STRUCTURAL STUDIES OF HIGH & LOW-FIDELITY OF DNA POLYMERASES BY SAXS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。DNA复制是细胞繁殖所必需的基本生物学过程。DNA复制的核心特征是由DNA聚合酶介导的模板诱导的核苷酸转移反应。该功能的关键是在聚合酶介导的引物伸长事件中维持核苷酸插入的高保真度。人们普遍认为，核苷酸插入的高保真度是由DNA聚合酶核苷酸结合亚域的关闭来控制的，以响应正确的核苷酸结合。该模型还认为，不正确的核苷酸不会发生这种构象变化，但对于不匹配的三元配合物，没有结构研究支持该模型。在此，我们报道了沿着高保真Pol β（哺乳动物DNA聚合酶β）和低保真Pol X（非洲发誓热病毒DNA聚合酶X）反应途径监测不同构象状态的溶液结构研究。利用一维小角度X射线溶液数据重建的各种形态Pol β和Pol X的三维密度图不仅与已有报道的Pol β和Pol X的高分辨率晶体结构很好地叠加，而且还为许多其他无结构的Pol β和Pol X配合物提供了新的信息。我们目前的研究结果表明，通过核苷酸结合亚结构域，错配的三元配合物Pol β的构象发生了小而明显的变化。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. DNA replication is a fundamental biological process required for cellular reproduction. The central feature of DNA replication is the template-induced nucleotidyl transfer reaction mediated by DNA polymerases. Critical to this function is the maintenance of high fidelity in the insertion of nucleotide in the event of polymerases-mediated elongation of the primer. It is widely accepted that the high fidelity of nucleotide insertion is controlled by closure of the DNA polymerases nucleotide-binding subdomain in response to binding the correct nucleotide. The model also holds that no such conformational change occurs in response to the incorrect nucleotides, yet no structural studies in support of the model exists for mismatched ternary complexes. Herein, we report the solution structural studies on monitoring different conformational states along the reaction pathway of high-fidelity of Pol beta (mammalian DNA polymerase beta) and of low-fidelity of Pol X (DNA polymerase X from African sworn fever virus). The reconstructed three-dimensional density maps on various forms of Pol beta and Pol X from one-dimensional small-angle X-ray solution data are found to be not only well-superimposed to the reported high-resolution crystal structures of Pol beta and Pol X, but also provide the novel information on many other complexes of Pol beta and Pol X with no structures available. Our current results suggest that a small but clear conformational change via the nucleotide-binding subdomain for the mismatched ternary complex of Pol beta.