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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。DNA复制是细胞繁殖所需的基本生物学过程。DNA复制的中心特征是由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延伸的情况下保持核苷酸插入的高保真度。广泛接受的是，核苷酸插入的高保真度是通过响应于结合正确的核苷酸而关闭DNA聚合酶核苷酸结合亚结构域来控制的。该模型还认为，没有这样的构象变化发生在响应不正确的核苷酸，但没有结构研究支持该模型存在的不匹配的三元复合物。在此，我们报告的解决方案的结构研究监测不同的构象状态沿着的反应途径的高保真度的Pol β（哺乳动物DNA聚合酶β）和高保真度的Pol X（DNA聚合酶X从非洲宣誓热病毒）。从一维小角X射线溶液数据重建的各种形式的Pol beta和Pol X的三维密度图被发现不仅很好地叠加到报告的高分辨率晶体结构的Pol beta和Pol X，但也提供了新的信息，对许多其他复杂的Pol beta和Pol X没有可用的结构。我们目前的研究结果表明，一个小的，但明确的构象变化，通过核苷酸结合亚结构域的错配三元复合物的Pol β。

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.