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Overhang polarity of chromosomal double-strand breaks impacts kinetics and fidelity of yeast non-homologous end joining.
Nucleic Acids Res. 2016 Apr 07; 44(6):2769-81.NA

Abstract

Non-homologous end joining (NHEJ) is the main repair pathway for DNA double-strand breaks (DSBs) in cells with limited 5' resection. To better understand how overhang polarity of chromosomal DSBs affects NHEJ, we made site-specific 5'-overhanging DSBs (5' DSBs) in yeast using an optimized zinc finger nuclease at an efficiency that approached HO-induced 3' DSB formation. When controlled for the extent of DSB formation, repair monitoring suggested that chromosomal 5' DSBs were rejoined more efficiently than 3' DSBs, consistent with a robust recruitment of NHEJ proteins to 5' DSBs. Ligation-mediated qPCR revealed that Mre11-Rad50-Xrs2 rapidly modified 5' DSBs and facilitated protection of 3' DSBs, likely through recognition of overhang polarity by the Mre11 nuclease. Next-generation sequencing revealed that NHEJ at 5' DSBs had a higher mutation frequency, and validated the differential requirement of Pol4 polymerase at 3' and 5' DSBs. The end processing enzyme Tdp1 did not impact joining fidelity at chromosomal 5' DSBs as in previous plasmid studies, although Tdp1 was recruited to only 5' DSBs in a Ku-independent manner. These results suggest distinct DSB handling based on overhang polarity that impacts NHEJ kinetics and fidelity through differential recruitment and action of DSB modifying enzymes.

Authors+Show Affiliations

Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA.Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA.Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA wilsonte@umich.edu.

Pub Type(s)

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

Language

eng

PubMed ID

26773053

Citation

Liang, Zhuobin, et al. "Overhang Polarity of Chromosomal Double-strand Breaks Impacts Kinetics and Fidelity of Yeast Non-homologous End Joining." Nucleic Acids Research, vol. 44, no. 6, 2016, pp. 2769-81.
Liang Z, Sunder S, Nallasivam S, et al. Overhang polarity of chromosomal double-strand breaks impacts kinetics and fidelity of yeast non-homologous end joining. Nucleic Acids Res. 2016;44(6):2769-81.
Liang, Z., Sunder, S., Nallasivam, S., & Wilson, T. E. (2016). Overhang polarity of chromosomal double-strand breaks impacts kinetics and fidelity of yeast non-homologous end joining. Nucleic Acids Research, 44(6), 2769-81. https://doi.org/10.1093/nar/gkw013
Liang Z, et al. Overhang Polarity of Chromosomal Double-strand Breaks Impacts Kinetics and Fidelity of Yeast Non-homologous End Joining. Nucleic Acids Res. 2016 Apr 7;44(6):2769-81. PubMed PMID: 26773053.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Overhang polarity of chromosomal double-strand breaks impacts kinetics and fidelity of yeast non-homologous end joining. AU - Liang,Zhuobin, AU - Sunder,Sham, AU - Nallasivam,Sivakumar, AU - Wilson,Thomas E, Y1 - 2016/01/14/ PY - 2016/01/05/accepted PY - 2015/08/10/received PY - 2016/1/17/entrez PY - 2016/1/17/pubmed PY - 2016/8/25/medline SP - 2769 EP - 81 JF - Nucleic acids research JO - Nucleic Acids Res VL - 44 IS - 6 N2 - Non-homologous end joining (NHEJ) is the main repair pathway for DNA double-strand breaks (DSBs) in cells with limited 5' resection. To better understand how overhang polarity of chromosomal DSBs affects NHEJ, we made site-specific 5'-overhanging DSBs (5' DSBs) in yeast using an optimized zinc finger nuclease at an efficiency that approached HO-induced 3' DSB formation. When controlled for the extent of DSB formation, repair monitoring suggested that chromosomal 5' DSBs were rejoined more efficiently than 3' DSBs, consistent with a robust recruitment of NHEJ proteins to 5' DSBs. Ligation-mediated qPCR revealed that Mre11-Rad50-Xrs2 rapidly modified 5' DSBs and facilitated protection of 3' DSBs, likely through recognition of overhang polarity by the Mre11 nuclease. Next-generation sequencing revealed that NHEJ at 5' DSBs had a higher mutation frequency, and validated the differential requirement of Pol4 polymerase at 3' and 5' DSBs. The end processing enzyme Tdp1 did not impact joining fidelity at chromosomal 5' DSBs as in previous plasmid studies, although Tdp1 was recruited to only 5' DSBs in a Ku-independent manner. These results suggest distinct DSB handling based on overhang polarity that impacts NHEJ kinetics and fidelity through differential recruitment and action of DSB modifying enzymes. SN - 1362-4962 UR - https://www.unboundmedicine.com/medline/citation/26773053/Overhang_polarity_of_chromosomal_double_strand_breaks_impacts_kinetics_and_fidelity_of_yeast_non_homologous_end_joining_ L2 - https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gkw013 DB - PRIME DP - Unbound Medicine ER -