SAXS STUDIES ON REACTION PATHWAY OF DNA POLYMERASE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 将对DNA聚合酶及其复合体进行SAXS研究，验证其可行性，并优化反应条件。前期的实验是成功的，并对导致聚集最小的条件也进行了优化。在蛋白质制备的最后一步，所有实验样品都经过凝胶过滤层析纯化，并用银染色法用SDS-PAGE检测纯度。上述生化方法均未检测到这些组分的聚集现象。DNA底物用凝胶电泳法纯化。只有最纯净的部分被汇集在一起进行SAXS测量。我们对蛋白质-DNA和蛋白质-DNA-dNTP复合体的结构很感兴趣，这些数据将用于核磁共振的补充结构研究。

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.