TOPOLOGY OF DNA BINDING TO DNA POLYMERASES

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。我们的目标是进一步表征与水热菌（Taq）和大肠杆菌Pol 1型DNA聚合酶结合的DNA结构拓扑结构。“Klenow”聚合酶是大肠杆菌Pol 1的“大片段”，“Klentaq”聚合酶是全长Taq聚合酶的等效大片段结构域。我们对大肠杆菌的Klenow和Klentaq片段和Taq聚合酶进行了大量直接平行的功能研究，并发现了这两种聚合酶以不同的初始“结合模式”结合DNA的差异（Datta & LiCata， J. Biol）。化学。278:5694；Datta & LiCata，核。酸Res. 31, 5590)。这些发现与人们普遍认为的DNA结合机制类似于这两种聚合酶的观点相矛盾。然而，目前还没有关于Klenow以这种初始或聚合位点结合模式与DNA结合的晶体结构数据。基于小角度x射线散射数据的从头算模型的新进展，使构建溶液中大分子的拓扑形状包络成为可能。我们在APS的BioCAT光束线上进行了最初的SAXS测量，随后对聚合酶结合的DNA进行了分子建模，产生了非常高分辨率的模型结构。这些模型结构表明聚合酶结合的拓扑结构不同于现有的晶体结构。为了进一步证实从之前的BIO-CAT SAXS数据中获得的假定拓扑结构，我们未来的计划是收集全长Taq聚合酶的等效数据，以及其他DNA结构的数据。基于SAXS的这些复合物的形状建模将提供溶液中DNA与聚合酶结合的结合拓扑结构，并将提供这两种聚合酶是否以相同方式结合相同DNA的答案。

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. We aim to further characterize the structural topology of DNA bound to Thermus aquaticus (Taq) and E. coli Pol 1 type 1 DNA polymerases. "Klenow" polymerase is the "large fragment" of E.coli Pol 1, and "Klentaq" polymerase is the equivalent large fragment domain from full length Taq polymerase. We have performed a number of directly parallel functional studies of the Klenow and Klentaq fragments of E. coli and Taq polymerases, and have identified differences that suggest that these two polymerases bind DNA with differing initial "binding modes" (Datta & LiCata, J. Biol. Chem. 278: 5694; Datta & LiCata, Nucl. Acids Res. 31, 5590). These findings contradict the widely held belief that the DNA binding mechanisms are analogous for the two polymerases. However, no crystal structure data exists for Klenow bound to DNA in this initial or polymerization-site binding mode. New advances in ab initio modeling from small angle X-ray scattering data are making it possible to construct topological shape envelopes for macromolecules in solution. Our initial SAXS measurments on the BioCAT beamline at APS, and subsequent molecular modeling of DNA bound the the polymerases have prodcued extraordinarily high resolution modeled structures. These modeled structures suggest an topology of polymerase bound the differs from the available crystal structures. In order to further confirm the putative toplogy obtained from the previous BIO-CAT SAXS data, our future plans are to collect equivalent data for full length Taq polymerase, and data with other DNA constructs. SAXS based shape modeling of these complexes will provide the binding topology for DNA bound to the polymerase in solution, and will provide an answer to whether these two polymerases bind the same DNA in the same way.