Characterization of Budding Yeast Nonhomologous End-Joining at DNA Double-Strand Breaks and Telomeres

Sammanfattning: The yeast K. lactis efficiently integrates DNA by illegitimate recombination (IR). IR was completely dependent upon nonhomologous end-joining (NHEJ). In contrast to S. cerevisiae, NHEJ in K. lactis repaired a wide variety of DNA ends, and was not regulated by cell-type. IR events occurred primarily in 5’ intergenic regions. Deletion of RAD52 did not affect the distribution of IR sites. IR events could be targeted to an ectopic DNA double-strand break (DSB). We interpret the genomic distribution of IR events as a map of mitotic DSBs.K. lactis mre11 and rad50 strains displayed a shortened telomere phenotype, and a ku80 strain displayed shortened telomeres within a background of telomere elongation. Deletion of KU80 also resulted in excess 3’ overhangs, indicative of a role in telomere end protection. Substantial increases in subtelomeric recombination were observed in rad50 and mre11 strains, while ku80 and lig4 strains displayed modest increases. Telomere-telomere fusions (T-Tfs) induced by loss of Rap1 binding at telomeres were dependent on LIG4 and KU80, but occurred independently of NEJ1. Circularized chromosomes severely inhibited passage of a diploid strain through meiosis. We conclude that K. lactis NHEJ proteins both mediate T-Tfs and contribute to telomere capping. The Srs2 helicase was identified as a Nej1 interaction partner. Mutational analysis of Nej1 suggested that the interaction was dependent on phosphorylation of Nej1 serines 297/298 by the Dun1 kinase. Deletion of NEJ1 or DUN1 impaired Srs2 recruitment to an HO induced DSB. Srs2 recruitment was unaffected by deletion of SIZ1, implicated in promoting Srs2 recruitment to replication forks. Both srs2 and nej1 strains were deficient in a RAD52-dependent repair event similar to single-strand annealing. Additionally, srs2 and dun1 strains performed NHEJ less efficiently than a wild-type strain. We propose that Nej1 promotes Srs2 recruitment to DSBs, and supports efficient NHEJ/SSA by antagonizing Rad51.