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Encounters between replication forks and unrepaired DNA single-strand breaks (SSBs) can generate both single-ended and double-ended double-strand breaks (seDSBs and deDSBs). seDSBs can be repaired by break-induced replication (BIR), which is a highly mutagenic pathway that is thought to be responsible for many of the mutations and genome rearrangements that drive cancer development. However, the frequency of BIR's deployment and its ability to be triggered by both leading and lagging template strand SSBs were unclear. Using site- and strand-specific SSBs generated by nicking enzymes, including CRISPR-Cas9 nickase (Cas9n), we demonstrate that leading and lagging template strand SSBs in fission yeast are typically converted into deDSBs that are repaired by homologous recombination. However, both types of SSBs can also trigger BIR, and the frequency of these events increases when fork convergence is delayed and the non-homologous end joining protein Ku70 is deleted., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Reprogramming to pluripotency is associated with DNA damage and requires the functions of the BRCA1 tumor suppressor. Here, we leverage separation-of-function mutations in BRCA1/2 as well as the physical and/or genetic interactions between BRCA1 and its associated repair proteins to ascertain the relevance of homology-directed repair (HDR), stalled fork protection (SFP), and replication gap suppression (RGS) in somatic cell reprogramming. Surprisingly, loss of SFP and RGS is inconsequential for the transition to pluripotency. In contrast, cells deficient in HDR, but proficient in SFP and RGS, reprogram with reduced efficiency. Conversely, the restoration of HDR function through inactivation of 53bp1 rescues reprogramming in Brca1-deficient cells, and 53bp1 loss leads to elevated HDR and enhanced reprogramming in mouse and human cells. These results demonstrate that somatic cell reprogramming is especially dependent on repair of replication-associated double-strand breaks (DSBs) by the HDR activity of BRCA1 and BRCA2 and can be improved in the absence of 53BP1., Competing Interests: Declaration of interests D.G. and D.E. have applied for a patent on interfering with 53BP1 during somatic cell reprogramming., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

DNA double-strand breaks (DSBs) pose a constant threat to genomic integrity. Such DSBs need to be repaired to preserve homeostasis at both the cellular and organismal levels. Hence, the DNA damage response (DDR) has evolved to repair these lesions and limit their toxicity. The initiation of DNA repair depends on the activation of the DDR, and we know that the strength of DDR signaling may differentially affect cellular viability. However, we do not fully understand what determines the cytotoxicity of a DSB. Recent work has identified genomic location, (in)correct DNA repair pathway usage, and cell-cycle position as contributors to DSB-induced cytotoxicity. In this review, we discuss how these determinants affect cytotoxicity, highlight recent discoveries, and identify open questions that could help to improve our understanding about cell fate decisions after a DNA DSB. Krenning et al. give an overview of determinants that affect the cellular toxicity of DNA double-strand breaks and discuss how these determinants affect cytotoxicity. The authors highlight recent discoveries and identify open questions that could help to improve our understanding about cell fate decisions after a DNA double-strand break. [ABSTRACT FROM AUTHOR]

MYC oncoproteins are key drivers of tumorigenesis. As transcription factors, MYC proteins regulate transcription by all three nuclear polymerases and gene expression. Accumulating evidence shows that MYC proteins are also crucial for enhancing the stress resilience of transcription. MYC proteins relieve torsional stress caused by active transcription, prevent collisions between the transcription and replication machineries, resolve R-loops, and repair DNA damage by participating in a range of protein complexes and forming multimeric structures at sites of genomic instability. We review the key complexes and multimerization properties of MYC proteins that allow them to mitigate transcription-associated DNA damage, and propose that the oncogenic functions of MYC extend beyond the modulation of gene expression., Competing Interests: Declaration of interests M.E. is the founder and a stockholder of Tucana Biosciences., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Human cleavage-stage embryos frequently acquire chromosomal aneuploidies during mitosis due to unknown mechanisms. Here, we show that S phase at the 1-cell stage shows replication fork stalling, low fork speed, and DNA synthesis extending into G2 phase. DNA damage foci consistent with collapsed replication forks, DSBs, and incomplete replication form in G2 in an ATR- and MRE11-dependent manner, followed by spontaneous chromosome breakage and segmental aneuploidies. Entry into mitosis with incomplete replication results in chromosome breakage, whole and segmental chromosome errors, micronucleation, chromosome fragmentation, and poor embryo quality. Sites of spontaneous chromosome breakage are concordant with sites of DNA synthesis in G2 phase, locating to gene-poor regions with long neural genes, which are transcriptionally silent at this stage of development. Thus, DNA replication stress in mammalian preimplantation embryos predisposes gene-poor regions to fragility, and in particular in the human embryo, to the formation of aneuploidies, impairing developmental potential. [Display omitted] • 1-cell embryos show replication fork stalling, with replication extending into G2 phase • Incompletely replicated DNA is converted to chromosome breaks and aneuploidy in mitosis • Spontaneous chromosome breaks and G2 DNA synthesis occur in congruent gene-poor regions • Chromosome fragility in human embryos occurs independently of embryonic genome activation In human preimplantation embryos, DNA replication in G2 phase results in chromosome breakage, segmental aneuploidies, and poor embryo quality. [ABSTRACT FROM AUTHOR]

Previously, we showed that CDYL1 is recruited to DNA double-strand breaks (DSBs) to promote homologous recombination (HR) repair and foster transcriptional silencing. However, how CDYL1 elicits DSB-induced silencing is not fully understood. Here, we identify a CDYL1-dependent local decrease in the transcriptionally active marks histone lysine crotonylation (Kcr) and crotonylated lysine 9 of H3 (H3K9cr) at Asi SI-induced DSBs, which correlates with transcriptional silencing. Mechanistically, we reveal that CDYL1 crotonyl-CoA hydratase activity counteracts Kcr and H3K9cr at DSB sites, which triggers the eviction of the transcription elongation factor ENL and fosters transcriptional silencing. Furthermore, genetic inhibition of CDYL1 hydratase activity blocks the reduction in H3K9cr and alleviates DSB-induced silencing, whereas HR efficiency unexpectedly remains intact. Therefore, our results functionally uncouple the repair and silencing activity of CDYL1 at DSBs. In a broader context, we address a long-standing question concerning the functional relationship between HR repair and DSB-induced silencing, suggesting that they may occur independently. [Display omitted] • Local decrease in Kcr at DSBs correlates with transcriptional silencing • CDYL1 crotonyl-CoA hydratase activity downregulates Kcr at DSBs • Kcr reduction at DSBs promotes ENL eviction and fosters transcriptional silencing • CDYL1 roles in DSB-induced silencing and HR repair are functionally uncoupled Abu-Zhaiya et al. identify a CDYL1-dependent decrease in lysine crotonylation at DSB sites that fosters transcriptional silencing. It is widely accepted that DSB-induced silencing is prerequisite for timely DSB repair. Here, the authors challenge the current dogma and provide evidence that functionally uncouple DSB-induced silencing and repair, suggesting that they occur independently. [ABSTRACT FROM AUTHOR]

Immunoglobulin heavy chain (IgH) locus-associated G-rich long noncoding RNA (SμGLT) is important for physiological and pathological B cell DNA recombination. We demonstrate that the METTL3 enzyme-catalyzed N6-methyladenosine (m6A) RNA modification drives recognition and 3′ end processing of SμGLT by the RNA exosome, promoting class switch recombination (CSR) and suppressing chromosomal translocations. The recognition is driven by interaction of the MPP6 adaptor protein with nuclear m6A reader YTHDC1. MPP6 and YTHDC1 promote CSR by recruiting AID and the RNA exosome to actively transcribe SμGLT. Direct suppression of m6A modification of SμGLT or of m6A reader YTHDC1 reduces CSR. Moreover, METTL3, an essential gene for B cell development in the bone marrow and germinal center, suppresses IgH-associated aberrant DNA breaks and prevents genomic instability. Taken together, we propose coordinated and central roles for MPP6, m6A modification, and m6A reader proteins in controlling long noncoding RNA processing, DNA recombination, and development in B cells. [Display omitted] • N6-methyladenosine (m6A) guides IgH lncRNA SμGLT 3′ end processing by RNA exosome • M6A RNA modification is important for IgH class switch recombination • M6A RNA modification is important for genomic stability of B cells • YTHDC1 and MPP6 guide m6A recognition of G-rich SμGLT B-lymphocytes generate a vast number of antibodies to neutralize pathogenic insults. Somatic mutations of antibody genes are important for antibody diversity but also cause lymphomagenesis. Here, Nair et al. demonstrate that N6-methyladenosine modification of the SμGLT long noncoding RNA expressed from antibody gene locus drives somatic mutations but suppresses lymphomagenic genomic alterations. [ABSTRACT FROM AUTHOR]

Single-stranded DNA (ssDNA) arising as an intermediate of cellular processes on DNA is a potential vulnerability of the genome unless it is appropriately protected. Recent evidence suggests that R-loops, consisting of ssDNA and DNA-RNA hybrids, can form in the proximity of DNA double-strand breaks (DSBs) within transcriptionally active regions. However, how the vulnerability of ssDNA in R-loops is overcome during DSB repair remains unclear. Here, we identify RAP80 as a factor suppressing the vulnerability of ssDNA in R-loops, chromosome translocations, and deletions during DSB repair. Mechanistically, RAP80 prevents unscheduled nucleolytic processing of ssDNA in R-loops by CtIP. This mechanism promotes efficient DSB repair via transcription-associated end joining dependent on BRCA1, Polθ, and LIG1/3. Thus, RAP80 suppresses the vulnerability of R-loops during DSB repair, thereby precluding genomic abnormalities in a critical component of the genome caused by deleterious R-loop processing., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Homologous recombination is essential to maintain genome stability in response to DNA damage. Here, we have used genome-wide sequencing to quantitatively analyze at nucleotide resolution the dynamics of DNA end resection, re-synthesis, and gene conversion at a double-strand break. Resection initiates asymmetrically in an MRX-independent manner before proceeding steadily in both directions. Sgs1, Exo1, Rad51, and Srs2 differently regulate the rate and symmetry of early and late resection. Exo1 also ensures the coexistence of resection and re-synthesis, while Srs2 guarantees a constant and symmetrical DNA re-polymerization. Gene conversion is MMR independent, spans only a minor fraction of the resected region, and its unidirectionality depends on Srs2. Finally, these repair factors prevent the development of alterations remote from the DNA lesion, such as subtelomeric instability, duplication of genomic regions, and over-replication of Ty elements. Altogether, this approach allows a quantitative analysis and a direct genome-wide visualization of DNA repair by homologous recombination., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

DNA damage impedes replication fork progression and threatens genome stability. Upon encounter with most DNA adducts, the replicative CMG helicase (CDC45-MCM2-7-GINS) stalls or uncouples from the point of synthesis, yet eventually resumes replication. However, little is known about the effect on replication of single-strand breaks or "nicks," which are abundant in mammalian cells. Using Xenopus egg extracts, we reveal that CMG collision with a nick in the leading strand template generates a blunt-ended double-strand break (DSB). Moreover, CMG, which encircles the leading strand template, "runs off" the end of the DSB. In contrast, CMG collision with a lagging strand nick generates a broken end with a single-stranded overhang. In this setting, CMG translocates along double-stranded DNA beyond the break and is then ubiquitylated and removed from chromatin by the same pathway used during replication termination. Our results show that nicks are uniquely dangerous DNA lesions that invariably cause replisome disassembly, and they suggest that CMG cannot be stored on dsDNA while cells resolve replication stress. [Display omitted] • The structures of leading and lagging strand collapsed forks are different • CMG passively "runs off" the broken DNA end during leading strand fork collapse • CMG removal from duplex DNA after lag collapse depends on CRL2Lrr1 and p97 • Nicks are uniquely toxic lesions that cause fork collapse and replisome disassembly Vrtis et al. show that when a replisome encounters a single-strand DNA break in the leading or lagging strand template, the replication fork collapses, generating a double-strand break. Collapse is accompanied by loss of the replicative helicase from DNA, implying that fork restart would require recruitment of a new helicase. [ABSTRACT FROM AUTHOR]

The RAD51 recombinase forms nucleoprotein filaments to promote double-strand break repair, replication fork reversal, and fork stabilization. The stability of these filaments is highly regulated, as both too little and too much RAD51 activity can cause genome instability. RADX is a single-strand DNA (ssDNA) binding protein that regulates DNA replication. Here, we define its mechanism of action. We find that RADX inhibits RAD51 strand exchange and D-loop formation activities. RADX directly and selectively interacts with ATP-bound RAD51, stimulates ATP hydrolysis, and destabilizes RAD51 nucleofilaments. The RADX interaction with RAD51, in addition to its ssDNA binding capability, is required to maintain replication fork elongation rates and fork stability. Furthermore, BRCA2 can overcome the RADX-dependent RAD51 inhibition. Thus, RADX functions in opposition to BRCA2 in regulating RAD51 nucleofilament stability to ensure the right level of RAD51 function during DNA replication. • RADX interacts directly with ATP-bound RAD51 • RADX antagonizes RAD51 functions by destabilizing RAD51 nucleofilaments • Interaction between RADX and RAD51 is required to maintain fork elongation rates • BRC motifs of BRCA2 neutralize RADX inhibition of RAD51 Adolph et al. identify a direct interaction between RADX and ATP-bound RAD51. The interaction and RADX ssDNA binding combine to destabilize RAD51 nucleofilaments. This activity prevents inappropriate RAD51 action at elongating replication forks to maintain fork speed and stability. [ABSTRACT FROM AUTHOR]

Crossing over is essential for chromosome segregation during meiosis. Protein modification by SUMO is implicated in crossover control, but pertinent targets have remained elusive. Here we identify Msh4 as a target of SUMO-mediated crossover regulation. Msh4 and Msh5 constitute the MutSγ complex, which stabilizes joint-molecule (JM) recombination intermediates and facilitates their resolution into crossovers. Msh4 SUMOylation enhances these processes to ensure that each chromosome pair acquires at least one crossover. Msh4 is directly targeted by E2 conjugase Ubc9, initially becoming mono-SUMOylated in response to DNA double-strand breaks, then multi/poly-SUMOylated forms arise as homologs fully engage. Mechanistically, SUMOylation fosters interaction between Msh4 and Msh5. We infer that initial SUMOylation of Msh4 enhances assembly of MutSγ in anticipation of JM formation, while secondary SUMOylation may promote downstream functions. Regulation of Msh4 by SUMO is distinct and independent of its previously described stabilization by phosphorylation, defining MutSγ as a hub for crossover control., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

To address how genetic variation alters gene expression in complex cell mixtures, we developed direct nuclear tagmentation and RNA sequencing (DNTR-seq), which enables whole-genome and mRNA sequencing jointly in single cells. DNTR-seq readily identified minor subclones within leukemia patients. In a large-scale DNA damage screen, DNTR-seq was used to detect regions under purifying selection and identified genes where mRNA abundance was resistant to copy-number alteration, suggesting strong genetic compensation. mRNA sequencing (mRNA-seq) quality equals RNA-only methods, and the low positional bias of genomic libraries allowed detection of sub-megabase aberrations at ultra-low coverage. Each cell library is individually addressable and can be re-sequenced at increased depth, allowing multi-tiered study designs. Additionally, the direct tagmentation protocol enables coverage-independent estimation of ploidy, which can be used to identify cell singlets. Thus, DNTR-seq directly links each cell's state to its corresponding genome at scale, enabling routine analysis of heterogeneous tumors and other complex tissues. • Scalable and flexible format for joint mRNA and whole-genome sequencing in single cells • RNA and DNA library quality equals single-modality methods • Applied to primary tissue and thousands of artificially altered genomes • Highlights essential genomic regions and transcripts resistant to copy-number change Precise measurements of genotype and phenotype in single cells are key in understanding complex tissues and clonal disease. Zachariadis et al. present a method for joint genomic and mRNA sequencing in single cells at scale and demonstrate its application to tissues and artificially altered genomes. [ABSTRACT FROM AUTHOR]

To determine whether double-strand break (DSB) mobility enhances the physical search for an ectopic template during homology-directed repair (HDR), we tested the effects of factors that control chromatin dynamics, including cohesin loading and kinetochore anchoring. The former but not the latter is altered in response to DSBs. Loss of the nonhistone high-mobility group protein Nhp6 reduces histone occupancy and increases chromatin movement, decompaction, and ectopic HDR. The loss of nucleosome remodeler INO80-C did the opposite. To see whether enhanced HDR depends on DSB mobility or the global chromatin response, we tested the ubiquitin ligase mutant uls1Δ , which selectively impairs local but not global movement in response to a DSB. Strand invasion occurs in uls1Δ cells with wild-type kinetics, arguing that global histone depletion rather than DSB movement is rate limiting for HDR. Impaired break movement in uls1Δ correlates with elevated MRX and cohesin loading, despite normal resection and checkpoint activation. • Intrinsic and break-induced chromatin dynamics show cell-cycle dependence • Uls1 STUbL regulates local DSB dynamics through MRX and cohesin turnover • Chromatin expansion and ectopic movement are critical for DSB repair by HR • Loss of centromere tethering is not a major DNA damage response Cheblal et al. show that DNA damage checkpoint-induced histone depletion enhances the homology search for a resected chromosome break for its homologous donor. This is achieved through chromatin expansion and ectopic locus mobility, but it is independent of local DSB movement. Local DSB dynamics are cell-cycle dependent and are regulated by cohesin turnover at the break, under control of a SUMO-dependent ubiquitin ligase. [ABSTRACT FROM AUTHOR]

Rad52 is a key factor for homologous recombination (HR) in yeast. Rad52 helps assemble Rad51-ssDNA nucleoprotein filaments that catalyze DNA strand exchange, and it mediates single-strand DNA annealing. We find that Rad52 has an even earlier function in HR in restricting DNA double-stranded break ends resection that generates 3′ single-stranded DNA (ssDNA) tails. In fission yeast, Exo1 is the primary resection nuclease, with the helicase Rqh1 playing a minor role. We demonstrate that the choice of two extensive resection pathways is regulated by Rad52. In rad52 cells, the resection rate increases from ∼3–5 kb/h up to ∼10–20 kb/h in an Rqh1-dependent manner, while Exo1 becomes dispensable. Budding yeast Rad52 similarly inhibits Sgs1-dependent resection. Single-molecule analysis with purified budding yeast proteins shows that Rad52 competes with Sgs1 for DNA end binding and inhibits Sgs1 translocation along DNA. These results identify a role for Rad52 in limiting ssDNA generated by end resection. • Rad52 attenuates resection of DNA double-strand break (DSB) ends • Rad52 prevents hyper-resection by Rqh1 (S. pombe) or Sgs1 (S. cerevisiae) • Rad52 interferes with Sgs1 and Dna2-mediated resection in vitro • Rad52 inhibits Sgs1 loading at DSB ends and its translocation along DNA The ends of DNA breaks are processed to generate single-stranded DNA (ssDNA) in preparation for break repair. Excessive ssDNA may lead to genome instability. Yan et al. find that the DNA repair protein Rad52 restricts the length of ssDNA by limiting access and translocation of RecQ helicases on DNA. [ABSTRACT FROM AUTHOR]

Conventional methods for single-cell genome sequencing are limited with respect to uniformity and throughput. Here, we describe sci-L3, a single-cell sequencing method that combines combinatorial indexing (sci-) and linear (L) amplification. The sci-L3 method adopts a 3-level (3) indexing scheme that minimizes amplification biases while enabling exponential gains in throughput. We demonstrate the generalizability of sci-L3 with proof-of-concept demonstrations of single-cell whole-genome sequencing (sci-L3-WGS), targeted sequencing (sci-L3-target-seq), and a co-assay of the genome and transcriptome (sci-L3-RNA/DNA). We apply sci-L3-WGS to profile the genomes of >10,000 sperm and sperm precursors from F1 hybrid mice, mapping 86,786 crossovers and characterizing rare chromosome mis-segregation events in meiosis, including instances of whole-genome equational chromosome segregation. We anticipate that sci-L3 assays can be applied to fully characterize recombination landscapes, to couple CRISPR perturbations and measurements of genome stability, and to other goals requiring high-throughput, high-coverage single-cell sequencing. • Flexible method for single-cell whole genomes, targeted sequencing, and RNA/DNA co-assay • Potential throughput of 1 million cells by 3-level single-cell combinatorial indexing • Linear amplification by in vitro transcription for uniform coverage of the genome • Discovery of whole-genome equational chromosome segregation in mouse meiosis I Yin et al. developed sci-L3, which combines a scalable single-cell barcoding scheme with linear amplification. The method flexibly enables single-cell whole-genome sequencing, targeted DNA sequencing, or concurrent profiling of the genome and transcriptome. With sci-L3, the authors discovered mitotic-like whole-genome chromosome segregation in male mouse meiosis I. [ABSTRACT FROM AUTHOR]

Genetic recombination in all kingdoms of life initiates when helicases and nucleases process (resect) the free DNA ends to expose single-stranded DNA (ssDNA) overhangs. Resection regulation in bacteria is programmed by a DNA sequence, but a general mechanism limiting resection in eukaryotes has remained elusive. Using single-molecule imaging of reconstituted human DNA repair factors, we identify phosphorylated RPA (pRPA) as a negative resection regulator. Bloom's syndrome (BLM) helicase together with exonuclease 1 (EXO1) and DNA2 nucleases catalyze kilobase-length DNA resection on nucleosome-coated DNA. The resulting ssDNA is rapidly bound by RPA, which further stimulates DNA resection. RPA is phosphorylated during resection as part of the DNA damage response (DDR). Remarkably, pRPA inhibits DNA resection in cellular assays and in vitro via inhibition of BLM helicase. pRPA suppresses BLM initiation at DNA ends and promotes the intrinsic helicase strand-switching activity. These findings establish that pRPA provides a feedback loop between DNA resection and the DDR. • Phosphorylated RPA (pRPA) inhibits DNA resection in vitro and in cells • RPA stimulates BLM initiation at DNA ends and inhibits strand-switching activity • BLM helicase stimulates EXO1/DNA2 resection past nucleosomes pRPA stalls DNA resection at nucleosomes Termination of DNA resection is critical for preserving genome stability during DNA break repair. Soniat et al. report that phosphorylated RPA is a negative regulator of BLM helicase and DNA resection. As phosphorylated RPA accumulates during resection, this work establishes a negative feedback loop between resection and its termination. [ABSTRACT FROM AUTHOR]

Although double-strand break (DSB) repair is essential for a cell's survival, little is known about how DSB repair mechanisms are affected by age. Here we characterize the impact of cellular aging on the efficiency of single-strand annealing (SSA), a DSB repair mechanism. We measure SSA repair efficiency in young and old yeast cells and report a 23.4% decline in repair efficiency. This decline is not due to increased use of non-homologous end joining. Instead, we identify increased G1 phase duration in old cells as a factor responsible for the decreased SSA repair efficiency. Expression of 3xCLN2 leads to higher SSA repair efficiency in old cells compared with expression of 1xCLN2, confirming the involvement of cell-cycle regulation in age-associated repair inefficiency. Examining how SSA repair efficiency is affected by sequence heterology, we find that heteroduplex rejection remains high in old cells. Our work provides insights into the links between single-cell aging and DSB repair efficiency., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Summary DNA double-strand break (DSB) repair is essential for maintaining our genomes. Mre11-Rad50-Nbs1 (MRN) and Ku70-Ku80 (Ku) direct distinct DSB repair pathways, but the interplay between these complexes at a DSB remains unclear. Here, we use high-throughput single-molecule microscopy to show that MRN searches for free DNA ends by one-dimensional facilitated diffusion, even on nucleosome-coated DNA. Rad50 binds homoduplex DNA and promotes facilitated diffusion, whereas Mre11 is required for DNA end recognition and nuclease activities. MRN gains access to occluded DNA ends by removing Ku or other DNA adducts via an Mre11-dependent nucleolytic reaction. Next, MRN loads exonuclease 1 (Exo1) onto the free DNA ends to initiate DNA resection. In the presence of replication protein A (RPA), MRN acts as a processivity factor for Exo1, retaining the exonuclease on DNA for long-range resection. Our results provide a mechanism for how MRN promotes homologous recombination on nucleosome-coated DNA. [ABSTRACT FROM AUTHOR]

Summary RAD51 promotes homology-directed repair (HDR), replication fork reversal, and stalled fork protection. Defects in these functions cause genomic instability and tumorigenesis but also generate hypersensitivity to cancer therapeutics. Here we describe the identification of RADX as an RPA-like, single-strand DNA binding protein. RADX is recruited to replication forks, where it prevents fork collapse by regulating RAD51. When RADX is inactivated, excessive RAD51 activity slows replication elongation and causes double-strand breaks. In cancer cells lacking BRCA2, RADX deletion restores fork protection without restoring HDR. Furthermore, RADX inactivation confers chemotherapy and PARP inhibitor resistance to cancer cells with reduced BRCA2/RAD51 pathway function. By antagonizing RAD51 at forks, RADX allows cells to maintain a high capacity for HDR while ensuring that replication functions of RAD51 are properly regulated. Thus, RADX is essential to achieve the proper balance of RAD51 activity to maintain genome stability. [ABSTRACT FROM AUTHOR]

Trinucleotide repeat expansions involving CTG/CAG triplets are responsible for several neurodegenerative disorders, including myotonic dystrophy and Huntington's disease. Because expansions trigger the disease, contracting repeat length could be a possible approach to gene therapy for these disorders. Here, we show that a TALEN-induced double-strand break was very efficient at contracting expanded CTG repeats in yeast. We show that RAD51, POL32, and DNL4 are dispensable for double-strand break repair within CTG repeats, the only required genes being RAD50, SAE2, and RAD52. Resection was totally abolished in the absence of RAD50 on both sides of the break, whereas it was reduced in a sae2Δ mutant on the side of the break containing the longest repeat tract, suggesting that secondary structures at double-strand break ends must be removed by the Mre11-Rad50 complex and Sae2. Following the TALEN double-strand break, single-strand annealing occurred between both sides of the repeat tract, leading to repeat contraction., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Summary Heritability and genome stability are shaped by meiotic recombination, which is initiated via hundreds of DNA double-strand breaks (DSBs). The distribution of DSBs throughout the genome is not random, but mechanisms molding this landscape remain poorly understood. Here, we exploit genome-wide maps of mouse DSBs at unprecedented nucleotide resolution to uncover previously invisible spatial features of recombination. At fine scale, we reveal a stereotyped hotspot structure—DSBs occur within narrow zones between methylated nucleosomes—and identify relationships between SPO11, chromatin, and the histone methyltransferase PRDM9. At large scale, DSB formation is suppressed on non-homologous portions of the sex chromosomes via the DSB-responsive kinase ATM, which also shapes the autosomal DSB landscape at multiple size scales. We also provide a genome-wide analysis of exonucleolytic DSB resection lengths and elucidate spatial relationships between DSBs and recombination products. Our results paint a comprehensive picture of features governing successive steps in mammalian meiotic recombination. [ABSTRACT FROM AUTHOR]

Chromatin moves with subdiffusive and spatially constrained dynamics within the cell nucleus. Here, we use single-locus tracking by time-lapse fluorescence microscopy to uncover information regarding the forces that influence chromatin movement following the induction of a persistent DNA double-strand break (DSB). Using improved time-lapse imaging regimens, we monitor trajectories of tagged DNA loci at a high temporal resolution, which allows us to extract biophysical parameters through robust statistical analysis. Polymer modeling based on these parameters predicts chromatin domain expansion near a DSB and damage extrusion from the domain. Both phenomena are confirmed by live imaging in budding yeast. Calculation of the anomalous exponent of locus movement allows us to differentiate forces imposed on the nucleus through the actin cytoskeleton from those that arise from INO80 remodeler-dependent changes in nucleosome organization. Our analytical approach can be applied to high-density single-locus trajectories obtained in any cell type., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)