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.

FIG. 1.

(A) Schematic representation of the chromosome fragmentation assay. The linear CFV undergoes de novo telomere addition to the tg tract at one end of the vector, and the other end invades the endogenous chromosomal locus duplicating sequences from the region of homology between the vector and native chromosome to the telomere. (B) Maps of the CFVs used for chromosome fragmentation. All contain URA3, SUP11, CEN4, and an ARS element. D8B refers to a 5-kb BglII fragment located 100 kb from the left telomere of chromosome III.

FIG. 2.

Identification of chromosome fragments by PFGE. Plugs were prepared from stable Ura⁺ transformants derived from CFV/YBR235-tg. The panel on the left shows the gel stained with SYBR gold, the panel on the right is a Southern blot of the same gel probed with a PCR product derived from YBR235 to indicate chromosome II and the CF.