The molecular mechanisms of mutagenesis will be examined at the enzymological level, utilizing recently developed techniques for manipulation and analysis of DNA. In one approach, recombinant DNA molecules, bearing genetic markers of Haemophilus influenzae, will be treated with mutagenic agents and then incubated with cell-free extracts under conditions in in vitro replication and/or repair. Establishment of mutations will be monitored by a sensitive genetic assay that utilizes transformation of H influenzae. This in vitro mutagenesis system will be used to purify proteins involved in mutagenesis, with emphasis on inducible activities required for certain types of mutagenesis in vivo. In another approach defined template primers, prepared by annealing [5'-32P] restriction fragments to single-stranded templates, will be incubated with purified DNa polymerases and other proteins that are potentially involved in mutagenesis. The lengths of elongated primers will be analyzed by high resolution gel electrophoresis/autoradiography. The accuracy of DNA synthesis will be assessed by measurement of chian elongation in the presence of only 3 of the 4 dNTPs. DNA synthesis of mutagen-damaged templates will be characterized to determine which nucleotides, if any, are inserted opporiste specific lesions.
