SAXS STUDIES ON REACTION PATHWAY OF DNA POLYMERASE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。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 研究中心，而研究中心不一定是研究者所在的机构。 将对DNA聚合酶及其复合物的小角X射线散射研究进行可行性检验和反应条件优化。早期的实验是成功的，并且导致聚集最小化的条件也被优化。在蛋白质制备的最后一步中，通过凝胶过滤色谱法纯化实验中的所有蛋白质样品，并通过SDS-PAGE通过银染色法检查纯度。 通过上述生物化学方法在这些级分中未检测到聚集。通过凝胶电泳纯化DNA底物。 仅合并最纯的级分用于SAXS测量。 我们感兴趣的是蛋白质-DNA和蛋白质-DNA-dNTP复合物的结构，这些数据将用于NMR的补充结构研究。

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. Will test the feasibility and optimize the reaction conditions on the SAXS studies of DNA polymerases and their complexes. Earlier experiments were successful, and the conditions led to minimize the aggregation were also optimized. All the protein samples in the experiments are purified by the gel- filtration chromatography in the final step of protein preparation, and the purity was checked by SDS-PAGE via silver stanning method. No aggregation were detected in these fractions by these above biochemical methods. DNA substrates are purified by gel electrophoresis. Only the puriest fractions were pooled for the SAXS measurements. We are interested in the structures of protein-DNA and protein-DNA-dNTP complexes, and the data will be used for the omplementary structural studies by NMR.