Biochemical and Biological in vivo Functions of Dna2p in Saccharomyces cerevisiae

摘要: We have characterized a temperature-sensitive yeast mutant, dna2ts, which is defective in DNA replication. DNA2 essential and encodes 172-kDa protein with helicase motif at its C-terminal portion. A homology search showed that Dna2p conserved structurally among species. Even Xenopus laevis Dna2 was able to complement an S. cerevisiae dna2-1 mutant strain for growth the nonpermissive temperature, suggesting also functionally. The site of mutation mapped using marker rescue technique turned out proline 504 serine, placing N-terminal portion protein, important activity Dna2p. Recombinant ScDna2p expressed insect cells purified. With purified we were demonstrate single-stranded endonuclease/helicase single stranded dependent ATPase, has various biochemical functions. found helicase-nuclease component telomeric chromatin. Both chromatin immunoprecipitation immunofluorescence associates telomeres but not bulk chromosomal G1 phase. In S phase, there dramatic redistribution ofDna2p from sites throughout replicating chromosomes. again localized late S, where it remains through G2 until next Telomeric localization required Sir3p, since amount by two different assays, one hybrid ChIP, severely reduced strains lacking Sir3p. distributed the nucleus growing presence DSB-inducing agents such as bleomycin. Finally, show functionally telomerase-dependent de novo telomere synthesis participates lengthening mutants lacking telomerase, genetic instability due dna2 mutations lead premature aging phenotype.