CHARACTERIZATION OF SACCHAROMYCES-CEREVISIAE DNA2 MUTANTS SUGGESTS A ROLE FOR THE HELICASE LATE IN S-PHASE

The TOR proteins, originally identified as targets of the immunosuppre ssant rapamycin, contain an ATM-like ''lipid kinase'' domain and are r equired for early G1 progression in eukaryotes. Using a screen to iden tify Saccharomyces cerevisiae mutants requiring overexpression of Tor1 p for viability, we have isolated mutations in a gene we call ROT1 (re quires overexpression of Tor1p). This gene is identical to DNA2, encod ing 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 mutant s. Dna2 mutants are also rescued by Tor2p and show synthetic lethality with tor1 deletion mutants under specific conditions. Temperature-sen sitive (Ts) dna2 mutants arrest irreversibly at G2/M in a RAD9- and ME C1-dependent manner, suggesting that Dna2p has a role in S phase. Freq uencies of mitotic recombination and chromosome loss are elevated in d na2 mutants, also supporting a role for the protein in DNA synthesis. Temperature-shift experiments indicate that Dna2p functions during lat e S phase, although dna2 mutants are not deficient in bulk DNA synthes is. These data suggest that Dna2p is not required for replication fork progression but may be needed for a later event such as Okazaki fragm ent maturation.