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.     

Nucleolin Participates in DNA Double-Strand Break-Induced Damage Response through MDC1-Dependent Pathway

H2AX is an important factor for chromatin remodeling to facilitate accumulation of DNA damage-related proteins at DNA double-strand break (DSB) sites. In order to further understand the role of H2AX in the DNA damage response (DDR), we attempted to identify H2AX-interacting proteins by proteomics analysis. As a result, we identified nucleolin as one of candidates. Here, we show a novel role of a major nucleolar protein, nucleolin, in DDR. Nucleolin interacted with γ-H2AX and accumulated to laser micro-irradiated DSB damage sites. Chromatin Immunoprecipitation assay also displayed the accumulation of nucleolin around DSB sites. Nucleolin-depleted cells exhibited repression of both ATM-dependent phosphorylation following exposure to γ-ray and subsequent cell cycle checkpoint activation. Furthermore, nucleolin-knockdown reduced HR and NHEJ activity and showed decrease in IR-induced chromatin accumulation of HR/NHEJ factors, agreeing with the delayed kinetics of γ-H2AX focus. Moreover, nucleolin-knockdown decreased MDC1-related events such as focus formation of 53 BP1, RNF168, phosphorylated ATM, and H2A ubiquitination. Nucleolin also showed FACT-like activity for DSB damage-induced histone eviction from chromatin. Taken together, nucleolin could promote both ATM-dependent cell cycle checkpoint and DSB repair by functioning in an MDC1-related pathway through its FACT-like function.     

Role of H2AX in DNA damage response and human cancers

H2AX, the evolutionarily conserved variant of histone H2A, has been identified as one of the key histones to undergo various post-translational modifications in response to DNA double-strand breaks (DSBs). By virtue of these modifications, that include acetylation, phosphorylation and ubiquitination, H2AX marks the damaged DNA double helix, facilitating local recruitment and retention of DNA repair and chromatin remodeling factors to restore genomic integrity. These modifications are essential for effective DSB repair, so is their removal for cell, to recover from checkpoint arrest. Because of these vital roles during DSB signaling and also its activation during early cancer stages, H2AX is emerging as an intriguing gene in tumor biology, supported further by frequent deletion of the region harboring this gene. This review focuses on the insights gained from recent studies on dynamic regulation of H2AX in DSB repair. Also, posing future challenges in the area of chromatin reorganization and retention of epigenetic signature post-DSB-repair with implication of its haploinsufficiency in human cancers.     

Removal of H2A.Z by INO80 promotes homologous recombination

The mammalian INO80 remodelling complex facilitates homologous recombination (HR), but the mechanism by which it does this is unclear. Budding yeast INO80 can remove H2A.Z/H2B dimers from chromatin and replace them with H2A/H2B dimers. H2A.Z is actively incorporated at sites of damage in mammalian cells, raising the possibility that H2A.Z may need to be subsequently removed for resolution of repair. Here, we show that H2A.Z in human cells is indeed rapidly removed from chromatin flanking DNA damage by INO80. We also report that the histone chaperone ANP32E, which is implicated in removing H2AZ from chromatin, similarly promotes HR and appears to work on the same pathway as INO80 in these assays. Importantly, we demonstrate that the HR defect in cells depleted of INO80 or ANP32E can be rescued by H2A.Z co‐depletion, suggesting that H2A.Z removal from chromatin is the primary function of INO80 and ANP32E in promoting homologous recombination.     

Histone Acetyltransferase 1 Promotes Homologous Recombination in DNA Repair by Facilitating Histone Turnover

Faithful repair of DNA double-strand breaks is vital to the maintenance of genome integrity and proper cell functions. Histone modifications, such as reversible acetylation, phosphorylation, methylation, and ubiquitination, which collectively contribute to the establishment of distinct chromatin states, play important roles in the recruitment of repair factors to the sites of double-strand breaks. Here we report that histone acetyltransferase 1 (HAT1), a classical B type histone acetyltransferase responsible for acetylating the N-terminal tail of newly synthesized histone H4 in the cytoplasm, is a key regulator of DNA repair by homologous recombination in the nucleus. We found that HAT1 is required for the incorporation of H4K5/K12-acetylated H3.3 at sites of double-strand breaks through its HIRA-dependent histone turnover activity. Incorporated histones with specific chemical modifications facilitate subsequent recruitment of RAD51, a key repair factor in mammalian cells, to promote efficient homologous recombination. Significantly, depletion of HAT1 sensitized cells to DNA damage compromised the global chromatin structure, inhibited cell proliferation, and induced cell apoptosis. Our experiments uncovered a role for HAT1 in DNA repair in higher eukaryotic organisms and provide a mechanistic insight into the regulation of histone dynamics by HAT1.     

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.     

Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining

The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmid-based NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals.     

Conditional deletion of Nbs1 in murine cells reveals its role in branching repair pathways of DNA double-strand breaks

NBS1 forms a complex with MRE11 and RAD50 (MRN) that is proposed to act on the upstream of two repair pathways of DNA double-strand break (DSB), homologous repair (HR) and non-homologous end joining (NHEJ). However, the function of Nbs1 in these processes has not fully been elucidated in mammals due to the lethal phenotype of cells and mice lacking Nbs1. Here, we have constructed mouse Nbs1-null embryonic fibroblasts and embryonic stem cells, through the Cre-loxP and sequential gene targeting techniques. We show that cells lacking Nbs1 display reduced HR of the single DSB in chromosomally integrated substrate, affecting both homology-directed repair (HDR) and single-stranded annealing pathways, and, surprisingly, increased NHEJ-mediated sequence deletion. Moreover, focus formation at DSBs and chromatin recruitment of the Nbs1 partners Rad50 and Mre11 as well as Rad51 and Brca1 are attenuated in these cells, whereas the NHEJ molecule Ku70 binding to chromatin is not affected. These data provide a novel insight into the function of MRN in the branching of DSB repair pathways.     

表观遗传调控：基于染色质环境的DNA双链断裂修复

DNA双链断裂（DNA double-strand breaks, DSBs）是威胁基因组完整性和细胞存活的最有害的DNA损伤类型。同源重组（homologous recombination,HR）和非同源末端连接（non-homologous end joining,NHEJ）是修复DNA双链断裂的两种主要途径。DSB修复涉及到损伤部位修复蛋白的募集和染色质结构的改变。在DNA双链断裂诱导下,染色质结构的动态变化在时间和空间上受到严格调控,进而对DNA双链断裂修复过程进行精细调节。特定的染色质修饰形成利于修复的染色质状态,有助于DNA双链断裂修复机器的招募、修复途径的选择和DNA损伤检查点的活化;其中修复途径的选择对于基因组稳定性至关重要。修复不当或失败可导致基因组不稳定性,甚至促进肿瘤的发生。本文综述了染色质结构和染色质修饰的动态变化在DSB修复中的重要作用。此外,文章还总结了在癌症治疗中靶向关键染色质调控因子在基因组稳定性维持、肿瘤发生发展以及潜在临床应用价值等方面的进展。     

Small Ubiquitin-like Modifier (SUMO) Isoforms and Conjugation-independent Function in DNA Double-strand Break Repair Pathways

Small ubiquitin-like modifier (SUMO) proteins act in DNA double-strand break (DSB) repair, but the pathway specificity of the three major isoforms has not been defined. In experiments in which we depleted the endogenous SUMO protein by RNAi, we found that SUMO1 functioned in all subpathways of either homologous recombination (HR) or non-homologous end joining (NHEJ), whereas SUMO2/3 was required for the major NHEJ pathway, called conservative NHEJ, but dispensable in other DSB repair pathways. To our surprise, we found that depletion of UBC9, the unique SUMO E2 enzyme, had no effect in HR or alternative NHEJ (Alt-NHEJ) but was required for conservative NHEJ. Consistent with this result, both non-conjugatable mutant and wild-type SUMO1 proteins functioned similarly in HR and Alt-NHEJ. These results detail the functional roles of specific SUMO isoforms in DSB repair in mammalian cells and reveal that SUMO1 functions in HR or Alt-NHEJ as a free protein and not as a protein conjugate.     

The histone methyltransferase SET8 is required for S-phase progression

Chromatin structure and function is influenced by histone posttranslational modifications. SET8 (also known as PR-Set7 and SETD8) is a histone methyltransferase that monomethylates histonfe H4-K20. However, a function for SET8 in mammalian cell proliferation has not been determined. We show that small interfering RNA inhibition of SET8 expression leads to decreased cell proliferation and accumulation of cells in S phase. This is accompanied by DNA double-strand break (DSB) induction and recruitment of the DNA repair proteins replication protein A, Rad51, and 53BP1 to damaged regions. SET8 depletion causes DNA damage specifically during replication, which induces a Chk1-mediated S-phase checkpoint. Furthermore, we find that SET8 interacts with proliferating cell nuclear antigen through a conserved motif, and SET8 is required for DNA replication fork progression. Finally, codepletion of Rad51, an important homologous recombination repair protein, abrogates the DNA damage after SET8 depletion. Overall, we show that SET8 is essential for genomic stability in mammalian cells and that decreased expression of SET8 results in DNA damage and Chk1-dependent S-phase arrest.