Santocanale C, Neecke H, Longhese M P, Lucchini G, Plevani P
Dipartimento di Genetica, Università degli Studi di Milano, Italy.
J Mol Biol. 1995 Dec 8;254(4):595-607. doi: 10.1006/jmbi.1995.0641.
The in vivo function of the 34 kDa subunit of yeast replication protein A (RPA), encoded by the RFA2 gene, has been studied by analyzing the effect of Rpa34 depletion and by producing and characterizing rfa2 temperature-sensitive mutants. We show that unbalanced stoichiometry of the RPA subunits does not affect cell growth and cell cycle progression until the level of Rpa34 becomes rate-limiting, at which point cells arrest with a late S/G2 DNA content. Rpa34 is involved in DNA replication in vivo, since rfa2 ts mutants are defective in S phase progression and ARS plasmid stability, and rfa2 pol1 double mutants are non-viable. Moreover, when shifted to the restrictive temperature, about 50% of the rfa2 mutant cells rapidly die while traversing the S phase and the surviving cells arrest in late S/G2 at the RAD9 checkpoint. Finally, rfa2 mutant cells have a mutator and hyper-recombination phenotype and are more sensitive to hydroxyurea and methyl-methane-sulfonate than wild-type cells.
通过分析Rpa34缺失的影响以及构建和鉴定rfa2温度敏感突变体，对酵母复制蛋白A（RPA）34 kDa亚基（由RFA2基因编码）的体内功能进行了研究。我们发现，直到Rpa34水平成为限速因素之前，RPA亚基的化学计量失衡并不影响细胞生长和细胞周期进程，此时细胞会停滞在S期晚期/G2期，DNA含量增加。Rpa34参与体内DNA复制，因为rfa2温度敏感突变体在S期进程和自主复制序列（ARS）质粒稳定性方面存在缺陷，并且rfa2 pol1双突变体无法存活。此外，当转移到限制温度时，约50%的rfa2突变体细胞在穿越S期时迅速死亡，而存活的细胞在RAD9检查点停滞在S期晚期/G2期。最后，rfa2突变体细胞具有突变和高重组表型，并且比野生型细胞对羟基脲和甲基磺酸甲酯更敏感。
