Tang Kuo-Hsiang, Niebuhr Marc, Tung Chang-Shung, Chan Hsiu-Chien, Chou Chia-Cheng, Tsai Ming-Daw
Departments of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
Nucleic Acids Res. 2008 May;36(9):2948-57. doi: 10.1093/nar/gkn138. Epub 2008 Apr 2.
Understanding how DNA polymerases control fidelity requires elucidation of the mechanisms of matched and mismatched dNTP incorporations. Little is known about the latter because mismatched complexes do not crystallize readily. In this report, we employed small-angle X-ray scattering (SAXS) and structural modeling to probe the conformations of different intermediate states of mammalian DNA polymerase beta (Pol beta) in its wild-type and an error-prone variant, I260Q. Our structural results indicate that the mismatched ternary complex lies in-between the open and the closed forms, but more closely resembles the open form for WT and the closed form for I260Q. On the basis of molecular modeling, this over-stabilization of mismatched ternary complex of I260Q is likely caused by formation of a hydrogen bonding network between the side chains of Gln(260), Tyr(296), Glu(295) and Arg(258), freeing up Asp(192) to coordinate MgdNTP. These results argue against recent reports suggesting that mismatched dNTP incorporations follow a conformational path distinctly different from that of matched dNTP incorporation, or that its conformational closing is a major contributor to fidelity.
了解DNA聚合酶如何控制保真度需要阐明匹配和错配的脱氧核苷酸三磷酸（dNTP）掺入机制。由于错配复合物不容易结晶，因此对后者了解甚少。在本报告中，我们采用小角X射线散射（SAXS）和结构建模来探究哺乳动物DNA聚合酶β（Polβ）野生型及其易错变体I260Q不同中间状态的构象。我们的结构结果表明，错配的三元复合物处于开放形式和封闭形式之间，但更类似于野生型的开放形式和I260Q的封闭形式。基于分子建模，I260Q错配三元复合物的这种过度稳定可能是由Gln（260）、Tyr（296）、Glu（295）和Arg（258）侧链之间形成氢键网络引起的，从而使Asp（192）游离出来以配位MgdNTP。这些结果与最近的报告相悖，那些报告表明错配的dNTP掺入遵循与匹配的dNTP掺入明显不同的构象路径，或者其构象闭合是保真度的主要贡献因素。
