DNA双链断裂损伤修复系统研究进展

多种内源或外源因素都能造成细胞基因组DNA损伤,细胞内建立了复杂的修复系统来应对不同形式的损伤。其中DNA双链断裂(DNA double-strand breaks,DSBs)作为最严重的损伤形式,主要激活同源重组修复(Homologous recombination repair)和非同源末端连接(Non-homologous end joining)通路。这两条通路都是由多个修复元件参与、经过多步反应的复杂过程。两者各具特点、协同作用,共同维护细胞基因组的稳定性。对其分子机制的阐明为肿瘤放化疗的辅助治疗提供了潜在的作用靶点。     

The CDK regulates repair of double-strand breaks by homologous recombination during the cell cycle

DNA double-strand breaks (DSBs) are dangerous lesions that can lead to genomic instability and cell death. Eukaryotic cells repair DSBs either by nonhomologous end-joining (NHEJ) or by homologous recombination. We investigated the ability of yeast cells (Saccharomyces cerevisiae) to repair a single, chromosomal DSB by recombination at different stages of the cell cycle. We show that cells arrested at the G1 phase of the cell cycle restrict homologous recombination, but are able to repair the DSB by NHEJ. Furthermore, we demonstrate that recombination ability does not require duplicated chromatids or passage through S phase, and is controlled at the resection step by Clb-CDK activity.     

Quality control of DNA break metabolism: in the 'end', it's a good thing

DNA ends pose specific problems in the control of genetic information quality. Ends of broken DNA need to be rejoined to avoid genome rearrangements, whereas natural DNA ends of linear chromosomes, telomeres, need to be stable and hidden from the DNA damage response. Efficient DNA end metabolism, either at induced DNA breaks or telomeres, does not result from the machine-like precision of molecular reactions, but rather from messier, more stochastic processes. The necessary molecular interactions are dynamically unstable, with constructive and destructive processes occurring in competition. In the end, quality control comes from the constant building up and tearing down of inappropriate, but also appropriate reaction steps in combination with factors that only slightly shift the equilibrium to eventually favour appropriate events. Thus, paradoxically, enzymes antagonizing DNA end metabolism help to ensure that genome maintenance becomes a robust process.     

Cytolethal distending toxin (CDT) is a radiomimetic agent and induces persistent levels of DNA double-strand breaks in human fibroblasts

Cytolethal distending toxin (CDT) is a unique genotoxin produced by several pathogenic bacteria. The tripartite protein toxin is internalized into mammalian cells via endocytosis followed by retrograde transport to the ER. Upon translocation into the nucleus, CDT catalyzes the formation of DNA double-strand breaks (DSBs) due to its intrinsic endonuclease activity. In the present study, we compared the DNA damage response (DDR) in human fibroblasts triggered by recombinant CDT to that of ionizing radiation (IR), a well-known DSB inducer. Furthermore, we dissected the pathways involved in the detection and repair of CDT-induced DNA lesions. qRT-PCR array-based mRNA and western blot analyses showed a partial overlap in the DDR pattern elicited by CDT and IR, with strong activation of both the ATM-Chk2 and the ATR-Chk1 axis. In line with its in vitro DNase I-like activity on plasmid DNA, neutral and alkaline Comet assay revealed predominant induction of DSBs in CDT-treated fibroblasts, whereas irradiation of cells generated higher amounts of SSBs and alkali-labile sites. Using confocal microscopy, the dynamics of the DSB surrogate marker γ-H2AX was monitored after pulse treatment with CDT or IR. In contrast to the fast induction and disappearance of γ-H2AX-foci observed in irradiated cells, the number of γ-H2AX-foci induced by CDT were formed with a delay and persisted. 53BP1 foci were also generated following CDT treatment and co-localized with γ-H2AX foci. We further demonstrated that ATM-deficient cells are very sensitive to CDT-induced DNA damage as reflected by increased cell death rates with concomitant cleavage of caspase-3 and PARP-1. Finally, we provided novel evidence that both homologous recombination (HR) and non-homologous end joining (NHEJ) protect against CDT-elicited DSBs. In conclusion, the findings suggest that CDT functions as a radiomimetic agent and, therefore, is an attractive tool for selectively inducing persistent levels of DSBs and unveiling the associated cellular responses.     

Single-cell microarray enables high-throughput evaluation of DNA double-strand breaks and DNA repair inhibitors

A key modality of non-surgical cancer management is DNA damaging therapy that causes DNA double-strand breaks that are preferentially toxic to rapidly dividing cancer cells. Double-strand break repair capacity is recognized as an important mechanism in drug resistance and is therefore a potential target for adjuvant chemotherapy. Additionally, spontaneous and environmentally induced DSBs are known to promote cancer, making DSB evaluation important as a tool in epidemiology, clinical evaluation and in the development of novel pharmaceuticals. Currently available assays to detect double-strand breaks are limited in throughput and specificity and offer minimal information concerning the kinetics of repair. Here, we present the CometChip, a 96-well platform that enables assessment of double-strand break levels and repair capacity of multiple cell types and conditions in parallel and integrates with standard high-throughput screening and analysis technologies. We demonstrate the ability to detect multiple genetic deficiencies in double-strand break repair and evaluate a set of clinically relevant chemical inhibitors of one of the major double-strand break repair pathways, non-homologous end-joining. While other high-throughput repair assays measure residual damage or indirect markers of damage, the CometChip detects physical double-strand breaks, providing direct measurement of damage induction and repair capacity, which may be useful in developing and implementing treatment strategies with reduced side effects.     

A proteome-wide visual screen identifies fission yeast proteins localizing to DNA double-strand breaks

DNA double-strand breaks (DSBs) are a major threat to genome integrity. Proteins involved in DNA damage checkpoint signaling and DSB repair often relocalize and concentrate at DSBs. Here, we used an ORFeome library of the fission yeast Schizosaccharomyces pombe to systematically identify proteins targeted to DSBs. We found 51 proteins that, when expressed from a strong exogenous promoter on the ORFeome plasmids, were able to form a distinct nuclear focus at an HO endonuclease-induced DSB. The majority of these proteins have known connections to DNA damage response, but few have been visualized at a specific DSB before. Among the screen hits, 37 can be detected at DSBs when expressed from native promoters. We classified them according to the focus emergence timing of the endogenously tagged proteins. Eight of these 37 proteins are yet unnamed. We named these eight proteins DNA-break-localizing proteins (Dbls) and performed preliminary functional analysis on two of them, Dbl1 (SPCC2H8.05c) and Dbl2 (SPCC553.01c). We found that Dbl1 and Dbl2 contribute to the normal DSB targeting of checkpoint protein Rad26 (homolog of human ATRIP) and DNA repair helicase Fml1 (homolog of human FANCM), respectively. As the first proteome-wide inventory of DSB-localizing proteins, our screen result will be a useful resource for understanding the mechanisms of eukaryotic DSB response.     

RAD51调控REV1参与DNA双链断裂修复

REV1是跨损伤聚合酶Y家族的重要成员之一,它不仅作为支架蛋白介导Y家族聚合酶招募至损伤位点完成跨损伤DNA合成(translesion DNA synthesis, TLS),还可利用自身的dCMP转移酶活性在一些损伤位点对侧整合dCMP参与TLS。此外,REV1也被报导参与调控同源重组修复。为进一步探讨REV1互作蛋白RAD51和RAD51C在其参与的同源重组修复通路中的调控作用,本研究采用脉冲氮激光微辐射实验,发现RAD51可调控REV1到双链断裂位点的募集。同时,免疫荧光实验结果证明REV1也反过来影响RAD51应答CPT损伤。然而敲低RAD51C并不影响REV1到DNA双链断裂位点的招募。结果表明,REV1和RAD51在HR通路中存在彼此相互调控的关系。     

Effect of double-strand break DNA sequence on the PARP-1 NHEJ pathway

Efficient repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity. In mammalian cells, DSBs are preferentially repaired by non-homologous end-joining (NHEJ). We have previously described a new DSBs microhomology end-joining pathway depending on PARP-1 and the XRCC1/DNA ligase III complex. In this study we analysed, with recombinant proteins and protein extracts, the effect of DSB end sequences: (i) on the DSB synapsis activity; (ii) on the end-joining activity. We report that PARP-1 DSB synapsis activity is independent of the DSB sequence and could be detected with non-complementary DSBs. We demonstrate also that the efficiency of DSBs repair by PARP-1 NHEJ is strongly dependent on the presence of G:C base pairs at microhomology termini. These results highlight a new role of the PARP-1 protein on the synapsis of DSBs and could explain why the PARP-1 NHEJ pathway is strongly dependent on the DSBs microhomology sequence.     

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.     

ATM-dependent chromatin remodeler Rsf-1 facilitates DNA damage checkpoints and homologous recombination repair

As a member of imitation switch (ISWI) family in ATP-dependent chromatin remodeling factors, RSF complex consists of SNF2h ATPase and Rsf-1. Although it has been reported that SNF2h ATPase is recruited to DNA damage sites (DSBs) in a poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent manner in DNA damage response (DDR), the function of Rsf-1 is still elusive. Here we show that Rsf-1 is recruited to DSBs confirmed by various cellular analyses. Moreover, the initial recruitment of Rsf-1 and SNF2h to DSBs shows faster kinetics than that of γH2AX after micro-irradiation. Signals of Rsf-1 and SNF2h are retained over 30 min after micro-irradiation, whereas γH2AX signals are gradually reduced at 10 min. In addition, Rsf-1 is accumulated at DSBs in ATM-dependent manner, and the putative pSQ motifs of Rsf-1 by ATM are required for its accumulation at DSBs. Furtheremore, depletion of Rsf-1 attenuates the activation of DNA damage checkpoint signals and cell survival upon DNA damage. Finally, we demonstrate that Rsf-1 promotes homologous recombination repair (HRR) by recruiting resection factors RPA32 and Rad51. Thus, these findings reveal a new function of chromatin remodeler Rsf-1 as a guard in DNA damage checkpoints and homologous recombination repair.     

Chromosomal breaks at the origin of small tandem DNA duplications

Small tandem DNA duplications in the range of 15 to 300 base-pairs play an important role in the aetiology of human disease and contribute to genome diversity. Here, we discuss different proposed mechanisms for their occurrence and argue that this type of structural variation mainly results from mutagenic repair of chromosomal breaks. This hypothesis is supported by both bioinformatical analysis of insertions occurring in the genome of different species and disease alleles, as well as by CRISPR/Cas9-based experimental data from different model systems. Recent work points to fill-in synthesis at double-stranded DNA breaks with complementary sequences, regulated by end-joining mechanisms, to account for small tandem duplications. We will review the prevalence of small tandem duplications in the population, and we will speculate on the potential sources of DNA damage that could give rise to this mutational signature. With the development of novel algorithms to analyse sequencing data, small tandem duplications are now more frequently detected in the human genome and identified as oncogenic gain-of-function mutations. Understanding their origin could lead to optimized treatment regimens to prevent therapy-induced activation of oncogenes and might expose novel vulnerabilities in cancer.     

Homologous recombination in DNA repair and DNA damage tolerance

Homologous recombination （HR） comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks （DSBs） and interstrand crosslinks （ICLs）. In addition, recombination provides critical support for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR： homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modalities of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.     

Ubiquitin and the DNA damage response

Comment on: Gatti M, et al. Cell Cycle 2012; 11:2538-44.     

Sequence and chromatin features guide DNA double-strand break resection initiation

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Interplay of two major repair pathways in the processing of complex double-strand DNA breaks

Radiation-induced complex double-strand breaks (DSBs) characterised by base lesions, abasic sites or single-strand breaks in close proximity to the break termini, are believed to be a major cause of the biological effects of ionising radiation exposure. It has been hypothesised that complex DSBs pose problems for the repair machinery of the cell. Using a biochemical approach, we have investigated the challenge to two major repair processes: base excision repair and ligation of DSB ends. Double-stranded oligonucleotides were synthesised with 8-oxo-7,8-dihydroguanine (8-oxoG) at defined positions relative to readily ligatable 3'-hydroxy or 5'-phosphate termini. The break termini interfere with removal of 8-oxoG during base excision repair as elucidated from the severely reduced efficiency of 8-oxoG removal by OGG1 with AP endonuclease-1 when in close proximity to break termini. NEIL-1, however, can partially restore processing of complex DSBs in an AP endonuclease-1 independent manner. The influence of 8-oxoG on ligation shows delayed rejoining if 8-oxoG is positioned two to three bases from the 3'-hydroxy or six bases from the 5'-phosphate termini. When two 8-oxoG lesions are positioned across the break junction ligation is severely retarded. This reduced efficiency of repair indicates that complex DSBs are likely to persist longer than simple DSBs in cells, and as a consequence are more significant in contributing to the biological effects of ionising radiation.     

Different fates of oocytes with DNA double-strand breaks in vitro and in vivo

In female mice, despite the presence of slight DNA double-strand breaks (DSBs), fully grown oocytes are able to undergo meiosis resumption as indicated by germinal vesicle breakdown (GVBD); however, severe DNA DSBs do reduce and delay entry into M phase through activation of the DNA damage checkpoint. But little is known about the effect of severe DNA DSBs on the spindle assembly checkpoint (SAC) during oocyte maturation. We showed that nearly no first polar body (PB1) was extruded at 12 h of in vitro maturation (IVM) in severe DNA DSBs oocytes, and the limited number of oocytes with PB1 were actually at telophase. However, about 60% of the severe DNA DSBs oocytes which underwent GVBD at 2 h of IVM released a PB1 at 18 h of IVM and these oocytes did reach the second metaphase (MII) stage. Chromosome spread at MI and MII stages showed that chromosomes fragmented after GVBD in severe DNA DSBs oocytes. The delayed PB1 extrusion was due to the disrupted attachment of microtubules to kinetochores and activation of the SAC. At the same time, misaligned chromosome fragments became obvious at the first metaphase (MI) in severe DNA DSBs oocytes. These data implied that the inactivation of SAC during the metaphase-anaphase transition of first meiosis was independent of chromosome integrity. Next, we induced DNA DSBs in vivo, and found that the number of superovulated oocytes per mouse was significantly reduced; moreover, this treatment increased the percentage of apoptotic oocytes. These results suggest that DNA DSBs oocytes undergo apoptosis in vivo.