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Ku DNA End-Binding Activity Promotes Repair Fidelity and Influences End-Processing During Nonhomologous End-Joining in Saccharomyces cerevisiae.
Genetics. 2018 05; 209(1):115-128.G

Abstract

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.

Authors+Show Affiliations

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030. Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030.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.Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030.Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030.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.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 abertuch@bcm.edu. Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

29500182

Citation

Emerson, Charlene H., et al. "Ku DNA End-Binding Activity Promotes Repair Fidelity and Influences End-Processing During Nonhomologous End-Joining in Saccharomyces Cerevisiae." Genetics, vol. 209, no. 1, 2018, pp. 115-128.
Emerson CH, Lopez CR, Ribes-Zamora A, et al. Ku DNA End-Binding Activity Promotes Repair Fidelity and Influences End-Processing During Nonhomologous End-Joining in Saccharomyces cerevisiae. Genetics. 2018;209(1):115-128.
Emerson, C. H., Lopez, C. R., Ribes-Zamora, A., Polleys, E. J., Williams, C. L., Yeo, L., Zaneveld, J. E., Chen, R., & Bertuch, A. A. (2018). Ku DNA End-Binding Activity Promotes Repair Fidelity and Influences End-Processing During Nonhomologous End-Joining in Saccharomyces cerevisiae. Genetics, 209(1), 115-128. https://doi.org/10.1534/genetics.117.300672
Emerson CH, et al. Ku DNA End-Binding Activity Promotes Repair Fidelity and Influences End-Processing During Nonhomologous End-Joining in Saccharomyces Cerevisiae. Genetics. 2018;209(1):115-128. PubMed PMID: 29500182.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Ku DNA End-Binding Activity Promotes Repair Fidelity and Influences End-Processing During Nonhomologous End-Joining in Saccharomyces cerevisiae. AU - Emerson,Charlene H, AU - Lopez,Christopher R, AU - Ribes-Zamora,Albert, AU - Polleys,Erica J, AU - Williams,Christopher L, AU - Yeo,Lythou, AU - Zaneveld,Jacques E, AU - Chen,Rui, AU - Bertuch,Alison A, Y1 - 2018/03/02/ PY - 2017/12/28/received PY - 2018/02/25/accepted PY - 2018/3/4/pubmed PY - 2018/10/3/medline PY - 2018/3/4/entrez KW - Ku KW - Saccharomyces cerevisiae KW - end-processing KW - nonhomologous end-joining KW - repair fidelity SP - 115 EP - 128 JF - Genetics JO - Genetics VL - 209 IS - 1 N2 - 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. SN - 1943-2631 UR - https://www.unboundmedicine.com/medline/citation/29500182/Ku_DNA_End_Binding_Activity_Promotes_Repair_Fidelity_and_Influences_End_Processing_During_Nonhomologous_End_Joining_in_Saccharomyces_cerevisiae_ L2 - http://www.genetics.org/cgi/pmidlookup?view=long&pmid=29500182 DB - PRIME DP - Unbound Medicine ER -