TOPOLOGY OF DNA BINDING TO DNA POLYMERASES

这个子项目是众多研究子项目之一

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.