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Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae.
Genetics. 1989 Jul; 122(3):519-34.G

Abstract

We have investigated HO endonuclease-induced double-strand break (DSB) recombination and repair in a LACZ duplication plasmid in yeast. A 117-bp MATa fragment, embedded in one copy of LACZ, served as a site for initiation of a DSB when HO endonuclease was expressed. The DSB could be repaired using wild-type sequences located on a second, promoterless, copy of LACZ on the same plasmid. In contrast to normal mating-type switching, crossing-over associated with gene conversion occurred at least 50% of the time. The proportion of conversion events accompanied by exchange was greater when the two copies of LACZ were in direct orientation (80%), than when inverted (50%). In addition, the fraction of plasmids lost was significantly greater in the inverted orientation. The kinetics of appearance of intermediates and final products were also monitored. The repair of the DSB is slow, requiring at least an hour from the detection of the HO-cut fragments to completion of repair. Surprisingly, the appearance of the two reciprocal products of crossing over did not occur with the same kinetics. For example, when the two LACZ sequences were in the direct orientation, the HO-induced formation of a large circular deletion product was not accompanied by the appearance of a small circular reciprocal product. We suggest that these differences may reflect two kinetically separable processes, one involving only one cut end and the other resulting from the concerted participation of both ends of the DSB.

Authors+Show Affiliations

Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254.No affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

2668114

Citation

Rudin, N, et al. "Genetic and Physical Analysis of Double-strand Break Repair and Recombination in Saccharomyces Cerevisiae." Genetics, vol. 122, no. 3, 1989, pp. 519-34.
Rudin N, Sugarman E, Haber JE. Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae. Genetics. 1989;122(3):519-34.
Rudin, N., Sugarman, E., & Haber, J. E. (1989). Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae. Genetics, 122(3), 519-34.
Rudin N, Sugarman E, Haber JE. Genetic and Physical Analysis of Double-strand Break Repair and Recombination in Saccharomyces Cerevisiae. Genetics. 1989;122(3):519-34. PubMed PMID: 2668114.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae. AU - Rudin,N, AU - Sugarman,E, AU - Haber,J E, PY - 1989/7/1/pubmed PY - 1989/7/1/medline PY - 1989/7/1/entrez SP - 519 EP - 34 JF - Genetics JO - Genetics VL - 122 IS - 3 N2 - We have investigated HO endonuclease-induced double-strand break (DSB) recombination and repair in a LACZ duplication plasmid in yeast. A 117-bp MATa fragment, embedded in one copy of LACZ, served as a site for initiation of a DSB when HO endonuclease was expressed. The DSB could be repaired using wild-type sequences located on a second, promoterless, copy of LACZ on the same plasmid. In contrast to normal mating-type switching, crossing-over associated with gene conversion occurred at least 50% of the time. The proportion of conversion events accompanied by exchange was greater when the two copies of LACZ were in direct orientation (80%), than when inverted (50%). In addition, the fraction of plasmids lost was significantly greater in the inverted orientation. The kinetics of appearance of intermediates and final products were also monitored. The repair of the DSB is slow, requiring at least an hour from the detection of the HO-cut fragments to completion of repair. Surprisingly, the appearance of the two reciprocal products of crossing over did not occur with the same kinetics. For example, when the two LACZ sequences were in the direct orientation, the HO-induced formation of a large circular deletion product was not accompanied by the appearance of a small circular reciprocal product. We suggest that these differences may reflect two kinetically separable processes, one involving only one cut end and the other resulting from the concerted participation of both ends of the DSB. SN - 0016-6731 UR - https://www.unboundmedicine.com/medline/citation/2668114/Genetic_and_physical_analysis_of_double_strand_break_repair_and_recombination_in_Saccharomyces_cerevisiae_ L2 - https://academic.oup.com/genetics/article-lookup/doi/10.1093/genetics/122.3.519 DB - PRIME DP - Unbound Medicine ER -