Nucleic Acids Research, 1994, Vol. 22, No. 14 2823-2829
© 1994
MOLECULAR BIOLOGY |
Single strand and double strand DNA damage-induced reciprocal recombination in yeast. Dependence on nucleotide excision repair and RAD1 recombination
Queens College, City University of New York, Department of Chemistry and Biochemistry 65-30 Kissena Blvd, Flushing, NY 11367, USA
*To whom correspondence should be addressed
Received February 28, 1994. Revised June 13, 1994. Accepted June 13, 1994.
Single strand and double strand DNA damage-induced recombination were compared in the yeast Saccharomyces cerevisiae. The non-replicating plasmid pUC18-HIS3 was damaged in vitro and introduced into yeast cells; plasmid - chromosome recombinants were selected as stable His+ transformants. Single strand damage was produced by UV irradiation at 254 nm or by psoralen photoreaction at 390 nm. Double strand damage was produced by psoralen photoreaction at 350 nm or by restriction endonuclease digestion. Recombinants were classified as resulting from gene conversion without crossing over, single plasmid integration, or multiple plasmid integration. Single and double strand DNA damage produced different patterns of recombination. In repair proficient cells double strand damage induced primarily multiple plasmid integrations, while single strand damage induced higher proportions of gene conversions and single integrations. Reciprocal recombination depended on the RAD1 gene, which is involved in both excision repair and recombination; plasmid integration induced by all forms of damage was decreased in a rad1 disruption strain. Mutation of the RAD3 excision repair gene decreased plasmid integration induced by far UV irradiation and psoralen crosslinks, but not by double strand breaks, which are not substrates of nucleotide excision repair. Double strand break-induced plasmid integration was also decreased by disruption of RAD10, which forms a complex with RAD1; disruption of RAD4 had no effect. Thus, while nucleotide excision repair genes are involved in the processing of damaged DNA to generate recombination intermediates, RAD1 and RAD10 are additionally involved in reciprocal exchange.
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