Functions and regulation of the MRX complex at DNA double-strand breaks

Gobbini, Elisa; Cassani, Corinne; Villa, Matteo; Bonetti, Diego; Longhese, Maria Pia

doi:10.15698/mic2016.08.517

Cited by 47 publications

(39 citation statements)

References 118 publications

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“…We next examined the role of the MRX complex in Ku DEBimpaired imprecise NHEJ. The MRX complex-composed of Mre11, Rad50, and Xrs2 subunits-contributes to several processes in S. cerevisiae, including initiating resection, homologous recombination (HR), NHEJ efficiency and fidelity, and telomere maintenance (Zhang and Paull 2005;Dewar and Lydall 2012;Balestrini et al 2013;Gobbini et al 2016;Iwasaki et al 2016). To determine whether MRX is required for error-prone repair with DEB-impaired mutant Ku, we analyzed imprecise NHEJ in mre11D yku70-R456E doublemutant strains.…”

Section: Efficient Imprecise Repair In a Ku Deb-impaired Mutant Does mentioning

confidence: 99%

Ku DNA End-Binding Activity Promotes Repair Fidelity and Influences End-Processing During Nonhomologous End-Joining in Saccharomyces cerevisiae

et al. 2018

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The Ku heterodimer acts centrally in nonhomologous end-joining (NHEJ) of DNA double-strand breaks (DSB). Ku, like mammalian Ku, binds and recruits NHEJ factors to DSB ends. Consequently, NHEJ is virtually absent in yeast Ku null (∆ or ∆) strains. Previously, we unexpectedly observed imprecise NHEJ proficiency in a yeast Ku mutant with impaired DNA end-binding (DEB). However, how DEB impairment supported imprecise NHEJ was unknown. Here, we found imprecise NHEJ proficiency to be a feature of a panel of DEB-impaired Ku mutants and that DEB impairment resulted in a deficiency in precise NHEJ. These results suggest that DEB-impaired Ku specifically promotes error-prone NHEJ. Epistasis analysis showed that classical NHEJ factors, as well as novel and previously characterized NHEJ-specific residues of Ku, are required for the distinct error-prone repair in a Ku DEB mutant. However, sequencing of repair junctions revealed that imprecise repair in Ku DEB mutants was almost exclusively characterized by small deletions, in contrast to the majority of insertions that define imprecise repair in wild-type strains. Notably, while sequencing indicated a lack of Pol4-dependent insertions at the site of repair, Pol2 exonuclease activity, which mediates small deletions in NHEJ, contributed to imprecise NHEJ in a Ku DEB mutant. The deletions were smaller than in Ku-independent microhomology-mediated end-joining (MMEJ) and were neither promoted by Mre11 nuclease activity nor Sae2 Thus, the quality of Ku's engagement at the DNA end influences end-processing during NHEJ and DEB impairment unmasks a Ku-dependent error-prone pathway of end-joining distinct from MMEJ.

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Section: Efficient Imprecise Repair In a Ku Deb-impaired Mutant Does mentioning

confidence: 99%

Ku DNA End-Binding Activity Promotes Repair Fidelity and Influences End-Processing During Nonhomologous End-Joining in Saccharomyces cerevisiae

et al. 2018

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“…The Mre11-Rad50-Xrs2 (MRX) complex plays a central role in the DNA damage response through detection and repair of cytotoxic DNA double-strand breaks (DSBs). Mutation of genes encoding the MRX complex in Saccharomyces cerevisiae causes genotoxin sensitivity, 3 shortening of telomeres and meiotic defects (Borde, 2007;Gobbini et al, 2016). Hypomorphic mutations of human MRN complex components (Nbs1 replaces Xrs2 in mammalian cells) result in the chromosome instability syndromes, Nijmegen breakage syndrome (NBS), NBS-like disease (NBSLD) and ataxia telangiectasia-like disease (ATLD), which are associated with radio-sensitivity, cancer predisposition and immune deficiencies (Carney et al, 1998;Stewart et al, 1999;Waltes et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Xrs2 and Tel1 independently contribute to MR-mediated DNA tethering and replisome stability

Lee

Rothstein

2018

Preprint

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The yeast Mre11-Rad50-Xrs2 (MRX) complex has structural, signaling and catalytic functions in the cellular response to DNA damage. Xrs2, the eukaryotic-specific component of the complex, is required for nuclear import of Mre11 and Rad50, and to recruit the Tel1 kinase to damage sites. We show that nuclear-localized MR complex (Mre11-NLS) catalyzes homology-dependent repair without Xrs2, but MR cannot activate Tel1 and it fails to tether DSBs resulting in sensitivity to genotoxins, replisome instability and increased gross chromosome rearrangements (GCRs). Fusing the Tel1 interaction domain from Xrs2 to Mre11-NLS is sufficient to restore telomere elongation and Tel1 signaling to Xrs2-deficient cells. Furthermore, Tel1 stabilizes Mre11-DNA association, and this stabilization function becomes important for DNA damage resistance in the absence of Xrs2. Enforcing Tel1 recruitment to the nuclear MR complex fully rescues end tethering, stalled replication fork stability and suppresses GCRs, highlighting important roles for Xrs2 and Tel1 to ensure optimal MR activity.

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“…The Mre11 nuclease, Rad50 ATPase, and FHA-containing Xrs2 protein can form a complex (“MRX”) that functions in DSB repair in mitotic cells, and has been implicated in both DSB formation and repair during meiosis ( Borde 2007 ; Gobbini et al 2016 ). We investigated the role of this complex in the repair of HO -mediated, meiotic DSBs in spo11 mutants by examining interhomolog recombination in spo11 spo13 mre11 and spo11 spo13 xrs2 meiotic cells that carry P SPO13 - HO and the HO cs5 .…”

Section: Resultsmentioning

confidence: 99%

HO Endonuclease-Initiated Recombination in Yeast Meiosis Fails To Promote Homologous Centromere Pairing and Is Not Constrained To Utilize the Dmc1 Recombinase

Yisehak

MacQueen

2018

G3 Genes|Genomes|Genetics

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Crossover recombination during meiosis is accompanied by a dramatic chromosome reorganization. In Saccharomyces cerevisiae, the onset of meiotic recombination by the Spo11 transesterase leads to stable pairwise associations between previously unassociated homologous centromeres followed by the intimate alignment of homologous axes via synaptonemal complex (SC) assembly. However, the molecular relationship between recombination and global meiotic chromosome reorganization remains poorly understood. In budding yeast, one question is why SC assembly initiates earliest at centromere regions while the DNA double strand breaks (DSBs) that initiate recombination occur genome-wide. We targeted the site-specific HO endonuclease to various positions on S. cerevisiae’s longest chromosome in order to ask whether a meiotic DSB’s proximity to the centromere influences its capacity to promote homologous centromere pairing and SC assembly. We show that repair of an HO-mediated DSB does not promote homologous centromere pairing nor any extent of SC assembly in spo11 meiotic nuclei, regardless of its proximity to the centromere. DSBs induced en masse by phleomycin exposure likewise do not promote homologous centromere pairing nor robust SC assembly. Interestingly, in contrast to Spo11, HO-initiated interhomolog recombination is not affected by loss of the meiotic kinase, Mek1, and is not constrained to use the meiosis-specific Dmc1 recombinase. These results strengthen the previously proposed idea that (at least some) Spo11 DSBs may be specialized in activating mechanisms that both 1) reinforce homologous chromosome alignment via homologous centromere pairing and SC assembly, and 2) establish Dmc1 as the primary strand exchange enzyme.

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Functions and regulation of the MRX complex at DNA double-strand breaks

Cited by 47 publications

References 118 publications

Ku DNA End-Binding Activity Promotes Repair Fidelity and Influences End-Processing During Nonhomologous End-Joining in Saccharomyces cerevisiae

Ku DNA End-Binding Activity Promotes Repair Fidelity and Influences End-Processing During Nonhomologous End-Joining in Saccharomyces cerevisiae

Xrs2 and Tel1 independently contribute to MR-mediated DNA tethering and replisome stability

HO Endonuclease-Initiated Recombination in Yeast Meiosis Fails To Promote Homologous Centromere Pairing and Is Not Constrained To Utilize the Dmc1 Recombinase

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