DeRose Eugene F, Li Dawei, Darden Thomas, Harvey Scott, Perrino Fred W, Schaaper Roel M, London Robert E
Laboratory of Structural Biology and Laboratory of Molecular Genetics, NIEHS, Box 12233, Research Triangle Park, North Carolina 27709, USA.
Biochemistry. 2002 Jan 8;41(1):94-110. doi: 10.1021/bi0114170.
The DNA polymerase III holoenzyme (HE) is the primary replicative polymerase of Escherichia coli. The epsilon subunit of the HE complex provides the 3'-exonucleolytic proofreading activity for this enzyme complex. epsilon consists of two domains: an N-terminal domain containing the proofreading exonuclease activity (residues 1-186) and a C-terminal domain required for binding to the polymerase (alpha) subunit (residues 187-243). Multidimensional NMR studies of (2)H-, (13)C-, and (15)N-labeled N-terminal domains (epsilon186) were performed to assign the backbone resonances and measure H(N)-H(N) nuclear Overhauser effects (NOEs). NMR studies were also performed on triple-lableled [U-(2)H,(13)C,(15)N]epsilon186 containing Val, Leu, and Ile residues with protonated methyl groups, which allowed for the assignment of H(N)-CH(3) and CH(3)-CH(3) NOEs. Analysis of the (13)C(alpha), (13)C(beta), and (13)CO shifts, using chemical shift indexing and the TALOS program, allowed for the identification of regions of the secondary structure. H(N)-H(N) NOEs provided information on the assembly of the extended strands into a beta-sheet structure and confirmed the assignment of the alpha helices. Measurement of H(N)-CH(3) and CH(3)-CH(3) NOEs confirmed the beta-sheet structure and assisted in the positioning of the alpha helices. The resulting preliminary characterization of the three-dimensional structure of the protein indicated that significant structural homology exists with the active site of the Klenow proofreading exonuclease domain, despite the extremely limited sequence homology. On the basis of this analogy, molecular modeling studies of epsilon186 were performed using as templates the crystal structures of the exonuclease domains of the Klenow fragment and the T4 DNA polymerase and the recently determined structure of the E. coli Exonuclease I. A multiple sequence alignment was constructed, with the initial alignment taken from the previously published hidden Markov model and NMR constraints. Because several of the published structures included complexed ssDNA, we were also able to incorporate an A-C-G trinucleotide into the epsilon186 structure. Nearly all of the residues which have been identified as mutators are located in the portion of the molecule which binds the DNA, with most of these playing either a catalytic or structural role.
DNA聚合酶III全酶（HE）是大肠杆菌的主要复制性聚合酶。HE复合物的ε亚基为该酶复合物提供3' - 核酸外切酶校对活性。ε由两个结构域组成：一个包含校对核酸外切酶活性的N端结构域（第1 - 186位氨基酸残基）和一个与聚合酶（α）亚基结合所需的C端结构域（第187 - 243位氨基酸残基）。对用（2）H、（13）C和（15）N标记的N端结构域（ε186）进行多维核磁共振研究，以确定主链共振并测量H（N）-H（N）核Overhauser效应（NOE）。还对含有质子化甲基的Val、Leu和Ile残基的三标记[U - （2）H，（13）C，（15）N]ε186进行了核磁共振研究，这使得能够确定H（N）-CH（3）和CH（3）-CH（3）NOE。使用化学位移索引和TALOS程序对（13）Cα、（13）Cβ和（13）CO化学位移进行分析，从而确定二级结构区域。H（N）-H（N）NOE提供了有关延伸链组装成β - 折叠结构的信息，并证实了α螺旋的归属。对H（N）-CH（3）和CH（3）-CH（3）NOE的测量证实了β - 折叠结构，并辅助确定了α螺旋的位置。所得蛋白质三维结构的初步特征表明，尽管序列同源性极低，但与Klenow校对核酸外切酶结构域的活性位点存在显著的结构同源性。基于这种相似性，以Klenow片段和T4 DNA聚合酶的核酸外切酶结构域的晶体结构以及最近确定的大肠杆菌核酸外切酶I的结构为模板，对ε186进行了分子建模研究。构建了一个多序列比对，初始比对取自先前发表的隐马尔可夫模型和核磁共振约束。由于一些已发表的结构包含复合单链DNA，我们还能够将一个A - C - G三核苷酸纳入ε186结构。几乎所有被鉴定为突变体的残基都位于分子中与DNA结合的部分，其中大多数发挥催化或结构作用。
