ATM limits incorrect end utilization during non-homologous end joining of multiple chromosome breaks

Chromosome rearrangements can form when incorrect ends are matched during end joining (EJ) repair of multiple chromosomal double-strand breaks (DSBs). We tested whether the ATM kinase limits chromosome rearrangements via suppressing incorrect end utilization during EJ repair of multiple DSBs. For this, we developed a system for monitoring EJ of two tandem DSBs that can be repaired using correct ends (Proximal-EJ) or incorrect ends (Distal-EJ, which causes loss of the DNA between the DSBs). In this system, two DSBs are induced in a chromosomal reporter by the meganuclease I-SceI. These DSBs are processed into non-cohesive ends by the exonuclease Trex2, which leads to the formation of I-SceI-resistant EJ products during both Proximal-EJ and Distal-EJ. Using this method, we find that genetic or chemical disruption of ATM causes a substantial increase in Distal-EJ, but not Proximal-EJ. We also find that the increase in Distal-EJ caused by ATM disruption is dependent on classical non-homologous end joining (c-NHEJ) factors, specifically DNA-PKcs, Xrcc4, and XLF. We present evidence that Nbs1-deficiency also causes elevated Distal-EJ, but not Proximal-EJ, to a similar degree as ATM-deficiency. In addition, to evaluate the roles of these factors on end processing, we examined Distal-EJ repair junctions. We found that ATM and Xrcc4 limit the length of deletions, whereas Nbs1 and DNA-PKcs promote short deletions. Thus, the regulation of end processing appears distinct from that of end utilization. In summary, we suggest that ATM is important to limit incorrect end utilization during c-NHEJ.     

DNA-PKcs and ATM co-regulate DNA double-strand break repair

DNA double-strand breaks (DSBs) are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR). The NHEJ/HR decision is under complex regulation and involves DNA-dependent protein kinase (DNA-PKcs). HR is elevated in DNA-PKcs null cells, but suppressed by DNA-PKcs kinase inhibitors, suggesting that kinase-inactive DNA-PKcs (DNA-PKcs-KR) would suppress HR. Here we use a direct repeat assay to monitor HR repair of DSBs induced by I-SceI nuclease. Surprisingly, DSB-induced HR in DNA-PKcs-KR cells was 2- to 3-fold above the elevated HR level of DNA-PKcs null cells, and ～4- to 7-fold above cells expressing wild-type DNA-PKcs. The hyperrecombination in DNA-PKcs-KR cells compared to DNA-PKcs null cells was also apparent as increased resistance to DNA crosslinks induced by mitomycin C. ATM phosphorylates many HR proteins, and ATM is expressed at a low level in cells lacking DNA-PKcs, but restored to wild-type level in cells expressing DNA-PKcs-KR. Several clusters of phosphorylation sites in DNA-PKcs, including the T2609 cluster, which is phosphorylated by DNA-PKcs and ATM, regulate access of repair factors to broken ends. Our results indicate that ATM-dependent phosphorylation of DNA-PKcs-KR contributes to the hyperrecombination phenotype. Interestingly, DNA-PKcs null cells showed more persistent ionizing radiation-induced RAD51 foci (but lower HR levels) compared to DNA-PKcs-KR cells, consistent with HR completion requiring RAD51 turnover. ATM may promote RAD51 turnover, suggesting a second (not mutually exclusive) mechanism by which restored ATM contributes to hyperrecombination in DNA-PKcs-KR cells. We propose a model in which DNA-PKcs and ATM coordinately regulate DSB repair by NHEJ and HR.     

Regulation of DNA double-strand break repair pathway choice

DNA double-strand breaks （DSBs） are critical lesions that can result in cell death or a wide variety of genetic alterations including largeor small-scale deletions, loss of heterozygosity, translocations, and chromosome loss. DSBs are repaired by non-homologous end-joining （NHEJ） and homologous recombination （HR）, and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous sources including reactive oxygen species generated during cellular metabolism, collapsed replication forks, and nucleases, and from exogenous sources including ionizing radiation and chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy. The DSB repair pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type. Here we review the regulatory factors that regulate DSB repair by NHEJ and HR in yeast and higher eukaryotes. These factors include regulated expression and phosphorylation of repair proteins, chromatin modulation of repair factor accessibility, and the availability of homologous repair templates. While most DSB repair proteins appear to function exclusively in NHEJ or HR, a number of proteins influence both pathways, including the MRE11/RAD50/NBS1（XRS2） complex, BRCA1, histone H2AX, PARP-1, RAD18, DNA-dependent protein kinase catalytic subunit （DNA-PKcs）, and ATM. DNA-PKcs plays a role in mammalian NHEJ, but it also influences HR through a complex regulatory network that may involve crosstalk with ATM, and the regulation of at least 12 proteins involved in HR that are phosphorylated by DNA-PKcs and/or ATM.     

Regulation of human polλ by ATM-mediated phosphorylation during non-homologous end joining

DNA double strand breaks (DSBs) trigger a variety of cellular signaling processes, collectively termed the DNA-damage response (DDR), that are primarily regulated by protein kinase ataxia-telangiectasia mutated (ATM). Among DDR activated processes, the repair of DSBs by non-homologous end joining (NHEJ) is essential. The proper coordination of NHEJ factors is mainly achieved through phosphorylation by an ATM-related kinase, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the molecular basis for this regulation has yet to be fully elucidated. In this study we identify the major NHEJ DNA polymerase, DNA polymerase lambda (Polλ), as a target for both ATM and DNA-PKcs in human cells. We show that Polλ is efficiently phosphorylated by DNA-PKcs in vitro and predominantly by ATM after DSB induction with ionizing radiation (IR) in vivo. We identify threonine 204 (T204) as a main target for ATM/DNA-PKcs phosphorylation on human Polλ, and establish that its phosphorylation may facilitate the repair of a subset of IR-induced DSBs and the efficient Polλ-mediated gap-filling during NHEJ. Molecular evidence suggests that Polλ phosphorylation might favor Polλ interaction with the DNA-PK complex at DSBs. Altogether, our work provides the first demonstration of how Polλ is regulated by phosphorylation to connect with the NHEJ core machinery during DSB repair in human cells.     

Telomere biology: integrating chromosomal end protection with DNA damage response

Telomeres play the key protective role at chromosomes. Many studies indicate that loss of telomere function causes activation of DNA damage response. Here, we review evidence supporting interdependence between telomere maintenance and DNA damage response and present a model in which these two pathways are combined into a single mechanism for protecting chromosomal integrity. Proteins directly involved in telomere maintenance and DNA damage response include Ku, DNA-PKcs, RAD51D, PARP-2, WRN and RAD50/MRE11/NBS1 complex. Since most of these proteins participate in the repair of DNA double-strand breaks (DSBs), this was perceived by many authors as a paradox, given that telomeres function to conceal natural DNA ends from mechanisms that detect and repair DSBs. However, we argue here that the key function of one particular DSB protein, Ku, is to prevent or control access of telomerase, the enzyme that synthesises telomeric sequences, to both internal DSBs and natural chromosomal ends. This view is supported by observations that Ku has a high affinity for DNA ends; it acts as a negative regulator of telomerase and that telomerase itself can target internal DSBs. Ku then directs other DSB repair/telomere maintenance proteins to either repair DSBs at internal chromosomal sites or prevent uncontrolled elongation of telomeres by telomerase. This model eliminates the above paradox and provides a testable scenario in which the role of DSB repair proteins is to protect chromosomal integrity by balancing repair activities and telomere maintenance. In our model, a close association between telomeres and different DNA damage response factors is not an unexpected event, but rather a logical result of chromosomal integrity maintenance activities. Review related to the 15th International Chromosome Conference (ICC XV), held in September 2004, Brunel University, London, UK     

Dynamics of the PI3K-like protein kinase members ATM and DNA-PKcs at DNA double strand breaks

The protein kinases ATM and DNA-PKcs play critical roles in the cellular response to DNA double strand breaks (DSBs). ATM and DNA-PKcs are activated in response to DSBs and play several important roles in propagation of the damage signal and for the repair of DNA damage. Recent work from several groups, including ours, has focused on studying the dynamics of each of these proteins at DSBs and the requirements and factors which play a role(s) in this process. The use of live cell imaging of fluorescently-tagged ATM and DNA-PKcs has allowed us to study the real-time response of these proteins to laser-generated DNA damage in vivo. Here, we will extensively discuss the behavior of the ATM and DNA-PKcs proteins at DSB sites.     

Repair of a minimal DNA double-strand break by NHEJ requires DNA-PKcs and is controlled by the ATM/ATR checkpoint

Mammalian cells primarily rejoin DNA double-strand breaks (DSBs) by the non-homologous end-joining (NHEJ) pathway. The joining of the broken DNA ends appears directly without template and accuracy is ensured by the NHEJ factors that are under ATM/ATR regulated checkpoint control. In the current study we report the engineering of a mono-specific DNA damaging agent. This was used to study the molecular requirements for the repair of the least complex DSB in vivo. Single-chain PvuII restriction enzymes fused to protein delivery sequences transduce cells efficiently and induce blunt end DSBs in vivo. We demonstrate that beside XRCC4/LigaseIV and KU, the DNA-PK catalytic subunit (DNA-PKcs) is also essential for the joining of this low complex DSB in vivo. The appearance of blunt end 3′-hydroxyl and 5′-phosphate DNA DSBs induces a significantly higher frequency of anaphase bridges in cells that do not contain functional DNA-PKcs, suggesting an absolute requirement for DNA-PKcs in the control of chromosomal stability during end joining. Moreover, these minimal blunt end DSBs are sufficient to induce a p53 and ATM/ATR checkpoint function.     

Analysis of DNA-dependent protein kinase-mediated DNA end joining by two-photon fluorescence cross-correlation spectroscopy

Nonhomologous end joining (NHEJ) is the primary mechanism by which mammalian cells repair DNA double-strand breaks (DSBs). Proteins known to play a role in NHEJ include the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), the Ku 70/Ku 80 heterodimer (Ku), XRCC4, and DNA ligase IV. One of the main roles of the DNA-PKcs-Ku complex is to bring the ends of the DSB together in a process termed synapsis, prior to end joining. Synapsis results in the autophosphorylation of DNA-PKcs, which is required to make the DNA ends available for ligation. Here, we describe a novel assay using two-photon fluorescence cross-correlation spectroscopy that allows for the analysis of DNA synapsis and end joining in solution using purified proteins. We demonstrate that although autophosphorylation-defective DNA-PKcs does not support DNA ligase-mediated DNA end joining, like wild-type (WT) DNA-PKcs, it is capable of Ku-dependent DNA synapsis in solution. Moreover, we show that, in the presence of Ku, both WT DNA-PKcs and autophosphorylation-defective DNA-PKcs promote the formation of multiple, large multi-DNA complexes in solution, suggesting that, rather than align two opposing DNA ends, multiple DNA-PK molecules may serve to bring multiple DNA ends into the NHEJ complex.     

Mechanisms of DNA double strand break repair and chromosome aberration formation

It is widely accepted that unrepaired or misrepaired DNA double strand breaks (DSBs) lead to the formation of chromosome aberrations. DSBs induced in the DNA of higher eukaryotes by endogenous processes or exogenous agents can in principle be repaired either by non-homologous endjoining (NHEJ), or homology directed repair (HDR). The basis on which the selection of the DSB repair pathway is made remains unknown but may depend on the inducing agent, or process. Evaluation of the relative contribution of NHEJ and HDR specifically to the repair of ionizing radiation (IR) induced DSBs is important for our understanding of the mechanisms leading to chromosome aberration formation. Here, we review recent work from our laboratories contributing to this line of inquiry. Analysis of DSB rejoining in irradiated cells using pulsed-field gel electrophoresis reveals a fast component operating with half times of 10-30 min. This component of DSB rejoining is severely compromised in cells with mutations in DNA-PKcs, Ku, DNA ligase IV, or XRCC4, as well as after chemical inhibition of DNA-PK, indicating that it reflects classical NHEJ; we termed this form of DSB rejoining D-NHEJ to signify its dependence on DNA-PK. Although chemical inhibition, or mutation, in any of these factors delays processing, cells ultimately remove the majority of DSBs using an alternative pathway operating with slower kinetics (half time 2-10 h). This alternative, slow pathway of DSB rejoining remains unaffected in mutants deficient in several genes of the RAD52 epistasis group, suggesting that it may not reflect HDR. We proposed that it reflects an alternative form of NHEJ that operates as a backup (B-NHEJ) to the DNA-PK-dependent (D-NHEJ) pathway. Biochemical studies confirm the presence in cell extracts of DNA end joining activities operating in the absence of DNA-PK and indicate the dominant role for D-NHEJ, when active. These observations in aggregate suggest that NHEJ, operating via two complementary pathways, B-NHEJ and D-NHEJ, is the main mechanism through which IR-induced DSBs are removed from the DNA of higher eukaryotes. HDR is considered to either act on a small fraction of IR induced DSBs, or to engage in the repair process at a step after the initial end joining. We propose that high speed D-NHEJ is an evolutionary development in higher eukaryotes orchestrated around the newly evolved DNA-PKcs and pre-existing factors. It achieves within a few minutes restoration of chromosome integrity through an optimized synapsis mechanism operating by a sequence of protein-protein interactions in the context of chromatin and the nuclear matrix. As a consequence D-NHEJ mostly joins the correct DNA ends and suppresses the formation of chromosome aberrations, albeit, without ensuring restoration of DNA sequence around the break. B-NHEJ is likely to be an evolutionarily older pathway with less optimized synapsis mechanisms that rejoins DNA ends with kinetics of several hours. The slow kinetics and suboptimal synapsis mechanisms of B-NHEJ allow more time for exchanges through the joining of incorrect ends and cause the formation of chromosome aberrations in wild type and D-NHEJ mutant cells.     

Dynamics of the PI3K-like protein kinase members ATM and DNA-PKcs at DNA double strand breaks

The protein kinases ATM and DNA-PKcs play critical roles in the cellular response to DNA double strand breaks (DSBs). ATM and DNA-PKcs are activated in response to DSBs and play several important roles in propagation of the damage signal and for the repair of DNA damage. Recent work from several groups, including ours, has focused on studying the dynamics of each of these proteins at DSBs and the requirements and factors which play a role(s) in this process. The use of live cell imaging of fluorescently-tagged ATM and DNA-PKcs has allowed us to study the real-time response of these proteins to laser-generated DNA damage in vivo. Here, we will extensively discuss the behavior of the ATM and DNA-PKcs proteins at DSB sites.Key words: ATM, DNA-PKcs, DNA double strand breaks, autophosphorylation, live cell imaging     

Limiting the Persistence of a Chromosome Break Diminishes Its Mutagenic Potential

To characterize the repair pathways of chromosome double-strand breaks (DSBs), one approach involves monitoring the repair of site-specific DSBs generated by rare-cutting endonucleases, such as I-SceI. Using this method, we first describe the roles of Ercc1, Msh2, Nbs1, Xrcc4, and Brca1 in a set of distinct repair events. Subsequently, we considered that the outcome of such assays could be influenced by the persistent nature of I-SceI-induced DSBs, in that end-joining (EJ) products that restore the I-SceI site are prone to repeated cutting. To address this aspect of repair, we modified I-SceI-induced DSBs by co-expressing I-SceI with a non-processive 3′ exonuclease, Trex2, which we predicted would cause partial degradation of I-SceI 3′ overhangs. We find that Trex2 expression facilitates the formation of I-SceI-resistant EJ products, which reduces the potential for repeated cutting by I-SceI and, hence, limits the persistence of I-SceI-induced DSBs. Using this approach, we find that Trex2 expression causes a significant reduction in the frequency of repair pathways that result in substantial deletion mutations: EJ between distal ends of two tandem DSBs, single-strand annealing, and alternative-NHEJ. In contrast, Trex2 expression does not inhibit homology-directed repair. These results indicate that limiting the persistence of a DSB causes a reduction in the frequency of repair pathways that lead to significant genetic loss. Furthermore, we find that individual genetic factors play distinct roles during repair of non-cohesive DSB ends that are generated via co-expression of I-SceI with Trex2.     

Factors determining DNA double-strand break repair pathway choice in G2 phase

DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.     

Biochemical evidence for Ku-independent backup pathways of NHEJ

Cells of higher eukaryotes process within minutes double strand breaks (DSBs) in their genome using a non-homologous end joining (NHEJ) apparatus that engages DNA-PKcs, Ku, DNA ligase IV, XRCC4 and other as of yet unidentified factors. Although chemical inhibition, or mutation, in any of these factors delays processing, cells ultimately remove the majority of DNA DSBs using an alternative pathway operating with an order of magnitude slower kinetics. This alternative pathway is active in mutants deficient in genes of the RAD52 epistasis group and frequently joins incorrect ends. We proposed, therefore, that it reflects an alternative form of NHEJ that operates as a backup (B-NHEJ) to the DNA-PK-dependent (D-NHEJ) pathway, rather than homology directed repair of DSBs. The present study investigates the role of Ku in the coordination of these pathways using as a model end joining of restriction endonuclease linearized plasmid DNA in whole cell extracts. Efficient, error-free, end joining observed in such in vitro reactions is strongly inhibited by anti-Ku antibodies. The inhibition requires DNA-PKcs, despite the fact that Ku efficiently binds DNA ends in the presence of antibodies, or in the absence of DNA-PKcs. Strong inhibition of DNA end joining is also mediated by wortmannin, an inhibitor of DNA-PKcs, in the presence but not in the absence of Ku, and this inhibition can be rescued by pre-incubating the reaction with double stranded oligonucleotides. The results are compatible with a role of Ku in directing end joining to a DNA-PK dependent pathway, mediated by efficient end binding and productive interactions with DNA-PKcs. On the other hand, efficient end joining is observed in extracts of cells lacking DNA-PKcs, as well as in Ku-depleted extracts in line with the operation of alternative pathways. Extracts depleted of Ku and DNA-PKcs rejoin blunt ends, as well as homologous ends with 3′ or 5′ protruding single strands with similar efficiency, but addition of Ku suppresses joining of blunt ends and homologous ends with 3′ overhangs. We propose that the affinity of Ku for DNA ends, particularly when cooperating with DNA-PKcs, suppresses B-NHEJ by quickly and efficiently binding DNA ends and directing them to D-NHEJ for rapid joining. A chromatin-based model of DNA DSB rejoining accommodating biochemical and genetic results is presented and deviations between in vitro and in vivo results discussed.     

Non-homologous DNA end joining

DNA double strand breaks (DSB) are the most serious form of DNA damage. Repair of DSBs is important to prevent chromosomal fragmentation, translocations and deletions. Non-homologous end joining (NHEJ) is one of three major pathways for the repair of DSBs in human cells. In this process two DNA ends are joined directly, usually with no sequence homology, although in the case of same polarity of the single stranded overhangs in DSBs, regions of microhomology are utilized. NHEJ is typically imprecise, a characteristic that is useful for immune diversification in lymphocytes in V(D)J recombination. The main components of the NHEJ system in eukaryotes are the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku proteins, XRCC4, DNA ligase IV, and Artemis. This review focuses on the mechanisms an dregulation of DSB repair by NHEJ in mammalian cells.     

Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends

Non-homologous end joining (NHEJ) is one of the primary pathways for the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) in mammalian cells. Proteins required for NHEJ include the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Ku, XRCC4 and DNA ligase IV. Current models predict that DNA-PKcs, Ku, XRCC4 and DNA ligase IV assemble at DSBs and that the protein kinase activity of DNA-PKcs is essential for NHEJ-mediated repair of DSBs in vivo. We previously identified a cluster of autophosphorylation sites between amino acids 2609 and 2647 of DNA-PKcs. Cells expressing DNA-PKcs in which these autophosphorylation sites have been mutated to alanine are highly radiosensitive and defective in their ability to repair DSBs in the context of extrachromosomal assays. Here, we show that cells expressing DNA-PKcs with mutated autophosphorylation sites are also defective in the repair of IR-induced DSBs in the context of chromatin. Purified DNA-PKcs proteins containing serine/threonine to alanine or aspartate mutations at this cluster of autophosphorylation sites were indistinguishable from wild-type (wt) protein with respect to protein kinase activity. However, mutant DNA-PKcs proteins were defective relative to wt DNA-PKcs with respect to their ability to support T4 DNA ligase-mediated intermolecular ligation of DNA ends. We propose that autophosphorylation of DNA-PKcs at this cluster of sites is important for remodeling of DNA-PK complexes at DNA ends prior to DNA end joining.     

DNA-PK autophosphorylation facilitates Artemis endonuclease activity

The Artemis nuclease is defective in radiosensitive severe combined immunodeficiency patients and is required for the repair of a subset of ionising radiation induced DNA double-strand breaks (DSBs) in an ATM and DNA-PK dependent process. Here, we show that Artemis phosphorylation by ATM and DNA-PK in vitro is primarily attributable to S503, S516 and S645 and demonstrate ATM dependent phosphorylation at serine 645 in vivo. However, analysis of multisite phosphorylation mutants of Artemis demonstrates that Artemis phosphorylation is dispensable for endonuclease activity in vitro and for DSB repair and V(D)J recombination in vivo. Importantly, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) autophosphorylation at the T2609-T2647 cluster, in the presence of Ku and target DNA, is required for Artemis-mediated endonuclease activity. Moreover, autophosphorylated DNA-PKcs stably associates with Ku-bound DNA with large single-stranded overhangs until overhang cleavage by Artemis. We propose that autophosphorylation triggers conformational changes in DNA-PK that enhance Artemis cleavage at single-strand to double-strand DNA junctions. These findings demonstrate that DNA-PK autophosphorylation regulates Artemis access to DNA ends, providing insight into the mechanism of Artemis mediated DNA end processing.     

Involvement of DNA-dependent protein kinase in normal cell cycle progression through mitosis

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) plays an important role in DNA double-strand break (DSB) repair as the underlying mechanism of the non-homologous end joining pathway. When DSBs occur, DNA-PKcs is rapidly phosphorylated at both the Thr-2609 and Ser-2056 residues, and such phosphorylations are critical for DSB repair. In this study we report that, in addition to responding to DSBs, DNA-PKcs is activated and phosphorylated in normal cell cycle progression through mitosis. Mitotic induction of DNA-PKcs phosphorylation is closely associated with the spindle apparatus at centrosomes and kinetochores. Furthermore, depletion of DNA-PKcs protein levels or inhibition of DNA-PKcs kinase activity results in the delay of mitotic transition because of chromosome misalignment. These results demonstrate for the first time that DNA-PKcs, in addition to its role in DSB repair, is a critical regulator of mitosis and could modulate microtubule dynamics in chromosome segregation.     

Mre11 dimers coordinate DNA end bridging and nuclease processing in double-strand-break repair

Mre11 forms the core of the multifunctional Mre11-Rad50-Nbs1 (MRN) complex that detects DNA double-strand breaks (DSBs), activates the ATM checkpoint kinase, and initiates homologous recombination (HR) repair of DSBs. To define the roles of Mre11 in both DNA bridging and nucleolytic processing during initiation of DSB repair, we combined small-angle X-ray scattering (SAXS) and crystal structures of Pyrococcus furiosus Mre11 dimers bound to DNA with mutational analyses of fission yeast Mre11. The Mre11 dimer adopts a four-lobed U-shaped structure that is critical for proper MRN complex assembly and for binding and aligning DNA ends. Further, mutations blocking Mre11 endonuclease activity impair cell survival after DSB induction without compromising MRN complex assembly or Mre11-dependant recruitment of Ctp1, an HR factor, to DSBs. These results show how Mre11 dimerization and nuclease activities initiate repair of DSBs and collapsed replication forks, as well as provide a molecular foundation for understanding cancer-causing Mre11 mutations in ataxia telangiectasia-like disorder (ATLD).     

ATM Release at Resected Double-Strand Breaks Provides Heterochromatin Reconstitution to Facilitate Homologous Recombination

Non-homologous end-joining (NHEJ) and homologous recombination (HR) represent the two main pathways for repairing DNA double-strand breaks (DSBs). During the G2 phase of the mammalian cell cycle, both processes can operate and chromatin structure is one important factor which determines DSB repair pathway choice. ATM facilitates the repair of heterochromatic DSBs by phosphorylating and inactivating the heterochromatin building factor KAP-1, leading to local chromatin relaxation. Here, we show that ATM accumulation and activity is strongly diminished at DSBs undergoing end-resection during HR. Such DSBs remain unrepaired in cells devoid of the HR factors BRCA2, XRCC3 or RAD51. Strikingly, depletion of KAP-1 or expression of phospho-mimic KAP-1 allows repair of resected DSBs in the absence of BRCA2, XRCC3 or RAD51 by an erroneous PARP-dependent alt-NHEJ process. We suggest that DSBs in heterochromatin elicit initial local heterochromatin relaxation which is reversed during HR due to the release of ATM from resection break ends. The restored heterochromatic structure facilitates HR and prevents usage of error-prone alternative processes.     

RAD50 Is Required for Efficient Initiation of Resection and Recombinational Repair at Random, γ-Induced Double-Strand Break Ends

Resection of DNA double-strand break (DSB) ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random “dirty-ended” DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE). We utilized this “PFGE-shift” to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after γ-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1–2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 null mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent.