Genetic analyses of Schizosaccharomyces pombe dna2(+) reveal that dna2 plays an essential role in Okazaki fragment metabolism.
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 delta (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.
Kang HY, Choi E, Bae SH, Lee KH, Gim BS, Kim HD, Park C, MacNeill SA, Seo YS