Genetics, Vol. 155, 1055-1067, July 2000, Copyright © 2000

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

Ho-Young Kanga,b, Eunjoo Choia, Sung-Ho Baea, Kyoung-Hwa Leea, Byung-Soo Gima, Hee-Dai Kima, Chankyu Parkb, Stuart A. MacNeillc, and Yeon-Soo Seoa
a 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,
b Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yusung-Ku, Taejon, 305-701, Korea
c Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom

Corresponding author: Yeon-Soo Seo, National Creative Research Initiative Center for Cell Cycle Control, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 300 Chunchun-Dong, Changan-Ku Suwon, Kyunggi-Do, 440-746, Korea., ysseo{at}medical.skku.ac.kr (E-mail)

Communicating editor: G. R. SMITH

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.




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