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

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.

Remarkably Long-Tract Gene Conversion Induced by Fragile Site Instability inSaccharomyces cerevisiae

“…Previously we demonstrated that BIR is strongly stimulated in cells under replication stress, and that many of these BIR events are initiated by breaks at FS2 . BIR events can initiate centromereproximal to the original break location due to extensive 59 to 39 end resection at DNA breaks to expose ssDNA (Chung et al 2010;Symington 2016).…”

“…Previously we demonstrated that BIR is strongly stimulated in cells under replication stress, and that many of these BIR events are initiated by breaks at FS2 . BIR events can initiate centromereproximal to the original break location due to extensive 59 to 39 end resection at DNA breaks to expose ssDNA (Chung et al 2010;Symington 2016).Both the extensive lengths and high abundance of bidirectional noncrossover tracts we observed can be explained if these tracts result not from canonical DSBR but from dBIR. The dBIR mechanism is also a better fit for our experimental system in which replication stress is likely to result in replication fork 124 S. A. Chumki et al…”

DNA End Resection: Nucleases Team Up with the Right Partners to Initiate Homologous Recombination

“…Subsequent work revealed that these assays largely overestimated the length of DNA that is resected in vivo under normal conditions when repair is possible. In mitotic cells, it has been determined that ϳ2,000 -4,000 nt are resected in allelic recombination and ϳ3,000 -6,000 nt are resected in ectopic recombination (38). In meiotic cells, where the long-range resection is largely dependent on Exo1, the resection tracks are even shorter (ϳ800 nt) (39).…”

Yeast Helicase Pif1 Unwinds RNA:DNA Hybrids with Higher Processivity than DNA:DNA Duplexes

“…In yeast, Pif1 helicase is suggested to be involved in mitochondrial DNA recombination, repair, and replication (6,11). Some of its nuclear functions include Okazaki fragment processing (12,13), inhibition of rDNA replication (14), unwinding of G-quadruplex structures (15)(16)(17)(18), telomere regulation (19 -21), and repair of double-stranded DNA breaks (22)(23)(24)(25). S. cerevisiae Pif1 belongs to the superfamily (SF) 2 1B class of helicases.…”