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. Bacteriophage RB69 DNA polymerase (RB69 pol) is an excellent model system to study the mechanisms of DNA synthesis and replication fidelity, because RB69 pol belongs to B family polymerases that include human replicative polymerases. We have recently determined the crystal structures of the ternary complex of RB69 pol containing each of the 12 mispaired dNTPs in addition to the 4 matched dNTPs at about 2.0 A resolution at the substrate ground state. These results suggest that the chemical step of the nucleotidyl transfer reaction is a key discrimination step where correct dNMPs incorporate efficiently and incorrect dNTPs do not. To provide the structural basis for this discrimination, we plan to determine the reaction trajectories of the two reactions (the 3?-OH of the primer of the DNA duplex and the alpha-phosphoryl atom of the incoming dNTPs) in the nucleotidyl transfer reaction for incorporation of both correct and incorrect dNTPs. We have chemically synthesized photolabile dNTP analogs and determined their efficiency of stalling the reaction catalyzed by RB69 pol and the ability of formation of the ternary complex of RB69 pol with modified dNTPs. We will co-crystallized the ternary complexes of RB69 pol containing the matched and mismatched dNTPs with a photolabile group attached. We have preliminarily characterized the structures containing the caged dNTPs using monochromatic synchrotron radiation sources. We will study the two parallel polymerase-catalyzed nucleotidyl transfer reaction, one for correct nucleotides, and the other for incorrect nucleotides. From these studies, we will establish the principles that govern the replicative RB69 pol as to how it accelerates the nucleotidyl transfer reaction for correct nucleotides and prevents it from incorrect nucleotides.
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