Genetic Analyses of Schizosaccharomyces pombe dna2+ Reveal That Dna2 Plays an Essential Role in Okazaki Fragment Metabolism

摘要

In this report, we investigated the phenotypes caused by temperature-sensitive (ts) mutant alleles of dna2 + of Schizosaccharomyces pombe , a homologue of DNA2 of budding yeast, in an attempt to further define its function in vivo with respect to lagging-strand synthesis during the S-phase of the cell cycle. At the restrictive temperature, dna2 (ts) cells arrested at late S-phase but were unaffected in bulk DNA synthesis. Moreover, they exhibited aberrant mitosis when combined with checkpoint mutations, in keeping with a role for Dna2 in Okazaki fragment maturation. Similarly, spores in which dna2 + was disrupted duplicated their DNA content during germination and also arrested at late S-phase. Inactivation of dna2 + led to chromosome fragmentation strikingly similar to that seen when cdc17 +, the DNA ligase I gene, is inactivated. The temperature-dependent lethality of dna2 (ts) mutants was suppressed by overexpression of genes encoding subunits of polymerase δ ( cdc1 + and cdc27 +), DNA ligase I ( cdc17 +), and Fen-1 ( rad2 +). Each of these gene products plays a role in the elongation or maturation of Okazaki fragments. Moreover, they all interacted with S. pombe Dna2 in a yeast two-hybrid assay, albeit to different extents. On the basis of these results, we conclude that dna2 + plays a direct role in the Okazaki fragment elongation and maturation. We propose that dna2 + acts as a central protein to form a complex with other proteins required to coordinate the multienzyme process for Okazaki fragment elongation and maturation.