Defective resection at DNA double-strand breaks leads to de novo telomere formation and enhances gene targeting
| العنوان: | Defective resection at DNA double-strand breaks leads to de novo telomere formation and enhances gene targeting |
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| المؤلفون: | Anna Malkova, Zhu Zhu, Alma Papusha, Woo-Hyun Chung, Grzegorz Ira |
| المصدر: | PLoS Genetics, Vol 6, Iss 5, p e1000948 (2010) PLoS Genetics |
| بيانات النشر: | Public Library of Science (PLoS), 2010. |
| سنة النشر: | 2010 |
| مصطلحات موضوعية: | Cancer Research, DNA Repair, lcsh:QH426-470, DNA repair, Telomere Pathway, genetic processes, DNA, Single-Stranded, 03 medical and health sciences, 0302 clinical medicine, Genetics, Gene conversion, Molecular Biology, Replication protein A, Genetics (clinical), Ecology, Evolution, Behavior and Systematics, Alleles, 030304 developmental biology, Molecular Biology/Recombination, Recombination, Genetic, 0303 health sciences, Molecular Biology/DNA Repair, biology, Genetics and Genomics/Gene Therapy, Helicase, Telomere, Molecular biology, Electrophoresis, Gel, Pulsed-Field, enzymes and coenzymes (carbohydrates), lcsh:Genetics, Gene Targeting, biology.protein, Homologous recombination, 030217 neurology & neurosurgery, Sgs1, Research Article, DNA Damage |
| الوصف: | 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. |
| اللغة: | English |
| تدمد: | 1553-7404 1553-7390 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::eb7ee87001db0f96c746f42074e091f0 http://europepmc.org/articles/PMC2869328?pdf=render |
| Rights: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....eb7ee87001db0f96c746f42074e091f0 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 15537404 15537390 |
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