Characterization of Saccharomyces cerevisiae dna2 mutants suggests a role for the helicase late in S phase
Characterization of Saccharomyces cerevisiae dna2 mutants suggests a role for the helicase late in S phase
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DOI：
10.1091/mbc.8.12.2519
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发表时间：
1997-12-01
影响因子：
3.3
通讯作者：
Crabtree, GR
中科院分区：
文献类型：
--
作者：
Fiorentino, DF;Crabtree, GR
TOR蛋白最初被鉴定为免疫抑制剂雷帕霉素的靶点，含有ATM样“脂质激酶”结构域，是真核生物早期G1期进展所必需的。使用筛选来鉴定需要过表达Tor 1 p以维持生存能力的酿酒酵母突变体，我们分离出了一个我们称为ROT 1的基因中的突变（需要过表达Tor 1 p）。该基因与DNA 2相同，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，Tor 1 p的N-末端和C-末端区域以及激酶结构域都是拯救DNA 2突变体所必需的。Dna 2突变体也被Tor 2 p拯救，并在特定条件下与tor 1缺失突变体显示合成致死性。温度敏感性（Ts）dna 2突变体以RAD 9和MEC 1依赖的方式不可逆地停滞在G2/M期，表明Dna 2 p在S期起作用。在dna 2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。温度变化实验表明，Dna 2 p的功能在后期S期，虽然dna 2突变体是不缺乏批量DNA合成。这些数据表明，Dna 2 p是不需要复制叉进展，但可能需要为以后的事件，如冈崎片段成熟。
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.