Ku DNA End-Binding Activity Promotes Repair Fidelity and Influences End-Processing During Nonhomologous End-Joining in Saccharomyces cerevisiae

Genetics: A Periodical Record of Investigations Bearing on Heredity and Variation

Charlene H. Emerson^1¹  Albert Ribes-Zamora^2²  Christopher R. Lopez^2³  Erica J. Polleys^2⁴ 

[1] Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030;Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030^2;Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030^1;Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030^3

关键词: Ku;    nonhomologous end-joining;    end-processing;    repair fidelity;    Saccharomyces cerevisiae;   

DOI  :  10.1534/genetics.117.300672

学科分类：医学（综合）

来源: Genetics Society of America

【 摘 要 】

The Ku heterodimer acts centrally in nonhomologous end-joining (NHEJ) of DNA double-strand breaks (DSB). Saccharomyces cerevisiae Ku, like mammalian Ku, binds and recruits NHEJ factors to DSB ends. Consequently, NHEJ is virtually absent in yeast Ku null ( yku70 ∆ or yku80 ∆) strains. Previously, we unexpectedly observed imprecise NHEJ proficiency in a yeast Ku mutant with impaired DNA end-binding (DEB). However, how DEB impairment supported imprecise NHEJ was unknown. Here, we found imprecise NHEJ proficiency to be a feature of a panel of DEB-impaired Ku mutants and that DEB impairment resulted in a deficiency in precise NHEJ. These results suggest that DEB-impaired Ku specifically promotes error-prone NHEJ. Epistasis analysis showed that classical NHEJ factors, as well as novel and previously characterized NHEJ-specific residues of Ku, are required for the distinct error-prone repair in a Ku DEB mutant. However, sequencing of repair junctions revealed that imprecise repair in Ku DEB mutants was almost exclusively characterized by small deletions, in contrast to the majority of insertions that define imprecise repair in wild-type strains. Notably, while sequencing indicated a lack of Pol4 -dependent insertions at the site of repair, Pol2 exonuclease activity, which mediates small deletions in NHEJ, contributed to imprecise NHEJ in a Ku DEB mutant. The deletions were smaller than in Ku-independent microhomology-mediated end-joining (MMEJ) and were neither promoted by Mre11 nuclease activity nor Sae2 . Thus, the quality of Ku’s engagement at the DNA end influences end-processing during NHEJ and DEB impairment unmasks a Ku-dependent error-prone pathway of end-joining distinct from MMEJ.

【 授权许可】

CC BY