The replication and repair of genetic material is essential for all organisms and the survival of each species requires that this be done in a timely and accurate fashion. The synthesis of DNA is a complex, highly regulated reaction catalyzed by DNA polymerases. Damage to the DNA caused by chemical or physical agents causes premutagenic lesions. The biological effects of mutagens or DNA damaging agents depend on both the efficiency of DNA repair and what occurs when a DNA polymerase encounters the DNA lesion. A lesion that blocks replication is potentially a lethal event unless it can be repaired or bypassed. At the same time, translesion replication by a DNA polymerase may lead to mutation. Since chemical carcinogenesis is generally associated with adducts or damaged sites in the DNA, the study of DNA polymerases is highly relevant to an understanding of carcinogenesis. The long range goal of the proposed research is a molecular understanding of DNA replication. Attaining this goal requires a knowledge of the relationship between DNA polymerase structure and its functions. These functions include the binding of primer and template DNA, binding of dNTP substrates, the catalysis of polymerization, mechanisms for improving the fidelity of replication and mechanisms for increasing processivity. At the present time our understanding of these processes is limited by the complete lack of structural information on the class B, or alpha-type, polymerases that include the eukaryotic replicative polymerases and by the very limited structural data on polymerases in general. The primary aim of the proposed research is the determination of the three dimensional structure of the E. coli DNA polymerase II through the use of X-ray crystallographic techniques. DNA polymerase II is induced by DNA dazzle as part of the SOS response and appears to have a role in DNA repair. DNA polymerase II is related to the eukaryotic a type replicative polymerases rather than the better characterized E. coli DNA polymerase I, consequently this enzyme is of particular interest.
