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      Structural biology of the Mre11:Nbs1 complex structure yields insights into ataxia–telangiectasia–like disease mutations and DNA damage signaling

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          Summary

          The Mre11–Rad50–Nbs1 (MRN) complex tethers, processes and signals DNA double strand breaks, promoting genomic stability. To understand the functional architecture of MRN, we determined the crystal structures of the Schizosaccharomyces pombe Mre11 dimeric catalytic domain alone and in complex with a fragment of Nbs1. Two Nbs1 subunits stretch around the outside of Mre11’s nuclease domains, with one subunit additionally bridging and locking the Mre11 dimer via a highly conserved asymmetrical binding motif. Our results reveal that Mre11 forms a flexible dimer and suggest that Nbs1 is not only a checkpoint adaptor, but also functionally impacts on Mre11-Rad50. Clinical mutations in Mre11 are located along the Nbs1 interaction sites and weaken the Mre11–Nbs1 interaction. However, they differentially affect DNA repair and telomere maintenance in Saccharomyces cerevisiae, potentially providing insight into their different human disease pathologies.

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          The DNA damage response: ten years after.

          The DNA damage response (DDR), through the action of sensors, transducers, and effectors, orchestrates the appropriate repair of DNA damage and resolution of DNA replication problems, coordinating these processes with ongoing cellular physiology. In the past decade, we have witnessed an explosion in understanding of DNA damage sensing, signaling, and the complex interplay between protein phosphorylation and the ubiquitin pathway employed by the DDR network to execute the response to DNA damage. These findings have important implications for aging and cancer.
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            Human CtIP promotes DNA end resection.

            In the S and G2 phases of the cell cycle, DNA double-strand breaks (DSBs) are processed into single-stranded DNA, triggering ATR-dependent checkpoint signalling and DSB repair by homologous recombination. Previous work has implicated the MRE11 complex in such DSB-processing events. Here, we show that the human CtIP (RBBP8) protein confers resistance to DSB-inducing agents and is recruited to DSBs exclusively in the S and G2 cell-cycle phases. Moreover, we reveal that CtIP is required for DSB resection, and thereby for recruitment of replication protein A (RPA) and the protein kinase ATR to DSBs, and for the ensuing ATR activation. Furthermore, we establish that CtIP physically and functionally interacts with the MRE11 complex, and that both CtIP and MRE11 are required for efficient homologous recombination. Finally, we reveal that CtIP has sequence homology with Sae2, which is involved in MRE11-dependent DSB processing in yeast. These findings establish evolutionarily conserved roles for CtIP-like proteins in controlling DSB resection, checkpoint signalling and homologous recombination.
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              Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends.

              Formation of single-strand DNA (ssDNA) tails at a double-strand break (DSB) is a key step in homologous recombination and DNA-damage signaling. The enzyme(s) producing ssDNA at DSBs in eukaryotes remain unknown. We monitored 5'-strand resection at inducible DSB ends in yeast and identified proteins required for two stages of resection: initiation and long-range 5'-strand resection. We show that the Mre11-Rad50-Xrs2 complex (MRX) initiates 5' degradation, whereas Sgs1 and Dna2 degrade 5' strands exposing long 3' strands. Deletion of SGS1 or DNA2 reduces resection and DSB repair by single-strand annealing between distant repeats while the remaining long-range resection activity depends on the exonuclease Exo1. In exo1Deltasgs1Delta double mutants, the MRX complex together with Sae2 nuclease generate, in a stepwise manner, only few hundred nucleotides of ssDNA at the break, resulting in inefficient gene conversion and G2/M damage checkpoint arrest. These results provide important insights into the early steps of DSB repair in eukaryotes.
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                Author and article information

                Journal
                101186374
                31761
                Nat Struct Mol Biol
                Nat. Struct. Mol. Biol.
                Nature structural & molecular biology
                1545-9993
                1545-9985
                31 May 2012
                17 June 2012
                01 January 2013
                : 19
                : 7
                : 693-700
                Affiliations
                [1 ]Gene Center and Department of Chemistry and Biochemistry, Ludwig–Maximilians–University Munich, Feodor–Lynen–Str. 25, 81377 Munich, Germany
                [2 ]Center for Integrated Protein Science, Ludwig–Maximilians–University Munich, Feodor–Lynen–Str. 25, 81377 Munich, Germany
                [3 ]The Gurdon Institute and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK
                Author notes
                To whom correspondence should be addressed: Karl Peter–Hopfner, Gene Center, Ludwig–Maximilians University, Feodor–Lynen–Str. 25, D–81377 Munich, Germany, Tel: +49 (0) 89 2180 76953, fax: +49 (0) 89 2180 76999, hopfner@ 123456lmb.uni–muenchen.de
                Article
                NIHMS377602
                10.1038/nsmb.2323
                3392456
                22705791

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                Funding
                Funded by: National Institute of Allergy and Infectious Diseases Extramural Activities : NIAID
                Award ID: U19 AI083025 || AI
                Categories
                Article

                Molecular biology

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