The EMBO journal, 2003-03-17, Vol.22 (6), p.1419-1430
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 rhp55Δ background involving ku70+ and rhp51+. Surprisingly, DSB‐induced minichromosome loss was significantly reduced in ku70Δ and lig4Δ 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.
Chromosomes, Fungal ; Deoxyribonucleases, Type II Site-Specific - genetics ; Deoxyribonucleases, Type II Site-Specific - metabolism ; DNA Damage ; DNA integrity checkpoint ; DNA Repair - genetics ; DNA, Fungal - genetics ; DNA, Fungal - metabolism ; Fungal Proteins - genetics ; Fungal Proteins - metabolism ; Gamma Rays ; Gene Conversion ; Genes, Fungal ; HO-endonuclease ; homologous recombination ; Models, Biological ; Mutation ; non-homologous end joining ; Recombination, Genetic ; Saccharomyces cerevisiae - genetics ; Saccharomyces cerevisiae Proteins - genetics ; Saccharomyces cerevisiae Proteins - metabolism ; Schizosaccharomyces - genetics ; Schizosaccharomyces - radiation effects ; site-specific DNA double-strand break
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