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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, Ch(16), 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 rhp55Delta background involving ku70(+) and rhp51(+). Surprisingly, DSB-induced minichromosome loss was significantly reduced in ku70Delta and lig4Delta 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.

Original publication

DOI

10.1093/emboj/cdg119

Type

Journal article

Journal

EMBO J

Publication Date

17/03/2003

Volume

22

Pages

1419 - 1430

Keywords

Chromosomes, Fungal, DNA Damage, DNA Repair, DNA, Fungal, Deoxyribonucleases, Type II Site-Specific, Fungal Proteins, Gamma Rays, Gene Conversion, Genes, Fungal, Models, Biological, Mutation, Recombination, Genetic, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Schizosaccharomyces