Stocki S A, Nonay R L, Reha-Krantz L J
Department of Biological Sciences, University of Alberta, Edmonton, Canada.
J Mol Biol. 1995 Nov 17;254(1):15-28. doi: 10.1006/jmbi.1995.0595.
Many DNA polymerases are multifunctional with the ability to replicate DNA as well as to proofread misincorporated nucleotides. Since polymerase and 3'--> 5' exonuclease activities appear to reside in spatially distinct active centers, there must be some mechanism for coordinating replication with proofreading and for transferring DNA between the two active centers. We have designed a genetic selection scheme to isolate bacteriophage T4 mutant DNA polymerases that are defective in "switching" between polymerase and exonuclease activities. Amino acid residues that affected active-site-switching were identified in four regions of the T4 DNA polymerase: two regions in the proposed exonuclease domain. Representative mutant DNA polymerases from each region were purified for biochemical studies. We propose that amino acid substitutions identified by mutational analysis affect critical contacts between T4 DNA polymerase and DNA that are required for transfer of DNA between the polymerase and exonuclease active centers.
许多DNA聚合酶具有多功能性，既能复制DNA，又能校对错误掺入的核苷酸。由于聚合酶活性和3'→5'核酸外切酶活性似乎存在于空间上不同的活性中心，因此必定存在某种机制来协调复制与校对，并在两个活性中心之间转移DNA。我们设计了一种遗传筛选方案，以分离在聚合酶活性和核酸外切酶活性之间“切换”存在缺陷的噬菌体T4突变DNA聚合酶。在T4 DNA聚合酶的四个区域中鉴定出了影响活性位点切换的氨基酸残基：在所提出的核酸外切酶结构域中有两个区域。对每个区域的代表性突变DNA聚合酶进行了纯化，用于生化研究。我们提出，通过突变分析鉴定出的氨基酸取代会影响T4 DNA聚合酶与DNA之间的关键接触，而这种接触是DNA在聚合酶活性中心和核酸外切酶活性中心之间转移所必需 的。
