Eissenberg J C, Ayyagari R, Gomes X V, Burgers P M
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University Health Sciences Center, Missouri 63104, USA.
Mol Cell Biol. 1997 Nov;17(11):6367-78. doi: 10.1128/MCB.17.11.6367.
The importance of the interdomain connector loop and of the carboxy-terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases delta (Poldelta) and epsilon (Pol epsilon) was investigated by site-directed mutagenesis. Two alleles, pol30-79 (IL126,128AA) in the interdomain connector loop and pol30-90 (PK252,253AA) near the carboxy terminus, caused growth defects and elevated sensitivity to DNA-damaging agents. These two mutants also had elevated rates of spontaneous mutations. The mutator phenotype of pol30-90 was due to partially defective mismatch repair in the mutant. In vitro, the mutant PCNAs showed defects in DNA synthesis. Interestingly, the pol30-79 mutant PCNA (pcna-79) was most defective in replication with Poldelta, whereas pcna-90 was defective in replication with Pol epsilon. Protein-protein interaction studies showed that pcna-79 and pcna-90 failed to interact with Pol delta and Pol epsilon, respectively. In addition, pcna-90 was defective in interaction with the FEN-1 endo-exonuclease (RTH1 product). A loss of interaction between pcna-79 and the smallest subunit of Poldelta, the POL32 gene product, implicates this interaction in the observed defect with the polymerase. Neither PCNA mutant showed a defect in the interaction with replication factor C or in loading by this complex. Processivity of DNA synthesis by the mutant holoenzyme containing pcna-79 was unaffected on poly(dA) x oligo(dT) but was dramatically reduced on a natural template with secondary structure. A stem-loop structure with a 20-bp stem formed a virtually complete block for the holoenzyme containing pcna-79 but posed only a minor pause site for wild-type holoenzyme, indicating a function of the POL32 gene product in allowing replication past structural blocks.
通过定点诱变研究了酿酒酵母增殖细胞核抗原（PCNA）的结构域间连接环和羧基末端结构域与DNA聚合酶δ（Poldelta）和ε（Pol epsilon）功能相互作用的重要性。两个等位基因，结构域间连接环中的pol30 - 79（IL126,128AA）和羧基末端附近的pol30 - 90（PK252,253AA），导致生长缺陷并增加了对DNA损伤剂的敏感性。这两个突变体的自发突变率也有所提高。pol30 - 90的诱变表型是由于突变体中错配修复部分缺陷所致。在体外，突变的PCNA在DNA合成中表现出缺陷。有趣的是，pol30 - 79突变体PCNA（pcna - 79）在与Poldelta的复制中缺陷最明显，而pcna - 90在与Pol epsilon的复制中存在缺陷。蛋白质 - 蛋白质相互作用研究表明，pcna - 79和pcna - 90分别无法与Pol delta和Pol epsilon相互作用。此外，pcna - 90在与FEN - 1内切 - 外切核酸酶（RTH1产物）的相互作用中存在缺陷。pcna - 79与Poldelta最小亚基POL32基因产物之间相互作用的丧失，表明这种相互作用与观察到的聚合酶缺陷有关。两种PCNA突变体在与复制因子C的相互作用或由该复合物加载方面均未表现出缺陷。含有pcna - 79的突变全酶在聚（dA）×寡聚（dT）上进行DNA合成的持续合成能力未受影响，但在具有二级结构的天然模板上则显著降低。具有20个碱基对茎的茎环结构对含有pcna - 79的全酶形成了几乎完全的阻碍，但对野生型全酶仅构成一个较小的暂停位点，这表明POL32基因产物在允许复制越过结构阻碍方面具有功能。
