Abstract

We have examined the genetic requirements for efficient repair of a site-specific DNA double-strand break (DSB) in Schizosaccharomyces pombe. Tech nology was developed in which a unique DSB could be generated in a non-essential minichromosome, Ch¹⁶, using the Saccharomyces cerevisiae HO-endonuclease and its target site, MATa. DSB repair in this context was predominantly through interchromosomal gene conversion. We found that the homologous recombination (HR) genes rhp51⁺, rad22A⁺, rad32⁺ and the nucleotide excision repair gene rad16⁺ were required for efficient interchromosomal gene conversion. Further, DSB-induced cell cycle delay and efficient HR required the DNA integrity checkpoint gene rad3⁺. Rhp55 was required for interchromosomal gene conversion; however, an alternative DSB repair mechanism was used in an rhp55 background involving ku70⁺ and rhp51⁺. Surprisingly, DSB-induced minichromosome loss was significantly reduced in ku70 and lig4 non-homologous end joining (NHEJ) mutant backgrounds compared with wild type. Furthermore, roles for Ku70 and Lig4 were identified in suppressing DSB-induced chromosomal rearrangements associated with gene conversion. These findings are consistent with both competitive and cooperative interactions between components of the HR and NHEJ pathways.