Silencing and DNA double-strand break repair in budding yeast

Sammanfattning: Transcriptional silencing that makes large chromosomal domains inaccessible for the transcriptional apparatus is nucleated at DNA elements called silencers. In K.lactis a 102bp HML? silencer was defined revealing three distinct protein-binding regions (A, B, and C) that were required for silencing of HML?. The B-sequence was a binding-site for the transcription factor Reb1. Temperature sensitive reb1 alleles displayed silencing defects at the restrictive temperature. In addition, point mutations in the B-sequence abolished both Reb1-binding and silencer function. Silencing in the related yeast S.cerevisiae does not require Reb1 indicating that silencer-binding proteins diverge rapidly in hemiascomysetous species. Yeast strains lacking Sir2, Sir3 or Sir4 display a defect in the DNA double-strand break repair pathway, called nonhomologous end joining (NHEJ). We identified the first haploid-specific gene (NEJ1) involved in DSB repair and demonstrated that the NHEJ defect of sir mutants was largely attributable to the transcriptional repression of NEJ1. Cells lacking Nej1 initially slowed down the 5´to 3´DNA degradation rate at a DSB suggesting that Nej1 has a direct role in end joining repair. The localization of Nej1 to the nucleus and a direct molecular interaction with the NHEJ protein Lif1 supported this notion. Indirect participation of Nej1 in end joining by regulating the transport or stability of Lif1 was excluded.In K.lactis integration of exogenous DNA via illegitimate recombination (IR) occurred with a high frequency and was completely dependent on the NHEJ pathway. NHEJ was capable of efficient repair of a wide variety of DNA ends and both haploid and diploid cells performed NHEJ with the same efficiency. Furthermore, IR preferentially took place within intergenic regions and ribosomal DNA. A rad52 mutant had no affect on targeting preference for IGRs indicating that DSBs in ORFs were not primarily repaired by homologous recombination (HR). Introduction of an ectopic DSB preferentially targeted IR to this site. Thus, we suggest that IR occurs at DSBs and by analysing IR-events spontaneously arising mitotic DSBs can be mapped.