The second subunit of DNA polymerase III (δ) is encoded by the HYS2 gene in Saccharomyces cerevisiae
The second subunit of DNA polymerase III (δ) is encoded by the HYS2 gene in Saccharomyces cerevisiae
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DOI：
10.1093/nar/26.2.477
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发表时间：
1998-01-15
影响因子：
14.9
通讯作者：
Sugino, A
中科院分区：
文献类型：
--
作者：
Hashimoto, K;Nakashima, N;Sugino, A
将酿酒酵母的DNA聚合酶III（δ）纯化为至少两种多肽的复合物，其分子量为125和55 kDa，通过SDS-PAGE判断。本文测定了125和55 kDa多肽的部分氨基酸序列，发现它们分别与CDC 2和HYS 2基因的核苷酸序列预测的部分氨基酸序列相匹配。我们还通过Western blotting证实，Hys 2蛋白与DNA聚合酶III活性以及Cdc 2多肽共纯化。在温敏的Hys 2突变细胞提取物中不能检测到DNA聚合酶III活性的复合物形式，尽管发现了另一种形式的DNA聚合酶III。这种形式的DNA聚合酶III也可以在野生型提取物中检测到，与Hys 2蛋白无关，并且不受增殖细胞核抗原（PCNA）的刺激，复制因子A（RF-A）或复制因子C（RF-C），在多拷贝质粒上的CDC 2基因可以抑制CDC 2 -1和CDC 2 -2突变的温度敏感性生长表型，这些数据表明，HYS 2基因编码的55 kDa多肽是酿酒酵母中DNA聚合酶III复合物的亚基之一，并且是由DNA聚合酶III催化的高度进行性DNA合成所必需的PCNA、RF-A和RF-C的表达。
DNA polymerase III (delta) of Saccharomyces cerevisiae is purified as a complex of at least two polypeptides with molecular masses of 125 and 55 kDa as judged by SDS-PAGE. In this paper we determine partial amino acid sequences of the 125 and 55 kDa polypeptides and find that they match parts of the amino acid sequences predicted from the nucleotide sequence of the CDC2 and HYS2 genes respectively, We also show by Western blotting that Hys2 protein co-purifies with DNA polymerase III activity as well as Cdc2 polypeptide. The complex form of DNA polymerase III activity could not be detected in thermosensitive hys2 mutant cell extracts, although another form of DNA polymerase III was found, This form of DNA polymerase III, which could also be detected in wild-type extracts, was not associated with Hys2 protein and was not stimulated by addition of proliferating cell nuclear antigen (PCNA), replication factor A (RF-A) or replication factor C (RF-C), The temperature-sensitive growth phenotype of hys2-1 and hys2-2 mutations could be suppressed by the CDC2 gene on a multicopy plasmid, These data suggest that the 55 kDa polypeptide encoded by the HYS2 gene is one of the subunits of DNA polymerase III complex in S.cerevisiae and is required for highly processive DNA synthesis catalyzed by DNA polymerase III in the presence of PCNA, RF-A and RF-C.