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Background Proper repair and restart of stressed replication forks requires intact homologous recombination (HR). HR at stressed replication forks can be initiated by the 5′ endonuclease EEPD1, which cleaves the stalled replication fork. Inherited or acquired defects in HR, such as mutations in breast cancer susceptibility protein-1 (BRCA1) or BRCA2, predispose to cancer, including breast and ovarian cancers. In order for these HR-deficient tumor cells to proliferate, they become addicted to a bypass replication fork repair pathway mediated by radiation repair protein 52 (RAD52). Depleting RAD52 can cause synthetic lethality in BRCA1/2 mutant cancers by an unknown molecular mechanism. Methods We hypothesized that cleavage of stressed replication forks by EEPD1 generates a fork repair intermediate that is toxic when HR-deficient cells cannot complete repair with the RAD52 bypass pathway. To test this hypothesis, we applied cell survival assays, immunofluorescence staining, DNA fiber and western blot analyses to look at the correlation between cell survival and genome integrity in control, EEPD1, RAD52 and EEPD1/RAD52 co-depletion BRCA1-deficient breast cancer cells. Results Our data show that depletion of EEPD1 suppresses synthetic lethality, genome instability, mitotic catastrophe, and hypersensitivity to stress of replication of RAD52-depleted, BRCA1 mutant breast cancer cells. Without HR and the RAD52-dependent backup pathway, the BRCA1 mutant cancer cells depleted of EEPD1 skew to the alternative non-homologous end-joining DNA repair pathway for survival. Conclusion This study indicates that the mechanism of synthetic lethality in RAD52-depleted BRCA1 mutant cancer cells depends on the endonuclease EEPD1. The data imply that EEPD1 cleavage of stressed replication forks may result in a toxic intermediate when replication fork repair cannot be completed. Electronic supplementary material The online version of this article (doi:10.1186/s13058-017-0912-8) contains supplementary material, which is available to authorized users.

The PHD finger protein 1 (PHF1) is essential in epigenetic regulation and genome maintenance. Here we show that the Tudor domain of human PHF1 binds to histone H3 trimethylated at Lys36 (H3K36me3). We report a 1.9-A resolution crystal structure of the Tudor domain in complex with H3K36me3 and describe the molecular mechanism of H3K36me3 recognition using NMR. Binding of PHF1 to H3K36me3 inhibits the ability of the Polycomb PRC2 complex to methylate Lys27 of histone H3 in vitro and in vivo. Laser microirradiation data show that PHF1 is transiently recruited to DNA double-strand breaks, and PHF1 mutants impaired in the H3K36me3 interaction exhibit reduced retention at double-strand break sites. Together, our findings suggest that PHF1 can mediate deposition of the repressive H3K27me3 mark and acts as a cofactor in early DNA-damage response.

DNA damage repair is crucial for the maintenance of genome integrity and cancer suppression. We found that loss of the mouse transcription factor YY1 resulted in polyploidy and chromatid aberrations, which are signatures of defects in homologous recombination. Further biochemical analyses identified a YY1 complex comprising components of the evolutionarily conserved INO80 chromatin-remodeling complex. Notably, RNA interference–mediated knockdown of YY1 and INO80 increased cellular sensitivity toward DNA-damaging agents. Functional assays revealed that both YY1 and INO80 are essential in homologous recombination–based DNA repair (HRR), which was further supported by the finding that YY1 preferentially bound a recombination-intermediate structure in vitro. Collectively, these observations reveal a link between YY1 and INO80 and roles for both in HRR, providing new insight into mechanisms that control the cellular response to genotoxic stress.

The repair of DNA double-strand breaks (DSBs) is critical for maintaining genome stability. Eukaryotic cells repair DSBs using both non-homologous end joining (NHEJ) and homologous recombination (HR). How chromatin structure is altered in response to DSBs and how such alterations influence DSB repair processes are important questions. In vertebrates, phosphorylation of the histone variant H2A.X (γ-H2A) occurs rapidly after formation of DSBs1, spreads over megabase chromatin domains, and is required for stable accumulation of DNA repair proteins at DNA damage foci2. In Saccharomyces cerevisiae, phosphorylation of the two major H2A species is also signaled by DSB formation, spreading ∼40 Kb in either direction from a DSB3. Here we show that near a DSB γ-H2A is followed by loss of histones H2B and H3 and increased sensitivity of chromatin to digestion by micrococcal nuclease. However, γ-H2A and nucleosome loss occur independently of one another. The DNA damage sensor MRX (Mre11-Rad50-Xrs2)4 is required for histone eviction, which additionally depends on the ATP-dependent nucleosome-remodeling complex, INO805. The repair protein Rad516 shows delayed recruitment to a DSB in the absence of histone loss, suggesting that MRX-dependent nucleosome remodeling regulates the accessibility of factors with direct roles in DNA damage repair by HR. We propose that MRX regulates two pathways of chromatin changes, including nucleosome displacement, required for efficient recruitment of HR proteins, and γ-H2A, which modulates checkpoint responses to DNA damage2.

Double-strand break (DSB)-induced gene conversion in yeast was studied in crosses between ura3 heteroalleles carrying phenotypically silent markers at approximately 100-bp intervals, which allow high-resolution analyses of tract structures. DSBs were introduced in vivo by HO nuclease at sites within shared homology and were repaired using information donated by unbroken alleles. Previous studies with these types of crosses showed that most tracts of Ura+ products are continuous, unidirectional, and extend away from frameshift mutations in donor alleles. Here we demonstrate that biased tract directionality is a consequence of selection pressure against Ura- products that results when frameshift mutations in donor alleles are transferred to recipient alleles. We also performed crosses in which frameshift mutations in recipient and donor alleles were arranged such that events initiated at DSBs could not convert broken alleles to Ura+ via a single gap repair event or a single long-tract mismatch repair event in heteroduplex DNA. This constraint led to low recombination frequencies relative to unconstrained crosses, and inhibited preferential conversion of broken alleles. Physical analysis of 51 DSB-induced products arising from multiple recombinational repair events suggested that hDNA formation is generally limiting, but that some hDNA regions may extend more than 600 bp. Among these products, markers separated by 20 bp were independently repaired about 40% of the time.

A simple and reproducible method for transferring low copy-number episomal plasmids from yeast to Escherichia coli has been developed. Although slightly more time-consuming than direct transfer methods, which are effective with high copy number plasmids, the method is significantly faster than methods that require purification of yeast DNA. Plasmid DNA is released from yeast cells during brief treatments involving grinding with glass beads and heating. The treated yeast are cooled, electrocompetent E. coli is added, the mixture is electroporated, and transformants are selected using standard conditions for E. coli electrotransformation. The procedure typically yields sufficient transformants for most applications.