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
H Y
HY
National Creative Research Initiative Center for Cell Cycle Control, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Changan-Ku Suwon, Kyunggi-Do, 440-746, Korea.
Choi
E
E
Bae
S H
SH
Lee
K H
KH
Gim
B S
BS
Kim
H D
HD
Park
C
C
MacNeill
S A
SA
Seo
Y S
YS
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002875:Chromosomes / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics; D004268:DNA-Binding Proteins; D004706:Endodeoxyribonucleases; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D018628:Gene Dosage; D005804:Genes, Lethal; D018816:Genes, cdc; D020662:Guanine Nucleotide Exchange Factors; D009152:Mutagenicity Tests; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D016196:S Phase / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013172:Spores, Fungal / Q000235:genetics / Q000254:growth & development; D013696:Temperature; D020798:Two-Hybrid System Techniques; D014466:Ultraviolet Rays
C090459:CDC1 protein, S cerevisiae; C073594:CDC24 protein, S cerevisiae; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; C022262:Okazaki fragments; D011518:Proto-Oncogene Proteins; D029701:Saccharomyces cerevisiae Proteins; C073998:RAD2 protein, S cerevisiae; D004247:DNA; D004256:DNA Polymerase I; C083536:POL1 protein, S cerevisiae; D004706:Endodeoxyribonucleases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C410222:DNA2 protein, S cerevisiae