Induction of multiple plasmid recombination in Saccharomyces cerevisiae by psoralen reaction and double strand breaks

Abstract

DNA damage-induced multiple recombination was studied by cotransforming yeast cells with pairs of nonreplicating plasmids carrying different genetic markers. Reaction of one of the plasmids with the interstrand crosslinking agent, psoralen, stimulated cellular transformation by the undamaged plasmid. The cotransformants carried copies of both plasmids cointegrated in tandem arrays at chromosomal sites homologous to either the damaged or the undamaged DNA. Plasmid linearization, by restriction endonuclease digestion, was also found to stimulate the cointegration of unmodified plasmids. Disruption of the RAD1 gene reduced the psoralen damage-induced cotransformation of intact plasmid, but had no effect on the stimulation by double strand breaks. Placement of the double strand breaks within yeast genes produced cointegration only at sequences homologous to the damaged plasmids, while digestion within vector sequences produced integration at chromosomal sites homologous to either the damaged or the undamaged plasmid molecules. These observations suggest a model for multiple recombination events in which an initial exchange occurs between the damaged DNA and homologous sequences on an undamaged molecule. Linked sequences on the undamaged molecule up to 870 base pairs distant from the break site participate in subsequent exchanges with other intact DNA molecules. These events result in recombinants produced by reciprocal exchange between three or more DNA molecules.