Fiorentino D F, Crabtree G R
Department of Developmental Biology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305, USA.
Mol Biol Cell. 1997 Dec;8(12):2519-37. doi: 10.1091/mbc.8.12.2519.
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.
TOR蛋白最初被鉴定为免疫抑制剂雷帕霉素的作用靶点，它含有一个类似ATM的“脂质激酶”结构域，是真核生物G1期早期进程所必需的。通过筛选鉴定出需要过表达Tor1p才能存活的酿酒酵母突变体，我们分离出了一个我们称为ROT1（需要过表达Tor1p）的基因中的突变。该基因与DNA2相同，DNA2编码DNA复制所需的解旋酶。与其在细胞周期进程中的作用一样，Tor1p的N端和C端区域以及激酶结构域都是拯救dna2突变体所必需的。Tor2p也能拯救dna2突变体，并且在特定条件下与tor1缺失突变体表现出合成致死性。温度敏感（Ts）的dna2突变体以依赖RAD9和MEC1的方式在G2/M期不可逆地停滞，这表明Dna2p在S期发挥作用。dna2突变体中，有丝分裂重组和染色体丢失的频率升高，这也支持了该蛋白在DNA合成中的作用。温度转换实验表明，Dna2p在S期后期发挥作用，尽管dna2突变体在总体DNA合成方面没有缺陷。这些数据表明，Dna2p不是复制叉前进所必需的，但可能是冈崎片段成熟等后续事件所必需的。
