BRCA1-deficient tumors are hypersensitive to PARP inhibitors (PARPi) due to impaired homologous recombination (HR). The 53BP1-Shieldin complex inhibits end resection at replication-independent DNA double-strand breaks (DSBs), promoting PARPi sensitivity in BRCA1-deficient cells. However, its role in replication-coupled DSBs is less clear. Here, we show that loss of Shld2 or Shld3, but not 53bp1, confers PARPi resistance in mouse embryonic stem cells lacking a functional BRCA1 BRCT domain. Unlike 53bp1 loss, deletion of Shld2 or Shld3 partially restores HR at replication-coupled DSBs, reduces BRCA1-linked insertion/deletion signatures, and promotes RAD51 loading. This 53BP1-independent function requires the CST complex and counteracts residual BRCA1 coiled-coil domain activity in RAD51 loading. Shld2 loss also confers PARPi resistance in Bard1-null cells retaining residual BRCA1, but not in cells expressing RING-less BRCA1 with degraded BARD1. These findings identify a 53BP1-independent

Genome editing using transcription-activator like effector nucleases or RNA guided nucleases allows one to precisely engineer desired changes within a given target sequence. The genome editing reagents introduce double stranded breaks (DSBs) at the target site which can then undergo DNA repair by non-homologous end joining (NHEJ) or homology directed recombination (HDR) when a template DNA molecule is available. NHEJ repair results in indel mutations at the target site. As PCR amplified products from mutant target regions are likely to exhibit different melting profiles than PCR products amplified from wild type target region, we designed a high resolution melting analysis (HRMA) for rapid identification of efficient genome editing reagents. We also designed TaqMan assays using probes situated across the cut site to discriminate wild type from mutant sequences present after genome editing. The experiments revealed that the sensitivity of the assays to detect NHEJ-mediated DNA repair co

Author summary DNA double-strand breaks (DSBs) pose a serious threat to genomic integrity, and their accurate repair is essential for cell survival. In bacteria, DSB repair largely depends on homologous recombination mediated by the RecA recombinase and supported by the SMC-like protein RecN. Although RecA and RecN are known to cooperate, how their interaction contributes to efficient repair has remained unclear. Here, we investigated the functional relationship between RecA and RecN by expressing proteins from Pseudomonas aeruginosa in Escherichia coli. We found that P. aeruginosa RecN (paRecN) exhibits limited activity in E. coli but regains full functionality when co-expressed with its cognate RecA, revealing strong species-specific compatibility. Furthermore, we identified gain-of-function paRecN mutants that restore compatibility with E. coli RecA while losing compatibility with their native partner. These findings demonstrate that the RecA–RecN interaction interface is shaped by species-specific coevolution. Our study provides mechanistic and evolutionary insights into how protein–protein interactions involved in genome maintenance are optimized within each species.

In meiosis, homologous recombination (HR) facilitates the halving of genomic content in diploid parents to produce haploid gametes, while also reshuffling genetic material. However, studies in yeast and mammals have suggested that HR during meiosis also makes the process mutagenic, and it has been proposed that the single-strand (ss) DNA formed by HR of programmed double-strand breaks (DSBs) contributes to the observed mutagenesis. To determine the full mutagenic potential of ssDNA formed during meiosis, we expressed human APOBEC3A (A3A), a deaminase that specifically attacks and therefore allows detection of ssDNA, in meiotic yeast cells. We demonstrate that meiotic cells accumulate long tracts of ssDNA manifested by A3A-mutation clusters, of up to 134 mutations spanning more than 25 kb. We show that the formation of mutation clusters during meiosis require Spo11-induced DSBs, and that break-induced replication and hyper-resection of DSBs are the primary mechanisms underlying the form

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Here we exploit long sequencing reads from sperm samples to study recombination processes in the male germline. In particular, the very high accuracy of Pacific Biosciences CCS base calls enables us to confidently identify NCO events, as recently done across primates26, and extend beyond previous genome-wide sperm sequencing studies that were limited to CO events8,21,25. Even in sperm, we expect to see the results of mitotic as well as meiotic recombination processes, arising from repair of DSBs occurring between the zygote and meiosis. Our observation that a substantial fraction of sperm NCOs are located away from meiotic CO hotspots, and that these are associated with fragile sites, as are the corresponding events seen in blood, is consistent with a significant fraction of sperm NCOs arising from pre-meiotic repair (Fig. 4). This is confirmed in paternal NCO events from pedigree data, and supported by a relative increase in this component in reads classified as germline from testis tissues26, which include reads from pre-meiotic cells. We do not identify what mechanistically gives rise to these events, but note that they are consistent in principle with gene conversion during recombination-mediated DSB repair, and also that if this is the source it must be...

DNA double-strand breaks function as promoter-like elements that drive transcription and protein synthesis of polyadenylated RNAs from otherwise silent DNA regions.

Organisms rely on coordinated networks of DNA repair pathways to protect genomes against toxic double-strand breaks (DSBs), particularly in germ cells. All repair mechanisms must successfully negotiat...

DNA double-strand breaks (DSBs) generated during meiotic prophase by the topoisomerase-like protein SPO11 are essential to create crossovers between homologous chromosomes. Since crossovers are requir...