AtPRD1 is required for meiotic double strand break formation in Arabidopsis thaliana

The initiation of meiotic recombination by the formation of DNA double-strand breaks (DSBs) catalysed by the Spo11 protein is strongly evolutionary conserved. In Saccharomyces cerevisiae, Spo11 requires nine other proteins for meiotic DSB formation, but, unlike Spo11, few of these proteins seem to be conserved across kingdoms. In order to investigate this recombination step in higher eukaryotes, we have isolated a new gene, AtPRD1, whose mutation affects meiosis in Arabidopsis thaliana. In Atprd1 mutants, meiotic recombination rates fall dramatically, early recombination markers (e.g., DMC1 foci) are absent, but meiosis progresses until achiasmatic univalents are formed. Besides, Atprd1 mutants suppress DSB repair defects of a large range of meiotic mutants, showing that AtPRD1 is involved in meiotic recombination and is required for meiotic DSB formation. Furthermore, we showed that AtPRD1 and AtSPO11-1 interact in a yeast two-hybrid assay, suggesting that AtPRD1 could be a partner of AtSPO11-1. Moreover, our study reveals similarity between AtPRD1 and the mammalian protein Mei1, suggesting that AtPRD1 could be a Mei1 functional homologue.     

MRG-1 is required for genomic integrity in Caenorhabditis elegans germ cells

During meiotic cell division, proper chromosome synapsis and accurate repair of DNA double strand breaks (DSBs) are required to maintain genomic integrity, loss of which leads to apoptosis or meiotic defects. The mechanisms underlying meiotic chromosome synapsis, DSB repair and apoptosis are not fully understood. Here, we report that the chromodomain-containing protein MRG-1 is an important factor for genomic integrity in meiosis in Caenorhabditis elegans. Loss of mrg-1 function resulted in a significant increase in germ cell apoptosis that was partially inhibited by mutations affecting DNA damage checkpoint genes. Consistently, mrg-1 mutant germ lines exhibited SPO-11-generated DSBs and elevated exogenous DNA damage-induced chromosome fragmentation at diakinesis. In addition, the excessive apoptosis in mrg-1 mutants was partially suppressed by loss of the synapsis checkpoint gene pch-2, and a significant number of meiotic nuclei accumulated at the leptotene/zygotene stages with an elevated level of H3K9me2 on the chromatin, which was similarly observed in mutants deficient in the synaptonemal complex, suggesting that the proper progression of chromosome synapsis is likely impaired in the absence of mrg-1. Altogether, these findings suggest that MRG-1 is critical for genomic integrity by promoting meiotic DSB repair and synapsis progression in meiosis.     

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.     

Recent advances in understanding of the DNA double-strand break repair machinery of plants

Living cells suffer numerous and varied alterations of their genetic material. Of these, the DNA double-strand break (DSB) is both particularly threatening and common. Double-strand breaks arise from exposure to DNA damaging agents, but also from cell metabolism-in a fortuitous manner during DNA replication or repair of other kinds of lesions and in a programmed manner, for example during meiosis or V(D)J gene rearrangement. Cells possess several overlapping repair pathways to deal with these breaks, generally designated as genetic recombination. Genetic and biochemical studies have provided considerable amounts of data about the proteins involved in recombination processes and their functions within these processes. Although they have long played a key role in building understanding of genetics, relatively little is known at the molecular level of the genetic recombination processes in plants. The use of reverse genetic approaches and the public availability of sequence tagged mutants in Arabidopsis thaliana have led to increasingly rapid progress in this field over recent years. The rapid progress of studies of recombination in plants is obviously not limited to the DSB repair machinery as such and we ask readers to understand that in order to maintain the focus and to rest within a reasonable length, we present only limited discussion of the exciting advances in the of plant meiosis field, which require a full review in their own right . We thus present here an update on recent advances in understanding of the DSB repair machinery of plants, focussing on Arabidopsis and making a particular effort to place these in the context of more general of understanding of these processes.     

The role of the BRCA1 tumor suppressor in DNA double-strand break repair

The tumor suppressor gene BRCA1 was cloned in 1994 based on its linkage to early-onset breast and ovarian cancer. Although the BRCA1 protein has been implicated in multiple cellular functions, the precise mechanism that determines its tumor suppressor activity is not defined. Currently, the emerging picture is that BRCA1 plays an important role in maintaining genomic integrity by protecting cells from double-strand breaks (DSB) that arise during DNA replication or after DNA damage. The DSB repair pathways available in mammalian cells are homologous recombination and nonhomologous end-joining. BRCA1 function seems to be regulated by specific phosphorylations in response to DNA damage and we will focus this review on the roles played by BRCA1 in DNA repair and cell cycle checkpoints. Finally, we will explore the idea that tumor suppression by BRCA1 depends on its control of DNA DSB repair, resulting in the promotion of error-free and the inhibition of error-prone recombinational repair.     

Multiple barriers to nonhomologous DNA end joining during meiosis in Drosophila

Repair of meiotic double-strand breaks (DSBs) uses the homolog and recombination to yield crossovers while alternative pathways such as nonhomologous end joining (NHEJ) are suppressed. Our results indicate that NHEJ is blocked at two steps of DSB repair during meiotic prophase: first by the activity of the MCM-like protein MEI-218, which is required for crossover formation, and, second, by Rad51-related proteins SPN-B (XRCC3) and SPN-D (RAD51C), which physically interact and promote homologous recombination (HR). We further show that the MCM-like proteins also promote the activity of the DSB repair checkpoint pathway, indicating an early requirement for these proteins in DSB processing. We propose that when a meiotic DSB is formed in the absence of both MEI-218 and SPN-B or SPN-D, a DSB substrate is generated that can enter the NHEJ repair pathway. Indeed, due to its high error rate, multiple barriers may have evolved to prevent NHEJ activity during meiosis.     

BRCA2 is epistatic to the RAD51 paralogs in response to DNA damage

Homologous recombination plays an important role in the high-fidelity repair of DNA double-strand breaks. A central player in this process, RAD51, polymerizes onto single-stranded DNA and searches for homology in a duplex donor DNA molecule, usually the sister chromatid. Homologous recombination is a highly regulated event in mammalian cells: some proteins have direct enzymatic functions, others mediate or overcome rate-limiting steps in the process, and still others signal cell cycle arrest to allow repair to occur. While the human BRCA2 protein has a clear role in delivering and loading RAD51 onto single-stranded DNA generated after resection of the DNA break, the mechanistic functions of the RAD51 paralogs remain unclear. In this study, we sought to determine the genetic interactions between BRCA2 and the RAD51 paralogs during DNA DSB repair. We utilized siRNA-mediated knockdown of these proteins in human cells to assess their impact on the DNA damage response. The results indicate that loss of BRCA2 alone imparts a more severe phenotype than the loss of any individual RAD51 paralog and that BRCA2 is epistatic to each of the four paralogs tested.     

BRCA1-Ku80 Protein Interaction Enhances End-joining Fidelity of Chromosomal Double-strand Breaks in the G1 Phase of the Cell Cycle

Quality control of DNA double-strand break (DSB) repair is vital in preventing mutagenesis. Non-homologous end-joining (NHEJ), a repair process predominant in the G₁ phase of the cell cycle, rejoins DSBs either accurately or with errors, but the mechanisms controlling its fidelity are poorly understood. Here we show that BRCA1, a tumor suppressor, enhances the fidelity of NHEJ-mediated DSB repair and prevents mutagenic deletional end-joining through interaction with canonical NHEJ machinery during G₁. BRCA1 binds and stabilizes Ku80 at DSBs through its N-terminal region, promotes precise DSB rejoining, and increases cellular resistance to radiation-induced DNA damage in a G₁ phase-specific manner. These results suggest that BRCA1, as a central player in genome integrity maintenance, ensures high fidelity repair of DSBs by not only promoting homologous recombination repair in G₂/M phase but also facilitating fidelity of Ku80-dependent NHEJ repair, thus preventing deletional end-joining of chromosomal DSBs during G₁.     

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.     

Cell cycle-dependent complex formation of BRCA1.CtIP.MRN is important for DNA double-strand break repair

BRCA1 plays an important role in the homologous recombination (HR)-mediated DNA double-strand break (DSB) repair, but the mechanism is not clear. Here we describe that BRCA1 forms a complex with CtIP and MRN (Mre11/Rad50/Nbs1) in a cell cycle-dependent manner. Significantly, the complex formation, especially the ionizing radiation-enhanced association of BRCA1 with MRN, requires cyclin-dependent kinase activity. CtIP directly interacts with Nbs1. The in vivo association of BRCA1 with MRN is largely dependent on the association of CtIP with the BRCT domains at the C terminus of BRCA1, whereas the N terminus of BRCA1 also contributes to its association with MRN. CtIP, as well as the interaction of BRCA1 with CtIP and MRN, is critical for IR-induced single-stranded DNA formation and cellular resistance to radiation. Consistently, CtIP itself is required for efficient HR-mediated DSB repair, like BRCA1 and MRN. These studies suggest that the complex formation of BRCA1.CtIP.MRN is important for facilitating DSB resection to generate single-stranded DNA that is needed for HR-mediated DSB repair. Because cyclin-dependent kinase is important for establishing IR-enhanced interaction of MRN with BRCA1, we propose that the cell cycle-dependent complex formation of BRCA1, CtIP, and MRN contributes to the activation of HR-mediated DSB repair in the S and G(2) phases of the cell cycle.     

CeBRC-2 stimulates D-loop formation by RAD-51 and promotes DNA single-strand annealing

The BRCA2 tumour suppressor regulates the RAD-51 recombinase during double-strand break (DSB) repair by homologous recombination (HR) but how BRCA2 executes its functions is not well understood. We previously described a functional homologue of BRCA2 in Caenorhabditis elegans (CeBRC-2) that binds preferentially to single-stranded DNA via an OB-fold domain and associates directly with RAD-51 via a single BRC domain. Consistent with a direct role in HR, Cebrc-2 mutants are defective for repair of meiotic and radiation-induced DSBs due to an inability to regulate RAD-51. Here, we explore the function of CeBRC-2 in HR processes using purified proteins. We show that CeBRC-2 stimulates RAD-51-mediated D-loop formation and reduces the rate of ATP hydrolysis catalysed by RAD-51. These functions of CeBRC-2 are dependent upon direct association with RAD-51 via its BRC motif and on its DNA-binding activity, as point mutations in the BRC domain that abolish RAD-51 binding or the BRC domain of CeBRC-2 alone, lacking the DNA-binding domain, fail to stimulate RAD-51-mediated D-loop formation and do not reduce the rate of ATP hydrolysis by RAD-51. Phenotypic comparison of Cebrc-2 and rad-51 mutants also revealed a role for CeBRC-2 in an error-prone DSB repair pathway independent of rad-51 and non-homologous end joining, raising the possibility that CeBRC-2 may have replaced the role of vertebrate Rad52 in DNA single-strand annealing (SSA), which is missing from C. elegans. Indeed, we show here that CeBRC-2 mediates SSA of RPA-oligonucleotide complexes similar to Rad52. These results reveal RAD-51-dependent and -independent functions of CeBRC-2 that provide an explanation for the difference in DNA repair defects observed in Cebrc-2 and rad-51 mutants, and define mechanistic roles for CeBRC-2 in HR and in the SSA pathway for DSB repair.     

BRCA1 modulates ionizing radiation-induced nuclear focus formation by the replication protein A p34 subunit

Mutations in BRCA1 account for a significant proportion of familial breast and ovarian cancers. BRCA1 has been implicated in DNA damage responses including double-strand break (DSB) repair. However, its exact role in DSB repair and its functional relationship with other known repair proteins remain to be elucidated. In this study, we carried out a cytological analysis of the effect of BRCA1 on damage-induced nuclear focus formation mediated by the replication protein A (RPA). RPA is a multi-functional protein that participates in both DNA replication and various types of DNA repair including DSB repair. Following ionizing radiation (IR), RPA and BRCA1 formed punctate nuclear staining patterns that co-localized with each other, consistent with the implicated roles of both proteins in the same repair process. The number of damage-induced RPA foci in BRCA1-deficient cells, however, was significantly greater than that in BRCA1-positive cells. Moreover, the effect of BRCA1 on the RPA staining pattern appeared to be specific for IR but not ultraviolet (UV) irradiation. These data suggest that BRCA1 plays an important role in processing the RPA-associated intermediates during DSB repair.     

The interplay between BRCA1 and 53BP1 influences death,aging, senescence and cancer

In proliferating cells DNA double strand breaks (DSBs) are a common occurrence during DNA replication. DSB repair using homologous recombination is essential for the error-free repair of such breaks and proliferating cells require some level of HR activity for their viability. The BRCA1 tumour suppressor has an important role in this process and is believed to channel the DSBs into the HR pathway. The related 53BP1 gene is known to positively regulate repair of DSBs outside of S phase, but via the NHEJ pathway. Two new studies suggest a new role for 53BP1 as an inhibitor of HR [1], [2]. These genetic studies establish that 53BP1, but not other components of the NHEJ machinery, can inhibit the early resection step of HR. In cells defective for BRCA1, which is required for efficient HR, the balance between promoting and inhibiting HR is thrown towards inhibition. Simultaneous loss of 53BP1 can rescue the HR defect of BRCA1-defective cells and restore cellular viability. Here, I provide an overview of these studies and discuss their implications for tumourigenesis.     

The Arabidopsis MEI1 gene encodes a protein with five BRCT domains that is involved in meiosis-specific DNA repair events independent of SPO11-induced DSBs

Arabidopsis thaliana MEI1 was first described as a gene involved in male meiosis, encoding a short protein showing homology with a human acrosin-trypsin inhibitor. We have isolated a new allele of mei1, and shown that in both mutants male and female meiosis are affected. In both reproductive pathways, meiosis proceeds while chromosomes become fragmented, resulting in aberrant meiotic products and in a strongly reduced fertility. We have shown that the gene mutated in mei1 mutants actually encodes a protein of 972 amino acids that contains five BRCA1 C-terminus (BRCT) domains and is similar to proteins involved in the response to DNA damage and replication blocks in eukaryotes. During meiosis, recombination is initiated by the formation of DNA double strand breaks (DSBs) induced by the protein SPO11. We analysed meiotic chromosome behaviour of the mei1 mutant in a spo11 mutant background and proved that the meiotic fragmentation observed in mei1 mutants was not the consequence of defects in the repair of meiotic DSBs induced by SPO11. We also analysed the effect of mei1 on the mitotic cell cycle but could not detect any sensitivity of mei1 seedlings to DNA-damaging agents like gamma-rays or UV. Therefore, MEI1 is a BRCT-domain-containing protein that could be specific to the meiotic cell cycle and that plays a crucial role in some DNA repair events independent of SPO11 DSB recombination repair.     

Efficient incorporation of large (>2 kb) heterologies into heteroduplex DNA: Pms1/Msh2-dependent and -independent large loop mismatch repair in Saccharomyces cerevisiae

DNA double-strand break (DSB) repair in yeast is effected primarily by gene conversion. Conversion can conceivably result from gap repair or from mismatch repair of heteroduplex DNA (hDNA) in recombination intermediates. Mismatch repair is normally very efficient, but unrepaired mismatches segregate in the next cell division, producing sectored colonies. Conversion of small heterologies (single-base differences or insertions <15 bp) in meiosis and mitosis involves mismatch repair of hDNA. The repair of larger loop mismatches in plasmid substrates or arising by replication slippage is inefficient and/or independent of Pms1p/Msh2p-dependent mismatch repair. However, large insertions convert readily (without sectoring) during meiotic recombination, raising the question of whether large insertions convert by repair of large loop mismatches or by gap repair. We show that insertions of 2.2 and 2.6 kbp convert efficiently during DSB-induced mitotic recombination, primarily by Msh2p- and Pms1p-dependent repair of large loop mismatches. These results support models in which Rad51p readily incorporates large heterologies into hDNA. We also show that large heterologies convert more frequently than small heterologies located the same distance from an initiating DSB and propose that this reflects Msh2-independent large loop-specific mismatch repair biased toward loop loss.     

BRCA1-mediated repression of mutagenic end-joining of DNA double-strand breaks requires complex formation with BACH1

BACH1 (BRCA1-associated C-terminal helicase 1), the product of the BRIP1 {BRCA1 [breast cancer 1, early onset]-interacting protein C-terminal helicase 1; also known as FANCJ [FA-J (Fanconi anaemia group J) protein]} gene mutated in Fanconi anaemia patients from complementation group J, has been implicated in DNA repair and damage signalling. BACH1 exerts DNA helicase activities and physically interacts with BRCA1 and MLH1 (mutL homologue 1), which differentially control DNA DSB (double-strand break) repair processes. The present study shows that BACH1 plays a role in both HR (homologous recombination) and MMEJ (microhomology-mediated non-homologous end-joining) and reveals discrete mechanisms underlying modulation of these pathways. Our results indicate that BACH1 stimulates HR, which depends on the integrity of the helicase domain. Disruption of the BRCA1-BACH1 complex through mutation of BACH1 compromised errorfree NHEJ (non-homologous end-joining) and accelerated error-prone MMEJ. Conversely, molecular changes in BACH1 abrogating MLH1 binding interfered neither with HR nor with MMEJ. Importantly, MMEJ is a mutagenic DSB repair pathway, which is derepressed in hereditary breast and ovarian carcinomas. Since BRCA1 and BACH1 mutations targeting the BRCA1-BACH1 interaction have been associated with breast cancer susceptibility, the results of the present study thus provide evidence for a novel role of BACH1 in tumour suppression.     

Loss of CHD1 causes DNA repair defects and enhances prostate cancer therapeutic responsiveness

The CHD1 gene, encoding the chromo‐domain helicase DNA‐binding protein‐1, is one of the most frequently deleted genes in prostate cancer. Here, we examined the role of CHD1 in DNA double‐strand break (DSB) repair in prostate cancer cells. We show that CHD1 is required for the recruitment of CtIP to chromatin and subsequent end resection during DNA DSB repair. Our data support a role for CHD1 in opening the chromatin around the DSB to facilitate the recruitment of homologous recombination (HR) proteins. Consequently, depletion of CHD1 specifically affects HR‐mediated DNA repair but not non‐homologous end joining. Together, we provide evidence for a previously unknown role of CHD1 in DNA DSB repair via HR and show that CHD1 depletion sensitizes cells to PARP inhibitors, which has potential therapeutic relevance. Our findings suggest that CHD1 deletion, like BRCA1/2 mutation in ovarian cancer, may serve as a marker for prostate cancer patient stratification and the utilization of targeted therapies such as PARP inhibitors, which specifically target tumors with HR defects.