defective resection at dna double-strand breaks leads to de novo telomere formation and enhances gene targeting有缺陷的切除导致新创端粒dna双链断裂形成和提高基因打靶.pdfVIP

Defective Resection at DNA Double-Strand Breaks Leads to De Novo Telomere Formation and Enhances Gene Targeting 1 1 1 2 1 Woo-Hyun Chung , Zhu Zhu , Alma Papusha , Anna Malkova , Grzegorz Ira * 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 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 sgs1D exo1D 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 sgs1D exo1D, and the requirement of the Ku complex for new telomere formation is partially bypasse