RAD51-Dependent Break-Induced Replication in Yeast

Abstract

A chromosome fragmentation assay was used to measure the efficiency and genetic control of break-induced replication (BIR) in Saccharomyces cerevisiae. Formation of a chromosome fragment by de novo telomere generation at one end of the linear vector and recombination-dependent replication of 100 kb of chromosomal sequences at the other end of the vector occurred at high frequency in wild-type strains. RAD51 was required for more than 95% of BIR events involving a single-end invasion and was essential when two BIR events were required for generation of a chromosome fragment. The similar genetic requirements for BIR and gene conversion suggest a common strand invasion intermediate in these two recombinational repair processes. Mutation of RAD50 or RAD59 conferred no significant defect in BIR in either RAD51 or rad51 strains. RAD52 was shown to be essential for BIR at unique chromosomal sequences, although rare recombination events were detected between the subtelomeric Y′ repeats.

We thank P. Hieter for providing pCF2 vectors and W. K. Holloman and members of the Symington laboratory for stimulating discussions and critical reading of the manuscript. We thank A. Lustig for advice on design and cloning of the oligonucleotides used for de novo telomere addition.

The research described in this article was supported by Public Health Service grant GM41784 from the National Institutes of Health.