TOPOLOGY OF DNA BINDING TO DNA POLYMERASES

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。我们的目的是进一步表征DNA结合到水生栖热菌（Taq）和大肠杆菌的结构拓扑。coliPol 1型DNA聚合酶。“Klenow”聚合酶是大肠杆菌Pol 1的“大片段”，而“Klentaq”聚合酶是来自全长Taq聚合酶的等效大片段结构域。我们对大肠杆菌的Klenow和Klentaq片段进行了一些直接平行的功能研究。coli和Taq聚合酶，并且已经鉴定了表明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.Chem.278：5694; Datta & LiCata，Nucl. Acids Res.31，5590）。这些发现与人们普遍认为的DNA结合机制与两种聚合酶类似的观点相矛盾。然而，没有晶体结构数据存在克列诺结合到DNA在这种初始或聚合位点结合模式。小角X射线散射数据的从头算模型的新进展使得在溶液中构建大分子的拓扑形状包络成为可能。我们最初在APS的BioCAT光束线上进行的SAXS测量，以及随后对DNA结合聚合酶的分子建模，产生了非常高分辨率的建模结构。这些模型化的结构表明聚合酶结合的拓扑结构不同于现有的晶体结构。为了进一步证实从先前的BIO-CAT SAXS数据中获得的推定拓扑学，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构建体的数据。这些复合物的基于SAXS的形状建模将提供溶液中与聚合酶结合的DNA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.