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