Ayyagari R, Impellizzeri K J, Yoder B L, Gary S L, Burgers P M
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Mol Cell Biol. 1995 Aug;15(8):4420-9. doi: 10.1128/MCB.15.8.4420.
The saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase delta holoenzyme activity was achieved at 37 degrees C but not at 14 degrees C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase delta but completely defective for interaction with DNA polymerase epsilon. Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication.
由POL30基因编码的酿酒酵母增殖细胞核抗原（PCNA）对于DNA复制和DNA修复过程至关重要。在POL30基因中构建了21个定点突变，每个突变将两个相邻的带电荷氨基酸替换为丙氨酸。尽管含有这些双丙氨酸突变作为PCNA唯一来源的突变菌株中没有一个对生长表现出温度敏感性或冷敏感性，但约三分之一的突变体对紫外线敏感。其中一些对紫外线敏感的突变体自发突变率升高。此外，几个突变体抑制了CDC44基因中的一个冷敏感突变，该基因编码复制因子C的大亚基。通过随机诱变分离出的一个冷敏感突变体在限制温度下表现出终末表型，这与DNA复制缺陷一致。从大肠杆菌中表达并纯化了几种突变型PCNA，并测定了它们的体外性质。冷敏感突变体（pol30 - 52，S115P）在溶液中是单体而非三聚体。该突变体在体外DNA合成方面存在缺陷。通过在测定中加入大分子拥挤剂聚乙二醇，在37℃时DNA聚合酶δ全酶活性部分恢复，但在14℃时未恢复。唯一表现出生长缺陷的另一个突变体（pol30 - 6，DD41,42AA）与复制因子C和DNA聚合酶δ的相互作用部分缺陷，但与DNA聚合酶ε的相互作用完全缺陷。另外两个对DNA损伤敏感的突变体在体外没有缺陷。这些结果表明，后一种突变体在一个或多个DNA修复过程中受到特异性损害，而pol30 - 6和pol30 - 52突变体在基本DNA复制机制中表现出主要缺陷，并可能在DNA修复中存在相关缺陷。因此，DNA修复需要修复特异性蛋白质与PCNA之间的相互作用，这与DNA复制所需的相互作用不同。
