DNA ligase I from Saccharomyces cerevisiae: physical and biochemical characterization of the CDC9 gene product.
DNA ligase I from Saccharomyces cerevisiae: physical and biochemical characterization of the CDC9 gene product.
复制标题
来自酿酒酵母的 DNA 连接酶 I：CDC9 基因产物的物理和生化特征。
DOI：
10.1021/bi00162a013
复制
发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Friedberg,EC
中科院分区：
文献类型：
--
作者：
Tomkinson,AE;Tappe,NJ;Friedberg,EC
1992年9月2日收到的修订稿摘要：遗传学研究表明，酿酒酵母CDC9基因产物在功能上与哺乳动物DNA连接酶I同源，是DNA复制所必需的，也参与DNA修复和基因重组。在本研究中，我们对酵母酶进行了纯化。在变性条件下测定，CDC9蛋白的多肽相对分子质量为87 kDa。这种酶的天然形式是一个80 kDa的不对称单体。这两个估算值都与CDC9基因翻译序列预测的M_1=84 406吻合较好。CDC9DNA连接酶通过与所有已知的依赖于ATP的DNA连接酶相同的基本反应机制发挥作用。催化功能位于70 kDa的C末端，在哺乳动物DNA连接酶I和裂殖酵母的CDCl7 DNA连接酶中保守。ATP类似物ATPaS抑制连接反应，尽管CDC9蛋白确实形成了酶-硫代腺苷中间体。由于CDC9 DNA连接酶与哺乳动物DNA连接酶I具有相同的底物特异性，因此该酶可被认为是酿酒酵母的DNA连接酶I。有遗传证据表明，DNA连接酶可能直接参与了容易出错的DNA修复。我们检测了CDC9 DNA连接酶连接末端错配的缺口的能力。NICKS 5‘端的错配对DNA连接的影响很小，而3’端与嘌呤相反的错配抑制了DNA连接。DNA连接酶将DNA分子与错配的末端连接起来，可能是导致突变的原因。
Revised Manuscript Received September 2, 1992 abstract: Genetic studies have previously demonstrated that the Saccharomyces cerevisiae CDC9 gene product, which is functionally homologous to mammalian DNA ligase I, is required forDNA replication and is also involved in DNA repair and genetic recombination. In the present study we have purified the yeast enzyme. When measured under denaturing conditions, Cdc9 protein has a polypeptide molecular mass of 87 kDa. The native form of the enzyme is an 80-kDa asymmetric monomer. Both estimates are in good agreement with the M,= 84 406 predicted from the translated sequence of the CDC9 gene. Cdc9 DNA ligase acts via the same basic reaction mechanism employed by all known ATP-dependent DNA ligases. The catalytic functions reside in a 70-kDa C-terminal domain that is conserved in mammalianDNA ligase I and in Cdcl7 DNA ligase from Schizosaccharomyces pombe. The ATP analog ATPaS inhibits the ligation reaction, although Cdc9 protein does form an enzyme-thioadenylate intermediate. Since Cdc9 DNA ligase exhibited the same substrate specifity as mammalian DNA ligase I, this enzyme can be considered to be the DNA ligase I of S. cerevisiae. There is genetic evidence suggesting that DNA ligase may be directly involved in error-prone DNA repair. We examined the ability of Cdc9 DNA ligase to join nicks with mismatches at the termini. Mismatches at the 5'termini of nicks had very little effect on ligation, whereas mismatches opposite a purine at 3'termini inhibited DNA ligation. The joiningof DNA molecules with mismatched termini by DNA ligase may be responsible for the generation of mutations.