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      Defective Resection at DNA Double-Strand Breaks Leads to De Novo Telomere Formation and Enhances Gene Targeting

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          Abstract

          The formation of single-stranded DNA (ssDNA) at double-strand break (DSB) ends is essential in repair by homologous recombination and is mediated by DNA helicases and nucleases. Here we estimated the length of ssDNA generated during DSB repair and analyzed the consequences of elimination of processive resection pathways mediated by Sgs1 helicase and Exo1 nuclease on DSB repair fidelity. In wild-type cells during allelic gene conversion, an average of 2–4 kb of ssDNA accumulates at each side of the break. Longer ssDNA is formed during ectopic recombination or break-induced replication (BIR), reflecting much slower repair kinetics. This relatively extensive resection may help determine sequences involved in homology search and prevent recombination within short DNA repeats next to the break. In sgs1Δ exo1Δ mutants that form only very short ssDNA, allelic gene conversion decreases 5-fold and DSBs are repaired by BIR or de novo telomere formation resulting in loss of heterozygosity. The absence of the telomerase inhibitor, PIF1, increases de novo telomere pathway usage to about 50%. Accumulation of Cdc13, a protein recruiting telomerase, at the break site increases in sgs1Δ exo1Δ, and the requirement of the Ku complex for new telomere formation is partially bypassed. In contrast to this decreased and alternative DSB repair, the efficiency and accuracy of gene targeting increases dramatically in sgs1Δ exo1Δ cells, suggesting that transformed DNA is very stable in these mutants. Altogether these data establish a new role for processive resection in the fidelity of DSB repair.

          Author Summary

          Chromosomal breaks occur spontaneously or are induced by ionizing radiation and many chemotherapeutic drugs. DNA double-strand breaks are processed by nucleases and helicases in yeast and human to generate single-stranded DNA that is then used for repair by recombination with homologous chromosome. Single-stranded DNA at chromosomal breaks also constitutes a signal for cells to arrest cell cycle progression until the DNA damage is repaired. This study examines the consequences of elimination of enzymes that process chromosomal breaks to single-stranded DNA on the fidelity of repair and genome stability in the model organism yeast. Mutants deficient in these enzymes often fail to repair the breaks by homologous recombination and instead add new telomeres at the breaks. Formation of new telomeres is associated with partial loss of the chromosome arm distal from the break. Such chromosomal aberrations were frequently observed in tumor cells and are responsible for about 10% of human genomic disorders resulting from chromosomal abnormalities. We also observed that elimination of enzymes that process chromosomal breaks into single-stranded DNA greatly stimulates genome manipulation by gene targeting, suggesting that transformed DNA is also a substrate for degradation by these enzymes. We discuss the possibility of using a similar approach in mammalian cells where gene targeting is inaccurate and less efficient when compared to yeast.

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          Most cited references 51

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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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              Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing.

              DNA ends exposed after introduction of double-strand breaks (DSBs) undergo 5'-3' nucleolytic degradation to generate single-stranded DNA, the substrate for binding by the Rad51 protein to initiate homologous recombination. This process is poorly understood in eukaryotes, but several factors have been implicated, including the Mre11 complex (Mre11-Rad50-Xrs2/NBS1), Sae2/CtIP/Ctp1 and Exo1. Here we demonstrate that yeast Exo1 nuclease and Sgs1 helicase function in alternative pathways for DSB processing. Novel, partially resected intermediates accumulate in a double mutant lacking Exo1 and Sgs1, which are poor substrates for homologous recombination. The early processing step that generates partly resected intermediates is dependent on Sae2. When Sae2 is absent, in addition to Exo1 and Sgs1, unprocessed DSBs accumulate and homology-dependent repair fails. These results suggest a two-step mechanism for DSB processing during homologous recombination. First, the Mre11 complex and Sae2 remove a small oligonucleotide(s) from the DNA ends to form an early intermediate. Second, Exo1 and/or Sgs1 rapidly process this intermediate to generate extensive tracts of single-stranded DNA that serve as substrate for Rad51.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                May 2010
                May 2010
                13 May 2010
                : 6
                : 5
                Affiliations
                [1 ]Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America
                [2 ]Department of Biology, School of Science, Indiana University–Purdue University Indianapolis, Indianapolis, Indiana, United States of America
                National Cancer Institute, United States of America
                Author notes

                Conceived and designed the experiments: WHC GI. Performed the experiments: WHC ZZ AP. Analyzed the data: WHC GI. Contributed reagents/materials/analysis tools: AM. Wrote the paper: WHC AM GI.

                Article
                09-PLGE-RA-2148R2
                10.1371/journal.pgen.1000948
                2869328
                20485519
                Chung et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 13
                Categories
                Research Article
                Genetics and Genomics/Gene Therapy
                Molecular Biology/DNA Repair
                Molecular Biology/Recombination

                Genetics

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