibition of DNA repair has been suggested as a mechanism of radiosensitization by WA. To test this, the effect of withaferin A on survival of DT40 chicken B-lymphocyte cell line and its repair deficient single gene mutants Rad54-/-, Ku70-/- and double mutant Ku70-/- /Rad54-/- after irradiation was studied. Exponentially growing cells were treated for 1 hr with 5 microM WA and then exposed to different doses of X-rays. Cell survival was studied by clonogenic assay. WA significantly reduced survival of DT40, Ku70-/- and Ku70-/- /Rad54-/-, but not Rad54-/- cells, suggesting that WA enhances radiosensitivity by interfering with homologous repair, the major pathway of DSB repair in these cells. Inhibition of DNA repair is further indicated in a significant decrease in surviving fraction of DT40 cells by post-irradiation incubation with WA. This could have relevance to cancer radiotherapy.     

The nonhomologous DNA end joining pathway is important for chromosome stability in primary fibroblasts

There are two types of chromosome instability, structural and numerical, and these are important in cancer. Many structural abnormalities are likely to involve double-strand DNA (dsDNA) breaks. Nonhomologous DNA end joining (NHEJ) and homologous recombination are the major pathways for repairing dsDNA breaks. NHEJ is the primary pathway for repairing dsDNA breaks throughout the G0, G1 and early S phases of the cell cycle [1]. Ku86 and DNA ligase IV are two major proteins in the NHEJ pathway. We examined primary dermal fibroblasts from mice (wild type, Ku86(+/-), Ku86(-/-), and DNA ligase IV(+/-)) for chromosome breaks. Fibroblasts from Ku86(+/-) or DNA ligase IV(+/-) mice have elevated frequencies of chromosome breaks compared with those from wild-type mice. Fibroblasts from Ku86(-/-) mice have even higher levels of chromosome breaks. Primary pre-B cells from the same animals did not show significant accumulation of chromosome breaks. Rather the pre-B cells showed increased cell death. These studies demonstrate that chromosome breaks arise frequently and that NHEJ is required to repair this constant spontaneous damage.     

RAD18 and poly(ADP-ribose) polymerase independently suppress the access of nonhomologous end joining to double-strand breaks and facilitate homologous recombination-mediated repair

The Saccharomyces cerevisiae RAD18 gene is essential for postreplication repair but is not required for homologous recombination (HR), which is the major double-strand break (DSB) repair pathway in yeast. Accordingly, yeast rad18 mutants are tolerant of camptothecin (CPT), a topoisomerase I inhibitor, which induces DSBs by blocking replication. Surprisingly, mammalian cells and chicken DT40 cells deficient in Rad18 display reduced HR-dependent repair and are hypersensitive to CPT. Deletion of nonhomologous end joining (NHEJ), a major DSB repair pathway in vertebrates, in rad18-deficient DT40 cells completely restored HR-mediated DSB repair, suggesting that vertebrate Rad18 regulates the balance between NHEJ and HR. We previously reported that loss of NHEJ normalized the CPT sensitivity of cells deficient in poly(ADP-ribose) polymerase 1 (PARP1). Concomitant deletion of Rad18 and PARP1 synergistically increased CPT sensitivity, and additional inactivation of NHEJ normalized this hypersensitivity, indicating their parallel actions. In conclusion, higher-eukaryotic cells separately employ PARP1 and Rad18 to suppress the toxic effects of NHEJ during the HR reaction at stalled replication forks.     

Lethality in PARP-1/Ku80 double mutant mice reveals physiological synergy during early embryogenesis

Ku is an abundant heterodimeric nuclear protein, consisting of 70- and 86-kDa tightly associated subunits that comprise the DNA binding component of DNA-dependent protein kinase. Poly(ADP-ribose) polymerase-1 (PARP-1) is a 113-kDa protein that catalyzes the synthesis of poly(ADP-ribose) on target proteins. Both Ku and PARP-1 recognize and bind to DNA ends. Ku functions in the non-homologous end joining (NHEJ) repair pathway whereas PARP-1 functions in the single strand break repair and base excision repair (BER) pathways. Recent studies have revealed that PARP-1 and Ku80 interact in vitro. To determine whether the association of PARP-1 and Ku80 has any physiological significance or synergistic function in vivo, mice lacking both PARP-1 and Ku80 were generated. The resulting offspring died during embryonic development displaying abnormalities around the gastrulation stage. In addition, PARP-1-/-/Ku80-/- cultured blastocysts had an increased level of apoptosis. These data suggest that the functions of both Ku80 and PARP-1 are essential for normal embryogenesis and that a loss of genomic integrity leading to cell death through apoptosis is likely the cause of the embryonic lethality observed in these mice.     

Persistently bound Ku at DNA ends attenuates DNA end resection and homologous recombination

DNA double strand breaks (DSBs) are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). The DNA cell cycle stage and resection of the DSB ends are two key mechanisms which are believed to push DSB repair to the HR pathway. Here, we show that the NHEJ factor Ku80 associates with DSBs in S phase, when HR is thought to be the preferred repair pathway, and its dynamics/kinetics at DSBs is similar to those observed for Ku80 in non-S phase in mammalian cells. A Ku homolog from Mycobacterium tuberculosis binds to and is retained at DSBs in S phase and was used as a tool to determine if blocking DNA ends affects end resection and HR in mammalian cells. A decrease in DNA end resection, as marked by IR-induced RPA, BrdU, and Rad51 focus formation, and HR are observed when Ku deficient rodent cells are complemented with Mt-Ku. Together, this data suggests that Ku70/80 binds to DSBs in all cell cycle stages and is likely actively displaced from DSB ends to free the DNA ends for DNA end resection and thus HR to occur.