The objective is to provide a better understanding of the molecular mechanisms of mutagenesis. By use of newly developed biochemical methodology we will investigate conditions that give rise to altered base pairing specificity during DNA synthesis. The results will bear directly on the molecular etiology of cancer, birth defects and other diseases of genetic origin. The following experimental approach will be used to characterize the specificity of dNMP incorporation during DNA synthesis on natural templates, catalyzed by purified DNA polymerases. High resolution polyacrylamide gel electrophoresis is used to monitor the successive addition of nucleotides onto the 3'-OH terminus of a [5'-32P]primer, annealed to a viral DNA template. The extent to which chain elongation occurs in the presence of only 3 of the 4 dNTPs (revealed by autoradiography) reflects the propensity of the DNA polymerase to incorporate noncomplementary nucleotides at different positions along the template. Results obtained thus far indicate that the fidelity of DNA synthesis and the nature of misincorporation vary considerably along the template and that conditions that alter the overall accuracy of incorporation generally do so in a sequence-dependent manner. To fully characterize the influence of nearest neighbor sequences on the formation of non-Watson-Crick base pairs during DNA synthesis, data will be collected over a large array of naturally occurring template sequences, under conditions that affect the overall accuracy of DNA synthesis, including (i) activation of polymerase by different divalent metal ions, (ii) catalysis by different DNA polymerases and (iii) the presence of auxiliary proteins of DNA replication (including proteins required for the """"""""SOS response"""""""" in E. coli). """"""""Dideoxy"""""""" sequencing of strands elongated in """"""""minus"""""""" reactions will be carried out to characterize the influence of template sequence on the nature of misincorporation during DNA synthesis.
