Genetic analyses of Schizosaccharomyces pombe dna2+ reveal that Dna2 plays an essential role in Okazaki fragment metabolism

HY Kang, E Choi, SH Bae, KH Lee, BS Gim, HD Kim, C Park, Stuart Andrew MacNeill, YS Seo

Research output: Contribution to journalArticlepeer-review

80 Citations (Scopus)

Abstract

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 xith 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 dna(2+) 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-l (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, ive 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.

Original languageEnglish
Pages (from-to)1055-1067
Number of pages13
JournalGenetics
Volume155
Issue number3
Publication statusPublished - Jul 2000

Keywords

  • CELL NUCLEAR ANTIGEN
  • BASE EXCISION-REPAIR
  • HIGH-EFFICIENCY TRANSFORMATION
  • SINGLE-STRANDED-DNA
  • SACCHAROMYCES-CEREVISIAE
  • FISSION YEAST
  • REPLICATION FORK
  • POLYMERASE-DELTA
  • LIGASE-I
  • ENDONUCLEASE ACTIVITY

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