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.     

PIK-75 promotes homology-directed DNA repair

Homo!ogy-directed repair(HDR)is one of two major DNA repair pathways to mend the double-strand breaks(DSBs)formed in the genome(Liang et al.,1998;Pardo et al.,2009).Although less efficient compared with another DNA repair pathway,nonhomologous end joining(NHEJ),HDR is a type of precise repair to restore DNA damage and sustain genomic stability(Pardo et al.,2009;Ceccaldi et al.,2016).By contrast,NHEJ usually introduces mutations into the repaired site,thus probably harming the genomic integrity(Lieber et al.,2003).The error-free property enables HDR to be harnessed to correct a faulty mutation for therapeutic purpose in cells or in the body(Wu et al.,2013).In add让ion,HDR possesses great potential in the generation of genome-edited animals with precise genetic modifications,such as point mutation,DNA replacement,and DNA insertion in a specific genomic site(Wang et al.,2013).However,the low repair frequency mediated by HDR significantly limits让s application for efficient gene correction or establishment of various genetically modified animal models.Currently,multiple site-specific endonucleases have emerged as highly efficient tools to create targeted DSBs and markedly promote subsequent DNA repair either via HDR or NHEJ(Gaj et al.,2013).Nonetheless,the HDR-mediated modifications following the cleavage of engineering nucleases are still inefficient,usually with an efficiency less than 20%in cultured mammalian cells and embryos(Mali et al..2013;Wang et al.,2013;Yang et al.,2013).     

DNA damage, homology-directed repair, and DNA methylation

To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.     

Structural requirements for the BARD1 tumor suppressor in chromosomal stability and homology-directed DNA repair

The BRCA1 tumor suppressor exists as a heterodimeric complex with BARD1, and this complex is thought to mediate many of the functions ascribed to BRCA1, including its role in tumor suppression. The two proteins share a common structural organization that features an N-terminal RING domain and two C-terminal BRCT motifs, whereas BARD1 alone also contains three tandem ankyrin repeats. In normal cells, the BRCA1/BARD1 heterodimer is believed to enhance chromosome stability by promoting homology-directed repair (HDR) of double strand DNA breaks. Here we have investigated the structural requirements for BARD1 in this process by complementation of Bard1-null mouse mammary carcinoma cells. Our results demonstrate that the ankyrin and BRCT motifs of BARD1 are each essential for both chromosome stability and HDR. Tandem BRCT motifs, including those found at the C terminus of BARD1, are known to form a phosphoprotein recognition module. Nonetheless, the HDR function of BARD1 was not perturbed by synthetic mutations predicted to ablate the phospho-recognition activity of its BRCT sequences, suggesting that some functions of the BRCT domains are not dependent on their ability to bind phosphorylated ligands. Also, cancer-associated missense mutations in the BRCT domains of BARD1 (e.g. C557S, Q564H, V695L, and S761N) have been observed in patients with breast, ovarian, and endometrial tumors. However, none of these was found to affect the HDR activity of BARD1, suggesting that any increased cancer risk conferred by these mutations is not because of defects in this repair mechanism.     

Mammalian Rif1 contributes to replication stress survival and homology-directed repair

Rif1, originally recognized for its role at telomeres in budding yeast, has been implicated in a wide variety of cellular processes in mammals, including pluripotency of stem cells, response to double-strand breaks, and breast cancer development. As the molecular function of Rif1 is not known, we examined the consequences of Rif1 deficiency in mouse cells. Rif1 deficiency leads to failure in embryonic development, and conditional deletion of Rif1 from mouse embryo fibroblasts affects S-phase progression, rendering cells hypersensitive to replication poisons. Rif1 deficiency does not alter the activation of the DNA replication checkpoint but rather affects the execution of repair. RNA interference to human Rif1 decreases the efficiency of homology-directed repair (HDR), and Rif1 deficiency results in aberrant aggregates of the HDR factor Rad51. Consistent with a role in S-phase progression, Rif1 accumulates at stalled replication forks, preferentially around pericentromeric heterochromatin. Collectively, these findings reveal a function for Rif1 in the repair of stalled forks by facilitating HDR.     

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 (g-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 g-H2AX in the in vitro phosphatase assays. Depletion of PP6c or PP6R2 led to persistent high levels of g-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 g-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.     

BARD1 participates with BRCA1 in homology-directed repair of chromosome breaks

The BRCA1 tumor suppressor has been implicated in the maintenance of chromosomal stability through homology-directed repair of DNA double-strand breaks. Much of the BRCA1 in cells forms a heterodimeric complex with a structurally related protein BARD1. We report that expression of truncated mouse or human BARD1 peptides capable of interacting with Brca1 results in a homologous-repair deficiency. Repair is mildly reduced in Brca1 wild-type cells and severely reduced in cells that harbor a Brca1 splice product deleted for exon 11. Nuclear localization of the Brca1 or BARD1 peptides is not compromised, implying that the repair deficiency is caused by a more direct effect on repair. The tumor suppressor activity of BRCA1 may require the participation of BARD1 to maintain chromosome integrity through the homologous-repair pathway.     

ATP hydrolysis by mammalian RAD51 has a key role during homology-directed DNA repair

Disruption of the gene encoding RAD51, the protein that catalyzes strand exchange during homologous recombination, leads to the accumulation of chromosome breaks and lethality in vertebrate cells. As RAD51 is implicated in BRCA1- and BRCA2-mediated tumor suppression as well as cellular viability, we have begun a functional analysis of a defined RAD51 mutation in mammalian cells. By using a dominant negative approach, we generated a mouse embryonic stem cell line that expresses an ATP hydrolysis-defective RAD51 protein, hRAD51-K133R, at comparable levels to the endogenous wild-type RAD51 protein, whose expression is retained in these cells. We found that these cells have increased sensitivity to the DNA-damaging agents mitomycin C and ionizing radiation and also exhibit a decreased rate of spontaneous sister-chromatid exchange. By using a reporter for the repair of a single chromosomal double-strand break, we also found that expression of the hRAD51-K133R protein specifically inhibits homology-directed double-strand break repair. Furthermore, expression of a BRC repeat from BRCA2, a peptide inhibitor of an early step necessary for strand exchange, exacerbates the inhibition of homology-directed repair in the hRAD51-K133R expressing cell line. Thus, ATP hydrolysis by RAD51 has a key role in various types of DNA repair in mammalian cells.     

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     

The dystonia gene THAP1 controls DNA double-strand break repair choice

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Stimulation of homology-directed repair at I-SceI-induced DNA breaks during the permissive life cycle of human cytomegalovirus

Human cytomegalovirus (HCMV) selectively relocalizes many DNA repair proteins, thereby avoiding a potentially detrimental damage response. In the present study, we evaluated interactions between HCMV and the homology-directed repair (HDR) pathway. In permissive human foreskin fibroblasts, a fluorescence-based double-stranded break repair assay was used to determine that HCMV stimulated HDR. Repair of both stably integrated and extrachromosomal reporter substrates was observed to increase. HDR was also stimulated through individual expression of the viral immediate-early protein IE1-72, mimicking full virus infection. These experiments further demonstrate HCMV's role in modulating critical cellular processes during a permissive infection.     

p53 controls low DNA damage-dependent premeiotic checkpoint and facilitates DNA repair during spermatogenesis.

Previously, it was implicated that p53 plays a role in spermatogenesis. Here we report that p53 knockout mice exhibit significantly less mature motile spermatozoa than their p53(+/+) counterparts. To better understand the role of p53 in spermatogenesis, we analyzed the response of spermatogenic cells to DNA insult during prophase. It was found that although low-level gamma-irradiation activated a p53-dependent premeiotic delay, higher levels of gamma-irradiation induced a p53-independent apoptosis during meiosis. Furthermore, p53 knockout mice exhibited reduced in vivo levels of unscheduled DNA synthesis, indicative of compromised DNA repair. Thus, p53 provides another level of stringency in addition to other spermatogenic "quality control" mechanisms.     

DNA2 and EXO1 in replication-coupled,homology-directed repair and in the interplay between HDR and the FA/BRCA network

During DNA replication, stalled replication forks and DSBs arise when the replication fork encounters ICLs (interstrand crosslinks), covalent protein/DNA intermediates or other discontinuities in the template. Recently, homologous recombination proteins have been shown to function in replication-coupled repair of ICLs in conjunction with the Fanconi anemia (FA) regulatory factors FANCD2-FANCI, and, conversely, the FA gene products have been shown to play roles in stalled replication fork rescue even in the absence of ICLs, suggesting a broader role for the FA network than previously appreciated. Here we show that DNA2 helicase/nuclease participates in resection during replication-coupled repair of ICLs and other replication fork stresses. DNA2 knockdowns are deficient in HDR (homology-directed repair) and the S phase checkpoint and exhibit genome instability and sensitivity to agents that cause replication stress. DNA2 is partially redundant with EXO1 in these roles. DNA2 interacts with FANCD2, and cisplatin induces FANCD2 ubiquitylation even in the absence of DNA2. DNA2 and EXO1 deficiency leads to ICL sensitivity but does not increase ICL sensitivity in the absence of FANCD2. This is the first demonstration of the redundancy of human resection nucleases in the HDR step in replication-coupled repair, and suggests that DNA2 may represent a new mediator of the interplay between HDR and the FA/BRCA pathway.     

Brca1 and differentiation

Breast cancer is one of the most frequent malignancies affecting women. The human breast cancer gene 1 (BRCA1) gene is mutated in a distinct proportion of hereditary breast and ovarian cancers. Tumourigenesis in individuals with germline BRCA1 mutations requires somatic inactivation of the remaining wild-type allelle. Although, this evidence supports a role for BRCA1 as a tumour suppressor, the mechanisms through which its loss leads to tumourigenesis remain to be determined. Neither the expression pattern nor the described functions of human BRCA1 and murine breast cancer gene 1 (Brca1) can explain the specific association of mutations in this gene with the development of breast and ovarian cancer. Investigation of the role of Brca1 in normal cell differentiation processes might provide the basis to understand the tissue-restricted properties.