Collaboration and competition between DNA double-strand break repair pathways

DNA double-strand breaks resulting from normal cellular processes including replication and exogenous sources such as ionizing radiation pose a serious risk to genome stability, and cells have evolved different mechanisms for their efficient repair. The two major pathways involved in the repair of double-strand breaks in eukaryotic cells are non-homologous end joining and homologous recombination. Numerous factors affect the decision to repair a double-strand break via these pathways, and accumulating evidence suggests these major repair pathways both cooperate and compete with each other at double-strand break sites to facilitate efficient repair and promote genomic integrity.     

DNA double-strand break repair signalling: the case of RAD51 post-translational regulation

DNA double-strand breaks (DSBs) are the major lethal lesion induced by ionizing radiation or by replication block. However, cells can take advantage of DSB-induced recombination in order to generate genetic diversity in physiological processes such as meiosis and V(D)J recombination. Two main alternative pathways compete for DSB repair: homologous recombination (HR) and non-homologous end-joining (NHEJ). This review will briefly present the mechanisms and the enzymatic complex for HR and NHEJ. The signalling of the DSB through the ATM pathway will be presented. Then, we will focus on the case of the RAD51 protein, which plays a pivotal role in HR and is conserved from bacteria to humans. Post-translational regulation of RAD51 is presented. Two contrasting situations are discussed: one with up-regulation (expression of the oncogene BCR/ABL) and one with a down-regulation (expression of the oncogene BCL-2) of RAD51, associated with apoptosis inhibition and tumour predisposition.     

Single-strand annealing mediates the conservative repair of double-strand DNA breaks in homologous recombination-defective germ cells of Caenorhabditis elegans

A missense mutation in C. elegans RAD-54, a homolog of RAD54 that operates in the homologous recombination (HR) pathway, was found to decrease ATPase activity in vitro. The hypomorphic mutation caused hypersensitivity of C. elegans germ cells to double-strand DNA breaks (DSBs). Although the formation of RAD-51 foci at DSBs was normal in both the mutant and knockdown worms, their subsequent dissipation was slow. The rad-54-deficient phenotypes were greatly aggravated when combined with an xpf-1 mutation, suggesting a conservative role of single-strand annealing (SSA) for DSB repair in HR-defective worms. The phenotypes of doubly-deficient rad-54;xpf-1 worms were partially suppressed by a mutation of lig-4, a nonhomologous end-joining (NHEJ) factor. In summary, RAD-54 is required for the dissociation of RAD-51 from DSB sites in C. elegans germ cells. Also, NHEJ and SSA exert negative and positive effects, respectively, on genome stability when HR is defective.     

Pol32 is required for Pol zeta-dependent translesion synthesis and prevents double-strand breaks at the replication fork

POL32 encodes a non-essential subunit of Polδ and plays a role in Polδ processivity and DNA repair. In order to understand how Pol32 is involved in these processes, we performed extensive genetic analysis and demonstrated that POL32 is required for Polζ-mediated translesion synthesis, but not for Polη-mediated activity. Unlike Polζ, inactivation of Pol32 does not result in decreased spontaneous mutagenesis, nor does it limit genome instability in the absence of the error-free postreplication repair pathway. In contrast, inactivation of Pol32 results in an increased rate of replication slippage and recombination. A genome-wide synthetic lethal screen revealed that in the absence of Pol32, homologous recombination repair and cell cycle checkpoints play crucial roles in maintaining cell survival and growth. These results are consistent with a model in which Pol32 functions as a coupling factor to facilitate a switch from replication to translesion synthesis when Polδ encounters replication-blocking lesions. When Pol32 is absent, the S-phase checkpoint complex Mrc1–Tof1 becomes crucial to stabilize the stalled replication fork and recruit Top3 and Sgs1. Lack of any of the above activities will cause double strand breaks at or near the replication fork that require recombination as well as Rad9 for cell survival.     

Double-strand break repair can lead to high frequencies of deletions within short CAG/CTG trinucleotide repeats

Trinucleotide repeats undergo contractions and expansions in humans, leading in some cases to fatal neurological disorders. The mechanism responsible for these large size variations is unknown, but replication-slippage events are often suggested as a possible source of instability. We constructed a genetic screen that allowed us to detect spontaneous expansions/contractions of a short trinucleotide repeat in yeast. We show that deletion of RAD27, a gene involved in the processing of Okazaki fragments, increases the frequency of contractions tenfold. Repair of a chromosomal double-strand break (DSB) using a trinucleotide repeat-containing template induces rearrangements of the repeat with a frequency 60 times higher than the natural rate of instability of the same repeat. Our data suggest that both gene conversion and single-strand annealing are major sources of trinucleotide repeat rearrangements. Received: 8 January 1999 / Accepted: 17 March 1999     

Replication protein A and gamma-H2AX foci assembly is triggered by cellular response to DNA double-strand breaks

Human replication protein A (RPA p34), a crucial component of diverse DNA excision repair pathways, is implicated in DNA double-strand break (DSB) repair. To evaluate its role in DSB repair, the intranuclear dynamics of RPA was investigated after DNA damage and replication blockage in human cells. Using two different agents [ionizing radiation (IR) and hydroxyurea (HU)] to generate DSBs, we found that RPA relocated into distinct nuclear foci and colocalized with a well-known DSB binding factor, gamma-H2AX, at the sites of DNA damage in a time-dependent manner. Colocalization of RPA and gamma-H2AX foci peaked at 2 h after IR treatment and subsequently declined with increasing postrecovery times. The time course of RPA and gamma-H2AX foci association correlated well with the DSB repair activity detected by a neutral comet assay. A phosphatidylinositol-3 (PI-3) kinase inhibitor, wortmannin, completely abolished both RPA and gamma-H2AX foci formation triggered by IR. Additionally, radiosensitive ataxia telangiectasia (AT) cells harboring mutations in ATM gene product were found to be deficient in RPA and gamma-H2AX colocalization after IR. Transfection of AT cells with ATM cDNA fully restored the association of RPA foci with gamma-H2AX illustrating the requirement of ATM gene product for this process. The exact coincidence of RPA and gamma-H2AX in response to HU specifically in S-phase cells supports their role in DNA replication checkpoint control. Depletion of RPA by small interfering RNA (SiRNA) substantially elevated the frequencies of IR-induced micronuclei (MN) and apoptosis in human cells suggestive of a role for RPA in DSB repair. We propose that RPA in association with gamma-H2AX contributes to both DNA damage checkpoint control and repair in response to strand breaks and stalled replication forks in human cells.     

RecJ nuclease is required for SOS induction after introduction of a double-strand break in a RecA loading deficient recB mutant of Escherichia coli

The SOS response is an important mechanism which allows Escherichia coli cells to maintain genome integrity. Two key proteins in SOS regulation are LexA (repressor) and RecA (coprotease). The signal for SOS induction is generated at the level of a RecA filament. Depending on the type of DNA damage, a RecA filament is produced by specific activities (helicase, nuclease and RecA loading) of either RecBCD, RecF or a hybrid recombination pathway. It was recently demonstrated that RecA loading activity is essential for the induction of the SOS response after UV-irradiation. In this paper we studied the genetic requirements for SOS induction after introduction of a double-strand break (DSB) by the I-SceI endonuclease in a RecA loading deficient recB mutant (recB1080). We monitored SOS induction by assaying beta-galactosidase activity and compared induction of the response between strains having one or more inactivated mechanisms of RecA loading and their derivatives. We found that simultaneous inactivation of both RecA loading functions (in recB1080 recO double mutant) partially impairs SOS induction after introduction of a DSB. However, we found that the RecJ nuclease is essential for SOS induction after the introduction of a DSB in the recB1080 mutant. This result indicates that RecJ is needed to prepare ssDNA for subsequent loading of RecA protein. It implies that an additional type of RecA loading could exist in the cell.     

Influence of non-homology between recombining DNA sequences on double-strand break repair in Saccharomyces cerevisiae

In this paper we study the influence of non-homology between plasmid and chromosomal DNA on the efficiency of recombinational repair of plasmid double-strand breaks and gaps in yeast. For this purpose we used different combinations of plasmids and yeast strains carrying various deletions within the yeast LYS2 gene. A 400 by deletion in plasmid DNA had no effect on recombinational plasmid repair. However, a 400 by deletion in chromosomal DNA dramatically reduced the efficiency of this repair mechanism, but recombinational repair of plasmids linearized by a double-strand break with cohesive ends still remained the dominant repair process. We have also studied the competition between recombination and ligation in the repair of linearized plasmids. Our experimental evidence suggests that recombinational repair is attempted but aborted if only one recombinogenic end with homology to chromosomal DNA is present in plasmid DNA. This situation results in a decreased probability of non-recombinational (i.e. ligation) repair of linearized plasmid DNA.     

ATM-mediated phosphorylation of polynucleotide kinase/phosphatase is required for effective DNA double-strand break repair

The cellular response to double-strand breaks (DSBs) in DNA is a complex signalling network, mobilized by the nuclear protein kinase ataxia-telangiectasia mutated (ATM), which phosphorylates many factors in the various branches of this network. A main question is how ATM regulates DSB repair. Here, we identify the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) as an ATM target. PNKP phosphorylates 5'-OH and dephosphorylates 3'-phosphate DNA ends that are formed at DSB termini caused by DNA-damaging agents, thereby regenerating legitimate ends for further processing. We establish that the ATM phosphorylation targets on human PNKP-Ser 114 and Ser 126-are crucial for cellular survival following DSB induction and for effective DSB repair, being essential for damage-induced enhancement of the activity of PNKP and its proper accumulation at the sites of DNA damage. These findings show a direct functional link between ATM and the DSB-repair machinery.     

DNA双链断裂修复的选择性调控机制

在各种DNA损伤中,DNA双链断裂(double-strand break,DSB)是最为严重的一种,快速准确地修复DSB对维持基因组稳定性起着至关重要的作用。真核生物细胞通过一系列复杂的信号转导途径激活对DSB的修复,其中最为重要的是同源重组和非同源末端连接机制。最近的研究表明,这两种方式在DSB修复的早期是相互竞争的关系,其选择在很大程度上受到53BP1及同源蛋白质的调控。将讨论53BP1作为DSB修复途径的核心因子,在染色质水平整合BRCA1、Ct IP等修复因子和多种组蛋白修饰构成的信号途径,介导同源重组和非同源末端连接通路选择的分子机制。     

Meiotic versus mitotic recombination: two different routes for double-strand break repair: the different functions of meiotic versus mitotic DSB repair are reflected in different pathway usage and different outcomes

Studies in the yeast Saccharomyces cerevisiae have validated the major features of the double-strand break repair (DSBR) model as an accurate representation of the pathway through which meiotic crossovers (COs) are produced. This success has led to this model being invoked to explain double-strand break (DSB) repair in other contexts. However, most non-crossover (NCO) recombinants generated during S. cerevisiae meiosis do not arise via a DSBR pathway. Furthermore, it is becoming increasingly clear that DSBR is a minor pathway for recombinational repair of DSBs that occur in mitotically-proliferating cells and that the synthesis-dependent strand annealing (SDSA) model appears to describe mitotic DSB repair more accurately. Fundamental dissimilarities between meiotic and mitotic recombination are not unexpected, since meiotic recombination serves a very different purpose (accurate chromosome segregation, which requires COs) than mitotic recombination (repair of DNA damage, which typically generates NCOs).     

耐辐射球菌DNA双链断裂修复机制研究新进展

耐辐射球菌对于电离辐射等DNA损伤剂具有极强的抗性,能够将同一个基因组中同时产生的高达100个以上的DNA双链断裂在数十小时内高效而精准地进行修复,是研究DNA双链断裂修复机制的重要模式生物。同源重组、非同源末端连接和单链退火途径作为3个主要的修复途径参与了耐辐射球菌基因组DNA双链断裂的修复过程。此外,一系列新发现的重要蛋白质,如Ppr I、Ddr B等对于耐辐射球菌基因组的修复过程同样至关重要。根据本实验室和国内外在这一研究领域近年来的报道,以不同的修复途径为线索,综述该菌DNA双链断裂修复机制的最新研究成果。     

SAW1 is required for SDSA double-strand break repair in S. cerevisiae

SAW1, coding for Saw1, is required for single-strand annealing (SSA) DNA double-strand break (DSB) repair in Saccharomycescerevisiae. Saw1 physically associates with Rad1 and Rad52 and recruits the Rad1–Rad10 endonuclease. Herein we show by fluorescence microscopy that SAW1 is similarly required for recruitment of Rad10 to sites of Synthesis-Dependent Strand Annealing (SDSA) and associates with sites of SDSA repair in a manner temporally overlapped with Rad10. The magnitude of induction of colocalized Saw1-CFP/Rad10-YFP/DSB-RFP foci in SDSA is more dramatic in S and G2 phase cells than in M phase, consistent with the known mechanism of SDSA. We observed a substantial fraction of foci in which Rad10 was localized to the repair site without Saw1, but few DSB sites that contained Saw1 without Rad10. Together these data are consistent with a model in which Saw1 recruits Rad1–Rad10 to SDSA sites, possibly even binding as a protein–protein complex, but departs the repair site in advance of Rad1–Rad10.     

RNA polymerase III is required for the repair of DNA double-strand breaks by homologous recombination

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Telomere-related functions of yeast KU in the repair of bleomycin-induced DNA damage

Bleomycins are small glycopeptide cancer chemotherapeutics that give rise to 3'-modified DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae, DSBs are predominantly repaired by RAD52-dependent homologous recombination (HR) with some support by Yku70/Yku80 (KU)-dependent pathways. The main DSB repair function of KU is believed to be as part of the non-homologous end-joining (NHEJ) pathway, but KU also functions in a "chromosome healing" pathway that seals DSBs by de novo telomere addition. We report here that rad52Deltayku70Delta double mutants are considerably more bleomycin hypersensitive than rad52Deltalig4Delta cells that lack the NHEJ-specific DNA ligase 4. Moreover, the telomere-specific KU mutation yku80-135i also dramatically increases rad52Delta bleomycin hypersensitivity, almost to the level of rad52Deltayku80Delta. The results indicate that telomere-specific functions of KU play a more prominent role in the repair of bleomycin-induced damage than its NHEJ functions, which could have important clinical implications for bleomycin-based combination chemotherapies.     

RNF4 regulates DNA double-strand break repair in a cell cycle-dependent manner

Both RNF4 and KAP1 play critical roles in the response to DNA double-strand breaks (DSBs), but the functional interplay of RNF4 and KAP1 in regulating DNA damage response remains unclear. We have previously demonstrated the recruitment and degradation of KAP1 by RNF4 require the phosphorylation of Ser824 (pS824) and SUMOylation of KAP1. In this report, we show the retention of DSB-induced pS824-KAP1 foci and RNF4 abundance are inversely correlated as cell cycle progresses. Following irradiation, pS824-KAP1 foci predominantly appear in the cyclin A (-) cells, whereas RNF4 level is suppressed in the G0-/G1-phases and then accumulates during S-/G2-phases. Notably, 53BP1 foci, but not BRCA1 foci, co-exist with pS824-KAP1 foci. Depletion of KAP1 yields opposite effect on the dynamics of 53BP1 and BRCA1 loading, favoring homologous recombination repair. In addition, we identify p97 is present in the RNF4-KAP1 interacting complex and the inhibition of p97 renders MCF7 breast cancer cells relatively more sensitive to DNA damage. Collectively, these findings suggest that combined effect of dynamic recruitment of RNF4 to KAP1 regulates the relative occupancy of 53BP1 and BRCA1 at DSB sites to direct DSB repair in a cell cycle-dependent manner.     

The role of RNA and RNA-related proteins in the regulation of DNA double strand break repair pathway choice

DNA end resection is a critical step in the repair of DNA double strand breaks. It controls the way the lesion is going to be repaired, thus its regulation has a great importance in maintaining genomic stability. In this review, we focus in recent discoveries in the field that point to a modulation of resection by RNA molecules and RNA-related proteins. Moreover, we aim to reconcile contradictory reports on the positive or negative effect of DNA:RNA hybrids in the resection process.     

Poly(ADP-ribose) polymerases in double-strand break repair: Focus on PARP1, PARP2 and PARP3

Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification of proteins catalysed by Poly(ADP-ribose) polymerases (PARP). A wealth of recent advances in the biochemical and functional characterization of the DNA-dependent PARP family members have highlighted their key contribution in the DNA damage response network, the best characterized being the role of PARP1 and PARP2 in the resolution of single-strand breaks as part of the BER/SSBR process. How PARylation contributes to the repair of double-strand breaks is less well defined but has become recently the subject of significant research in the field. The aim of this review is to provide an overview of the current knowledge concerning the role of the DNA-activated PARP1, PARP2 and PARP3 in cellular response to double-strand breaks (DSB). In addition, we outline the biological significance of these properties in response to programmed DNA lesions formed during physiological processes such as antibody repertoire assembly and diversification.     

Arg tyrosine kinase is involved in homologous recombinational DNA repair

c-Abl plays important roles in cellular response to DNA damage. However, possible roles for Arg (Abl-related gene) in DNA damage response are unknown. Here, we show that ionizing radiation (IR)-induced Rad51 focus formation is reduced in Arg-deficient cells generated from a chicken B cell line by targeted disruption. This is consistent with the findings that Arg-deficient cells display hypersensitivity to IR, elevated frequencies of IR-induced chromosomal aberrations, and reduced targeted integration frequencies. All of these abnormalities in DNA damage repair are also observed in ATM-deficient cells but not in c-Abl-deficient cells. Finally, we show that Arg interacts with and phosphorylates Rad51 in 293T cells. These results suggest that Arg plays a role in homologous recombinational (HR) DNA repair by phosphorylating Rad51.