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.
Mesh Terms:
Adenosine Triphosphatases, Cell Cycle Proteins, Cell Line, Chromosomes, DNA, DNA Helicases, DNA Polymerase I, DNA Replication, DNA-Binding Proteins, Endodeoxyribonucleases, Fungal Proteins, Gene Dosage, Genes, Lethal, Genes, cdc, Guanine Nucleotide Exchange Factors, Mutagenicity Tests, Proto-Oncogene Proteins, S Phase, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Schizosaccharomyces, Sequence Analysis, DNA, Spores, Fungal, Temperature, Two-Hybrid System Techniques, Ultraviolet Rays
Genetics
Date: Jul. 01, 2000
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