The interaction between CtIP and BRCA1 is not essential for resection-mediated DNA repair or tumor suppression

The CtIP protein facilitates homology-directed repair (HDR) of double-strand DNA breaks (DSBs) by initiating DNA resection, a process in which DSB ends are converted into 3′-ssDNA overhangs. The BRCA1 tumor suppressor, which interacts with CtIP in a phospho-dependent manner, has also been implicated in DSB repair through the HDR pathway. It was recently reported that the BRCA1–CtIP interaction is essential for HDR in chicken DT40 cells. To examine the role of this interaction in mammalian cells, we generated cells and mice that express Ctip polypeptides (Ctip-S326A) that fail to bind BRCA1. Surprisingly, isogenic lines of Ctip-S326A mutant and wild-type cells displayed comparable levels of HDR function and chromosomal stability. Although Ctip-S326A mutant cells were modestly sensitive to topoisomerase inhibitors, mice expressing Ctip-S326A polypeptides developed normally and did not exhibit a predisposition to cancer. Thus, in mammals, the phospho-dependent BRCA1–CtIP interaction is not essential for HDR-mediated DSB repair or for tumor suppression.     

DNA double-strand break repair pathway choice and cancer

Since DNA double-strand breaks (DSBs) contribute to the genomic instability that drives cancer development, DSB repair pathways serve as important mechanisms for tumor suppression. Thus, genetic lesions, such as BRCA1 and BRCA2 mutations, that disrupt DSB repair are often associated with cancer susceptibility. In addition, recent evidence suggests that DSB “mis-repair”, in which DSBs are resolved by an inappropriate repair pathway, can also promote genomic instability and presumably tumorigenesis. This notion has gained currency from recent cancer genome sequencing studies which have uncovered numerous chromosomal rearrangements harboring pathological DNA repair signatures. In this perspective, we discuss the factors that regulate DSB repair pathway choice and their consequences for genome stability and cancer.     

Recognition of DNA double strand breaks by the BRCA1 tumor suppressor network

DNA double-strand breaks (DSBs) occur in response to both endogenous and exogenous genotoxic stress. Inappropriate repair of DSBs can lead to either loss of viability or to chromosomal alterations that increase the likelihood of cancer development. In strong support of this assertion, many cancer predisposition syndromes stem from germline mutations in genes involved in DNA DSB repair. Among the most prominent of such tumor suppressor genes are the Breast Cancer 1 and Breast Cancer 2 genes (BRCA1 and BRCA2), which are mutated in familial forms of breast and ovarian cancer. Recent findings implicate BRCA1 as a central component of several distinct macromolecular protein complexes, each dedicated to distinct elements of DNA DSB repair and tumor suppression. Emerging evidence has shed light on some of the molecular recognition processes that are responsible for targeting BRCA1 and its associated partners to DNA and chromatin directly flanking DSBs. These events are required for BRCA1-dependent DNA repair and tumor suppression. Thus, a detailed temporal and spatial knowledge of how breaks are recognized and repaired has profound implications for understanding processes related to the genesis of malignancy and to its treatment.     

Mutation in Brca2 stimulates error-prone homology-directed repair of DNA double-strand breaks occurring between repeated sequences

Mutation of BRCA2 causes familial early onset breast and ovarian cancer. BRCA2 has been suggested to be important for the maintenance of genome integrity and to have a role in DNA repair by homology- directed double-strand break (DSB) repair. By studying the repair of a specific induced chromosomal DSB we show that loss of Brca2 leads to a substantial increase in error-prone repair by homology-directed single-strand annealing and a reduction in DSB repair by conservative gene conversion. These data demonstrate that loss of Brca2 causes misrepair of chromosomal DSBs occurring between repeated sequences by stimulating use of an error-prone homologous recombination pathway. Furthermore, loss of Brca2 causes a large increase in genome-wide error-prone repair of both spontaneous DNA damage and mitomycin C-induced DNA cross-links at the expense of error-free repair by sister chromatid recombination. This provides insight into the mechanisms that induce genome instability in tumour cells lacking BRCA2.     

The BRCA1-RAP80 complex regulates DNA repair mechanism utilization by restricting end resection

The tumor suppressor protein BRCA1 is a constituent of several different protein complexes and is required for homology-directed repair (HDR) of DNA double strand breaks (DSBs). The most recently discovered BRCA1-RAP80 complex is recruited to ubiquitin structures on chromatin surrounding the break. Deficiency of any member of this complex confers hypersensitivity to DNA-damaging agents by undefined mechanisms. In striking contrast to other BRCA1-containing complexes that are known to promote HDR, we demonstrate that the BRCA1-RAP80 complex restricts end resection in S/G(2) phase of the cell cycle, thereby limiting HDR. RAP80 or BRCC36 deficiency resulted in elevated Mre11-CtIP-dependent 5' end resection with a concomitant increase in HDR mechanisms that rely on 3' single-stranded overhangs. We propose a model in which the BRCA1-RAP80 complex limits nuclease accessibility to DSBs, thus preventing excessive end resection and potentially deleterious homology-directed DSB repair mechanisms that can impair genome integrity.     

DNA double-strand break repair in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Faithful repair of DNA double-strand breaks (DSBs) is vital for animal development, as inappropriate repair can cause gross chromosomal alterations that result in cellular dysfunction, ultimately leading to cancer, or cell death. Correct processing of DSBs is not only essential for maintaining genomic integrity, but is also required in developmental programs, such as gametogenesis, in which DSBs are deliberately generated. Accordingly, DSB repair deficiencies are associated with various developmental disorders including cancer predisposition and infertility. To avoid this threat, cells are equipped with an elaborate and evolutionarily well-conserved network of DSB repair pathways. In recent years, Caenorhabditis elegans has become a successful model system in which to study DSB repair, leading to important insights in this process during animal development. This review will discuss the major contributions and recent progress in the C. elegans field to elucidate the complex networks involved in DSB repair, the impact of which extends well beyond the nematode phylum.     

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.     

Solving the RIDDLE of 53BP1 recruitment to sites of damage

The cellular response to DNA double strand breaks is a complex, integrated network of pathways, coordinated by the PI-3-kinase-like family of kinases, which includes ATM, ATR and DNA-PK, that function to preserve the integrity of the genome. Mutations in genes that control these pathways are associated with increased genomic instability, neurodegeneration, immunodeficiency, premature aging and tumour predisposition. Indeed a significant proportion of our understanding regarding the mechanisms controlling DNA double strand break (DSB) repair has come from the study of cells derived from patients with inherited mutations in these genes. The discovery of the E3 ubiquitin ligase, RNF8, as a regulator of DNA DSB repair has brought to light a critical role for the ubiquitin system in regulating the cellular DSBs. Recently, identification of mutations in a second E3 ubiquitin ligase, RNF168, as the underlying genetic cause of the DNA repair deficiency disorder, RIDDLE syndrome, has provided the first link between ubiquitin-dependent DSB repair and immune system development in man. The finding that RNF168 functions downstream of RNF8 to orchestrate the recruitment of repair proteins, such as BRCA1 and 53BP1, to sites of DNA damage suggests that these two E3 ligases define a ubiquitylation cascade that regulates the spatial relocalisation of DSB repair proteins.     

Ring Finger Nuclear Factor RNF168 Is Important for Defects in Homologous Recombination Caused by Loss of the Breast Cancer Susceptibility Factor BRCA1

The RING finger nuclear factor RNF168 is required for recruitment of several DNA damage response factors to double strand breaks (DSBs), including 53BP1 and BRCA1. Because 53BP1 and BRCA1 function antagonistically during the DSB repair pathway homologous recombination (HR), the influence of RNF168 on HR has been unclear. We report that RNF168 depletion causes an elevated frequency of two distinct HR pathways (homology-directed repair and single strand annealing), suppresses defects in HR caused by BRCA1 silencing, but does not suppress HR defects caused by disruption of CtIP, RAD50, BRCA2, or RAD51. Furthermore, RNF168-depleted cells can form ionizing radiation-induced foci of the recombinase RAD51 without forming BRCA1 ionizing radiation-induced foci, indicating that this loss of BRCA1 recruitment to DSBs does not reflect a loss of function during HR. Additionally, we find that RNF168 and 53BP1 have a similar influence on HR. We suggest that RNF168 is important for HR defects caused by BRCA1 loss.     

BCCIP regulates homologous recombination by distinct domains and suppresses spontaneous DNA damage

Homologous recombination (HR) is critical for maintaining genome stability through precise repair of DNA double-strand breaks (DSBs) and restarting stalled or collapsed DNA replication forks. HR is regulated by many proteins through distinct mechanisms. Some proteins have direct enzymatic roles in HR reactions, while others act as accessory factors that regulate HR enzymatic activity or coordinate HR with other cellular processes such as the cell cycle. The breast cancer susceptibility gene BRCA2 encodes a critical accessory protein that interacts with the RAD51 recombinase and this interaction fluctuates during the cell cycle. We previously showed that a BRCA2- and p21-interacting protein, BCCIP, regulates BRCA2 and RAD51 nuclear focus formation, DSB-induced HR and cell cycle progression. However, it has not been clear whether BCCIP acts exclusively through BRCA2 to regulate HR and whether BCCIP also regulates the alternative DSB repair pathway, non-homologous end joining. In this study, we found that BCCIP fragments that interact with BRCA2 or with p21 each inhibit DSB repair by HR. We further show that transient down-regulation of BCCIP in human cells does not affect non-specific integration of transfected DNA, but significantly inhibits homology-directed gene targeting. Furthermore, human HT1080 cells with constitutive down-regulation of BCCIP display increased levels of spontaneous single-stranded DNA (ssDNA) and DSBs. These data indicate that multiple BCCIP domains are important for HR regulation, that BCCIP is unlikely to regulate non-homologous end joining, and that BCCIP plays a critical role in resolving spontaneous DNA damage.     

The involvement of human RECQL4 in DNA double‐strand break repair

Rothmund–Thomson syndrome (RTS) is an autosomal recessive hereditary disorder associated with mutation in RECQL4 gene, a member of the human RecQ helicases. The disease is characterized by genomic instability, skeletal abnormalities and predisposition to malignant tumors, especially osteosarcomas. The precise role of RECQL4 in cellular pathways is largely unknown; however, recent evidence suggests its involvement in multiple DNA metabolic pathways. This study investigates the roles of RECQL4 in DNA double‐strand break (DSB) repair. The results show that RECQL4‐deficient fibroblasts are moderately sensitive to γ‐irradiation and accumulate more γH2AX and 53BP1 foci than control fibroblasts. This is suggestive of defects in efficient repair of DSB’s in the RECQL4‐deficient fibroblasts. Real time imaging of live cells using laser confocal microscopy shows that RECQL4 is recruited early to laser‐induced DSBs and remains for a shorter duration than WRN and BLM, indicating its distinct role in repair of DSBs. Endogenous RECQL4 also colocalizes with γH2AX at the site of DSBs. The RECQL4 domain responsible for its DNA damage localization has been mapped to the unique N‐terminus domain between amino acids 363–492, which shares no homology to recruitment domains of WRN and BLM to the DSBs. Further, the recruitment of RECQL4 to laser‐induced DNA damage is independent of functional WRN, BLM or ATM proteins. These results suggest distinct cellular dynamics for RECQL4 protein at the site of laser‐induced DSB and that it might play important roles in efficient repair of DSB’s.     

Rapid recruitment of BRCA1 to DNA double-strand breaks is dependent on its association with Ku80

BRCA1 is the first susceptibility gene to be linked to breast and ovarian cancers. Although mounting evidence has indicated that BRCA1 participates in DNA double-strand break (DSB) repair pathways, its precise mechanism is still unclear. Here, we analyzed the in situ response of BRCA1 at DSBs produced by laser microirradiation. The amino (N)- and carboxyl (C)-terminal fragments of BRCA1 accumulated independently at DSBs with distinct kinetics. The N-terminal BRCA1 fragment accumulated immediately after laser irradiation at DSBs and dissociated rapidly. In contrast, the C-terminal fragment of BRCA1 accumulated more slowly at DSBs but remained at the sites. Interestingly, rapid accumulation of the BRCA1 N terminus, but not the C terminus, at DSBs depended on Ku80, which functions in the nonhomologous end-joining (NHEJ) pathway, independently of BARD1, which binds to the N terminus of BRCA1. Two small regions in the N terminus of BRCA1 independently accumulated at DSBs and interacted with Ku80. Missense mutations found within the N terminus of BRCA1 in cancers significantly changed the kinetics of its accumulation at DSBs. A P142H mutant failed to associate with Ku80 and restore resistance to irradiation in BRCA1-deficient cells. These might provide a molecular basis of the involvement of BRCA1 in the NHEJ pathway of the DSB repair process.     

Molecular mechanisms of DNA double-strand break repair

DNA double-strand breaks (DSBs) are major threats to the genomic integrity of cells. If not taken care of properly, they can cause chromosome fragmentation, loss and translocation, possibly resulting in carcinogenesis. Upon DSB formation, cell-cycle checkpoints are triggered and multiple DSB repair pathways can be activated. Recent research on the Nijmegen breakage syndrome, which predisposes patients to cancer, suggests a direct link between activation of cell-cycle checkpoints and DSB repair. Furthermore, the biochemical activities of proteins involved in the two major DSB repair pathways, homologous recombination and DNA end-joining, are now beginning to emerge. This review discusses these new findings and their implications for the mechanisms of DSB repair.     

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.     

The repair of environmentally relevant DNA double strand breaks caused by high linear energy transfer irradiation – No simple task

High linear energy transfer (LET) ionising radiation (IR) such as radon-derived alpha particles and high mass, high energy (HZE) particles of cosmic radiation are the predominant forms of IR to which humanity is exposed throughout life. High-LET forms of IR are established carcinogens relevant to human cancer, and their potent mutagenicity is believed, in part, to be due to a greater incidence of clustered DNA double strand breaks (DSBs) and associated lesions, as ionization events occur within a more confined genomic space. The repair of such DNA damage is now well-documented to occur with slower kinetics relative to that induced by low-LET IR, and to be more reliant upon homology-directed repair pathways. Underlying these phenomena is the relative inability of non-homologous end-joining (NHEJ) to adequately resolve high-LET IR-induced DSBs. Current findings suggest that the functionality of the DNA-dependent protein kinase (DNA-PK), comprised of the Ku70-Ku80 heterodimer and the DNA-PK catalytic subunit (DNA-PKcs), is particularly perturbed by high-LET IR-induced clustered DSBs, rendering DNA-PK dependent NHEJ less relevant to resolving these lesions. By contrast, the NHEJ-associated DNA processing endonuclease Artemis shows a greater relevance to high-LET IR-induced DSB repair. Here, we will review the cellular response to high-LET irradiation, the implications of the chronic, low-dose modality of this exposure and molecular pathways that respond to high-LET irradiation induced DSBs, with particular emphasis on NHEJ factors.     

Proteomics of mouse BRCA1-deficient mammary tumors identifies DNA repair proteins with potential diagnostic and prognostic value in human breast cancer

Breast cancer 1, early onset (BRCA1) hereditary breast cancer, a type of cancer with defects in the homology-directed DNA repair pathway, would benefit from the identification of proteins for diagnosis, which might also be of potential use as screening, prognostic, or predictive markers. Sporadic breast cancers with defects in the BRCA1 pathway might also be diagnosed. We employed proteomics based on one-dimensional gel electrophoresis in combination with nano-LC-MS/MS and spectral counting to compare the protein profiles of mammary tumor tissues of genetic mouse models either deficient or proficient in BRCA1. We identified a total of 3,545 proteins, of which 801 were significantly differentially regulated between the BRCA1-deficient and -proficient breast tumors. Pathway and protein complex analysis identified DNA repair and related functions as the major processes associated with the up-regulated proteins in the BRCA1-deficient tumors. In addition, by selecting highly connected nodes, we identified a BRCA1 deficiency signature of 45 proteins that enriches for homology-directed DNA repair deficiency in human gene expression breast cancer data sets. This signature also exhibits prognostic power across multiple data sets, with optimal performance in a data set enriched in tumors deficient in homology-directed DNA repair. In conclusion, by comparing mouse proteomes from BRCA1-proficient and -deficient mammary tumors, we were able to identify several markers associated with BRCA1 deficiency and a prognostic signature for human breast cancer deficient in homology-directed DNA repair.     

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.     

Protein phosphatase PP6 is required for homology-directed repair of DNA double-strand breaks

DNA double-strand breaks (DSBs) are among the most lethal lesions associated with genome stability, which, when destabilized, predisposes organs to cancers. DSBs are primarily fixed either with little fidelity by non-homologous end joining (NHEJ) repair or with high fidelity by homology-directed repair (HDR). The phosphorylated form of H2AX on serine 139 (γ-H2AX) is a marker of DSBs. In this study, we explored if the protein phosphatase PP6 is involved in DSB repair by depletion of its expression in human cancer cell lines, and determined PP6 expression in human breast cancer tissues by immunohistochemistry staining. We found that bacterially produced PP6c (the catalytic subunit of PP6)-containing heterotrimeric combinations exhibit phosphatase activity against γ-H2AX in the in vitro phosphatase assays. Depletion of PP6c or PP6R2 led to persistent high levels of γ-H2AX after DNA damage and a defective HDR. Chromatin immunoprecipitation assays demonstrated that PP6c was recruited to the region adjacent to the DSB sites. Expression of PP6c, PP6R2 and PP6R3 in human breast tumors was significantly lower than those in benign breast diseases. Taken together, our results suggest that γ-H2AX is a physiological substrate of PP6 and PP6 is required for HDR and its expression may harbor a protective role during the development of breast cancer.Key words: protein phosphatase, PP6, γ-H2AX, DNA double-strand break, homology-directed repair