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PMID: 9398673 Published · ppublish English Journal Article

Characterization of Saccharomyces cerevisiae dna2 mutants suggests a role for the helicase late in S phase.

Molecular biology of the cell ·Vol. 8 ·No. 12 ·1997-12-00 ·Pages 2519-37

Fiorentino DF, Crabtree GR

Abstract

The TOR proteins, originally identified as targets of the immunosuppressant rapamycin, contain an ATM-like "lipid kinase" domain and are required for early G1 progression in eukaryotes. Using a screen to identify Saccharomyces cerevisiae mutants requiring overexpression of Tor1p for viability, we have isolated mutations in a gene we call ROT1 (requires overexpression of Tor1p). This gene is identical to DNA2, encoding a helicase required for DNA replication. As with its role in cell cycle progression, both the N-terminal and C-terminal regions, as well as the kinase domain of Tor1p, are required for rescue of dna2 mutants. Dna2 mutants are also rescued by Tor2p and show synthetic lethality with tor1 deletion mutants under specific conditions. Temperature-sensitive (Ts) dna2 mutants arrest irreversibly at G2/M in a RAD9- and MEC1-dependent manner, suggesting that Dna2p has a role in S phase. Frequencies of mitotic recombination and chromosome loss are elevated in dna2 mutants, also supporting a role for the protein in DNA synthesis. Temperature-shift experiments indicate that Dna2p functions during late S phase, although dna2 mutants are not deficient in bulk DNA synthesis. These data suggest that Dna2p is not required for replication fork progression but may be needed for a later event such as Okazaki fragment maturation.

MeSH Terms
Adenosine Triphosphatases/chemistry,genetics,metabolism Amino Acid Sequence Cell Cycle Proteins/genetics,metabolism Cloning, Molecular DNA Helicases/chemistry,genetics,metabolism DNA Replication/genetics DNA, Fungal/biosynthesis,chemistry,genetics,metabolism Fungal Proteins/chemistry,genetics,metabolism Gene Expression Genes, Essential/genetics Genes, Fungal/genetics,physiology Genes, Suppressor/genetics Intracellular Signaling Peptides and Proteins Molecular Sequence Data Mutation/genetics Phenotype Phosphatidylinositol 3-Kinases Phosphotransferases (Alcohol Group Acceptor)/chemistry,genetics,metabolism Protein Binding Protein Serine-Threonine Kinases Recombination, Genetic/genetics S Phase/genetics Saccharomyces cerevisiae/cytology,enzymology,genetics Saccharomyces cerevisiae Proteins Sequence Deletion/genetics Temperature
Chemicals
Cell Cycle Proteins DNA, Fungal Fungal Proteins Intracellular Signaling Peptides and Proteins Saccharomyces cerevisiae Proteins rad9 protein Phosphotransferases (Alcohol Group Acceptor) TOR1 protein, S cerevisiae TOR2 protein, S cerevisiae MEC1 protein, S cerevisiae Protein Serine-Threonine Kinases Adenosine Triphosphatases DNA Helicases DNA2 protein, S cerevisiae
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Fiorentino D F
Department of Developmental Biology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305, USA.
Crabtree G R
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Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1059-1524
DOI
10.1091/mbc.8.12.2519
Published
1997-12-00
Pages
2519-37
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC25725
Subset
IM
External Links
PubMed source DOI: 10.1091/mbc.8.12.2519 PMC Full Text View journal details
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