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Requirement of POL3 and POL4 on non-homologous and microhomology-mediated end joining in rad50/xrs2 mutants of Saccharomyces cerevisiae.
Mutagenesis. 2015 Nov; 30(6):841-9.M

Abstract

Non-homologous end joining (NHEJ) directly joins two broken DNA ends without sequence homology. A distinct pathway called microhomology-mediated end joining (MMEJ) relies on a few base pairs of homology between the recombined DNA. The majority of DNA double-strand breaks caused by endogenous oxygen species or ionizing radiation contain damaged bases that hinder direct religation. End processing is required to remove mismatched nucleotides and fill in gaps during end joining of incompatible ends. POL3 in Saccharomyces cerevisiae encodes polymerase δ that is required for DNA replication and other DNA repair processes. Our previous results have shown that POL3 is involved in gap filling at 3' overhangs in POL4-independent NHEJ. Here, we studied the epistatic interaction between POL3, RAD50, XRS2 and POL4 in NHEJ using a plasmid-based endjoining assay in yeast. We demonstrated that either rad50 or xrs2 mutation is epistatic for end joining of compatible ends in the rad50 pol3-t or xrs2 pol3-t double mutants. However, the pol3-t and rad50 or pol3-t and xrs2 mutants caused an additive decrease in the end-joining efficiency of incompatible ends, suggesting that POL3 and RAD50 or POL3 and XRS2 exhibit independent functions in NHEJ. In the rad50 pol4 mutant, end joining of incompatible ends was not detected. In the rad50 or xrs2 mutants, NHEJ events did not contain any microhomology at the rejoined junctions. The pol3-t mutation restored MMEJ in the rad50 or xrs2 mutant backgrounds. Moreover, we demonstrated that NHEJ of incompatible ends required RAD50 and POL4 more than POL3. In conclusion, POL3 and POL4 have differential functions in NHEJ, independent of the RAD50-mediated repair pathway.

Authors+Show Affiliations

No affiliation info availableDepartments of Pathology, Environmental Health, and Radiation Oncology, David Geffen School of Medicine at UCLA and UCLA School of Public Health, 71-295 CHS, 650 Charles E. Young Drive South, Los Angeles, CA, USA.Departments of Pathology, Environmental Health, and Radiation Oncology, David Geffen School of Medicine at UCLA and UCLA School of Public Health, 71-295 CHS, 650 Charles E. Young Drive South, Los Angeles, CA, USA.No affiliation info availableDepartments of Pathology, Environmental Health, and Radiation Oncology, David Geffen School of Medicine at UCLA and UCLA School of Public Health, 71-295 CHS, 650 Charles E. Young Drive South, Los Angeles, CA, USA.

Pub Type(s)

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

Language

eng

PubMed ID

26122113

Citation

Galli, Alvaro, et al. "Requirement of POL3 and POL4 On Non-homologous and Microhomology-mediated End Joining in Rad50/xrs2 Mutants of Saccharomyces Cerevisiae." Mutagenesis, vol. 30, no. 6, 2015, pp. 841-9.
Galli A, Chan CY, Parfenova L, et al. Requirement of POL3 and POL4 on non-homologous and microhomology-mediated end joining in rad50/xrs2 mutants of Saccharomyces cerevisiae. Mutagenesis. 2015;30(6):841-9.
Galli, A., Chan, C. Y., Parfenova, L., Cervelli, T., & Schiestl, R. H. (2015). Requirement of POL3 and POL4 on non-homologous and microhomology-mediated end joining in rad50/xrs2 mutants of Saccharomyces cerevisiae. Mutagenesis, 30(6), 841-9. https://doi.org/10.1093/mutage/gev046
Galli A, et al. Requirement of POL3 and POL4 On Non-homologous and Microhomology-mediated End Joining in Rad50/xrs2 Mutants of Saccharomyces Cerevisiae. Mutagenesis. 2015;30(6):841-9. PubMed PMID: 26122113.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Requirement of POL3 and POL4 on non-homologous and microhomology-mediated end joining in rad50/xrs2 mutants of Saccharomyces cerevisiae. AU - Galli,Alvaro, AU - Chan,Cecilia Y, AU - Parfenova,Liubov, AU - Cervelli,Tiziana, AU - Schiestl,Robert H, Y1 - 2015/06/29/ PY - 2015/7/1/entrez PY - 2015/7/1/pubmed PY - 2016/8/20/medline SP - 841 EP - 9 JF - Mutagenesis JO - Mutagenesis VL - 30 IS - 6 N2 - Non-homologous end joining (NHEJ) directly joins two broken DNA ends without sequence homology. A distinct pathway called microhomology-mediated end joining (MMEJ) relies on a few base pairs of homology between the recombined DNA. The majority of DNA double-strand breaks caused by endogenous oxygen species or ionizing radiation contain damaged bases that hinder direct religation. End processing is required to remove mismatched nucleotides and fill in gaps during end joining of incompatible ends. POL3 in Saccharomyces cerevisiae encodes polymerase δ that is required for DNA replication and other DNA repair processes. Our previous results have shown that POL3 is involved in gap filling at 3' overhangs in POL4-independent NHEJ. Here, we studied the epistatic interaction between POL3, RAD50, XRS2 and POL4 in NHEJ using a plasmid-based endjoining assay in yeast. We demonstrated that either rad50 or xrs2 mutation is epistatic for end joining of compatible ends in the rad50 pol3-t or xrs2 pol3-t double mutants. However, the pol3-t and rad50 or pol3-t and xrs2 mutants caused an additive decrease in the end-joining efficiency of incompatible ends, suggesting that POL3 and RAD50 or POL3 and XRS2 exhibit independent functions in NHEJ. In the rad50 pol4 mutant, end joining of incompatible ends was not detected. In the rad50 or xrs2 mutants, NHEJ events did not contain any microhomology at the rejoined junctions. The pol3-t mutation restored MMEJ in the rad50 or xrs2 mutant backgrounds. Moreover, we demonstrated that NHEJ of incompatible ends required RAD50 and POL4 more than POL3. In conclusion, POL3 and POL4 have differential functions in NHEJ, independent of the RAD50-mediated repair pathway. SN - 1464-3804 UR - https://www.unboundmedicine.com/medline/citation/26122113/Requirement_of_POL3_and_POL4_on_non_homologous_and_microhomology_mediated_end_joining_in_rad50/xrs2_mutants_of_Saccharomyces_cerevisiae_ L2 - https://academic.oup.com/mutage/article-lookup/doi/10.1093/mutage/gev046 DB - PRIME DP - Unbound Medicine ER -