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CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.

The MRE11-RAD50-NBS1 (MRN) complex is a central, multifunctional factor in the detection, signaling and nucleolytic processing of DNA double-strand breaks (DSBs). To clarify how human MRN binds generic and telomeric DNA ends and can separate DNA end sensing from nuclease activities, we determined cryo-electron microscopy (cryo-EM) structures of human MRN bound to DNA and to DNA and the telomere protection factor TRF2. MRN senses DSBs through a tight clamp-like sensing state with closed coiled-coil domains, but auto-inhibited MRE11 nuclease. NBS1 wraps around the MRE11 dimer, with NBS1’s ATM recruitment motif sequestered by binding to the regulatory RAD50 S site, necessitating a switch in the NBS1 C helix for ATM activation. At telomeric DNA, TRF2 blocks the second S site via the iDDR motif to prevent nuclease and ATM activation. Our results provide a structural framework for DNA sensing via a gating mechanism and separation of sensing, signaling and processing activities of mammalian MRN. The MRN complex plays a key role in detecting, signalling, and processing DNA double-strand breaks. This study reports the cryo-EM structures of human MRN bound to DNA, alone and with telomeric protection factor TRF2, revealing MRN’s structural transitions and regulatory mechanisms.

The MRE11–RAD50–NBS1 (MRN) protein complex plays a vital role in DNA double strand break sensing, signaling, and repair. Mutation in any component of this complex may lead to disease as disrupting DNA double strand break repair has the potential to cause translocations and loss of genomic information. Here, we have investigated an MRE11 mutation, F237C, identified in a breast cancer tumor. We found that the analogous mutant of Pyrococcus furiosus Mre11 diminishes both the exonuclease and endonuclease activities of Mre11 in vitro. Solution state NMR experiments show that this mutant causes structural changes in the DNA-bound Mre11 for both exo- and endonuclease substrates and causes the protein to become generally more rigid. Moreover, by comparing the NMR data for this cancer-associated mutant with two previously described Mre11 separation-of-nuclease function mutants, a potential allosteric network was detected within Mre11 that connects the active site to regions responsible for recognizing the DNA ends and for dimerization. Together, our data further highlight the dynamics required for Mre11 nuclease function and illuminate the presence of allostery within the enzyme.

Ectopic R-loop accumulation causes DNA replication stress and genome instability. To avoid these outcomes, cells possess a range of anti-R-loop mechanisms, including RNaseH that degrades the RNA moiety in R-loops. To comprehensively identify anti-R-loop mechanisms, we performed a genome-wide trigenic interaction screen in yeast lacking RNH1 and RNH201 . We identified >100 genes critical for fitness in the absence of RNaseH, which were enriched for DNA replication fork maintenance factors including the MRE11-RAD50-NBS1 (MRN) complex. While MRN has been shown to promote R-loops at DNA double-strand breaks, we show that it suppresses R-loops and associated DNA damage at transcription–replication conflicts. This occurs through a non-nucleolytic function of MRE11 that is important for R-loop suppression by the Fanconi Anemia pathway. This work establishes a novel role for MRE11-RAD50-NBS1 in directing tolerance mechanisms at transcription–replication conflicts. Accumulations of R-loops can lead to genome instability. Here the authors reveal a role for the MRN complex in suppressing R-loops and associated DNA damage at transcription–replication conflicts.

DNA double-strand breaks arise accidentally upon exposure of DNA to radiation and chemicals or result from faulty DNA metabolic processes. DNA breaks can also be introduced in a programmed manner, such as during the maturation of the immune system, meiosis, or cancer chemo- or radiotherapy. Cells have developed a variety of repair pathways, which are fine-tuned to the specific needs of a cell. Accordingly, vegetative cells employ mechanisms that restore the integrity of broken DNA with the highest efficiency at the lowest cost of mutagenesis. In contrast, meiotic cells or developing lymphocytes exploit DNA breakage to generate diversity. Here, we review the main pathways of eukaryotic DNA double-strand break repair with the focus on homologous recombination and its various subpathways. We highlight the differences between homologous recombination and end-joining mechanisms including non-homologous end-joining and microhomology-mediated end-joining and offer insights into how these pathways are regulated. Finally, we introduce noncanonical functions of the recombination proteins, in particular during DNA replication stress.

Oncogene-induced senescence (OIS) arrests cell proliferation in response to replication stress (RS) induced by oncogenes. OIS depends on the DNA damage response (DDR), but also on the cGAS-STING pathway, which detects cytosolic DNA and induces type I interferons (IFNs). Whether and how RS and IFN responses cooperate to promote OIS remains unknown. Here, we show that the induction of OIS by the H-RAS V12 oncogene in immortalized human fibroblasts depends on the MRE11 nuclease. Indeed, treatment with the MRE11 inhibitor Mirin prevented RS, micronuclei formation and IFN response induced by RAS V12 . Overexpression of the cytosolic nuclease TREX1 also prevented OIS. Conversely, overexpression of a dominant negative mutant of TREX1 or treatment with IFN-β was sufficient to induce RS and DNA damage, independent of RAS V12 induction. These data suggest that the IFN response acts as a positive feedback loop to amplify DDR in OIS through a process regulated by MRE11 and TREX1. Oncogene-induced senescence is a key tumor suppressor mechanism. Here, the authors show that replication stress induced by the RASV12 oncogene activates the cGAS-STING pathway, which in turn acts as a positive feedback loop to promote senescence.

The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3′-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing. Cas9 remains bound to DNA after cleavage and its removal is required for DNA double-strand break repair. Here, the authors show that the HLTF translocase disrupts the Cas9- DNA post-cleavage complexes in a process that requires the HLTF HIRAN domain and ATPase activity.

Nucleolytic resection of DNA ends is critical for homologous recombination, but its mechanism is not fully understood, particularly in mammalian meiosis. Here we examine roles of the conserved MRN complex (MRE11, RAD50, and NBS1) through genome-wide analysis of meiotic resection during spermatogenesis in mice with various MRN mutations, including several that cause chromosomal instability in humans. Meiotic DSBs form at elevated levels but remain unresected if Mre11 is conditionally deleted, thus MRN is required for both resection initiation and regulation of DSB numbers. Resection lengths are reduced to varying degrees in MRN hypomorphs or if MRE11 nuclease activity is attenuated in a conditional nuclease-dead Mre11 model. These findings unexpectedly establish that MRN is needed for longer-range extension of resection beyond that carried out by the orthologous proteins in budding yeast meiosis. Finally, resection defects are additively worsened by combining MRN and Exo1 mutations, and mice that are unable to initiate resection or have greatly curtailed resection lengths experience catastrophic spermatogenic failure. Our results elucidate MRN roles in meiotic DSB end processing and establish the importance of resection for mammalian meiosis. The DNA breaks that initiate recombination during meiosis are resected by nucleases, but the mechanism of this resection in mammals is poorly understood. Here, the authors examine the roles of the MRE11-RAD50- NBS1 complex in beginning and extending resection during meiosis in male mice.

Chronic hepatitis B virus (HBV) infection can significantly increase the incidence of cirrhosis and liver cancer, and there is no curative treatment. The persistence of HBV covalently closed circular DNA (cccDNA) is the major obstacle of antiviral treatments. cccDNA is formed through repairing viral partially double-stranded relaxed circular DNA (rcDNA) by varies host factors. However, the detailed mechanisms are not well characterized. To dissect the biogenesis of cccDNA, we took advantage of an in vitro rcDNA repair system to precipitate host factors interacting with rcDNA and identified co-precipitated proteins by mass spectrometry. Results revealed the MRE11–RAD50–NBS1 (MRN) complex as a potential factor. Transiently or stably knockdown of MRE11, RAD50 or NBS1 in hepatocytes before HBV infection significantly decreased viral markers, including cccDNA, while reconstitution reversed the effect. Chromatin immunoprecipitation assay further validated the interaction of MRN complex and HBV DNA. However, MRN knockdown after HBV infection showed no effect on viral replication, which indicated that MRN complex inhibited the formation of cccDNA without affecting its stability or transcriptional activity. Interestingly, Mirin, a MRN complex inhibitor which can inhibit the exonuclease activity of MRE11 and MRN-dependent activation of ATM, but not ATM kinase inhibitor KU55933, could decrease cccDNA level. Likewise, the MRE11 endonuclease activity inhibitor PFM01 treatment decreased cccDNA. MRE11 nuclease assays indicated that rcDNA is a substrate of MRE11. Furthermore, the inhibition of ATR-CHK1 pathway, which is known to be involved in cccDNA formation, impaired the effect of MRN complex on cccDNA. Similarly, inhibition of MRE11 endonuclease activity mitigated the effect of ATR-CHK1 pathway on cccDNA. These findings indicate that MRN complex cooperates with ATR-CHK1 pathway to regulate the formation of HBV cccDNA. In summary, we identified host factors, specifically the MRN complex, regulating cccDNA formation during HBV infection. These findings provide insights into how HBV hijacks host enzymes to establish chronic infection and reveal new therapeutic opportunities.

The preferential response to PARP inhibitors (PARPis) in BRCA-deficient and Schlafen 11 (SLFN11)-expressing ovarian cancers has been documented, yet the underlying molecular mechanisms remain unclear. As the accumulation of single-strand DNA (ssDNA) gaps behind replication forks is key for the lethality effect of PARPis, we investigated the combined effects of SLFN11 expression and BRCA deficiency on PARPi sensitivity and ssDNA gap formation in human cancer cells. PARPis increased chromatin-bound RPA2 and ssDNA gaps in SLFN11-expressing cells and even more in cells with BRCA1 or BRCA2 deficiency. SLFN11 was co-localized with chromatin-bound RPA2 under PARPis treatment, with enhanced recruitment in BRCA2-deficient cells. Notably, the chromatin-bound SLFN11 under PARPis did not block replication, contrary to its function under replication stress. SLFN11 recruitment was attenuated by the inactivation of MRE11. Hence, under PARPi treatment, MRE11 expression and BRCA deficiency lead to ssDNA gaps behind replication forks, where SLFN11 binds and increases their accumulation. As ovarian cancer patients who responded (progression-free survival >2 years) to olaparib maintenance therapy had a significantly higher SLFN11-positivity than short-responders (<6 months), our findings provide a mechanistic understanding of the favorable responses to PARPis in SLFN11-expressing and BRCA-deficient tumors. It highlight the clinical implications of SLFN11.