Yeast recombination: the association between double-strand gap repair and crossing-over

Abstract

In previous experiments, we have used yeast transformation to study the recombinogenic repair of double-strand breaks and gaps. A plasmid containing a double-strand gap within sequences homologous to the yeast genome integrates efficiently by crossing-over. During the process of integration, the double-strand gap is repaired, using chromosomal information as a template. This repair reaction results in the transfer of genetic information from one DNA duplex to another and is therefore a pathway for gene conversion. Because meiotic gene conversion is associated with a high frequency (up to 50%) of crossing-over, we wished to determine the degree of association of double-strand gap repair with crossing-over. Only the class of repair events resulting in crossovers (plasmid integration) were detected in our earlier experiments with nonreplicating plasmids. In this paper, we describe the outcome of double-strand gap repair in plasmids that are capable of autonomous replication and therefore allow recovery of both crossover and noncrossover products. After the correct repair of a double-strand gap, we recover approximately equal numbers of integrated and nonintegrated plasmids. Thus, gene conversion by double-strand gap repair can occur either with or without crossing-over, and it is similar in this respect to meiotic gene conversion. Circularization of linear plasmid DNA by ligation is also observed, suggesting that yeast has an additional repair pathway for double-strand breaks that is independent of recombination. Gap repair on replicating plasmids permits rapid cloning of chromosomal alleles.