The long-term objectives of this grant are a molecular understanding of two related aspects of mutation rate and its control in E. coli: (1) mechanisms that provide for the fidelity of normal DNA replication; (2) basis for the enhanced mutation rate and replicational repair induced by the SOS response to DNA damage. The major specific aims for this grant period are to understand: (1) the relative contribution and mechanism of base selection and editing for the fidelity of DNA polymerase III holoenzyme; (2) the biochemistry of the multi-protein SOS-induced pathway for point mutations (UmuCD pathway); (3) the relationship between the point-mutation pathway and other SOS pathways involving nonmutagenic replicational repair and duplication mutations. The goal in all three areas is to define the contribution of each individual protein to the overall pathway and to correlate the biochemical experiments using pure proteins with the results of genetic and physiological analysis in vivo. An understanding of mutation rate and the mutagenic effects of DNA lesions has relevance to carcinogenesis in humans. Oncogenes can clearly be activated by mutation, and there is a correlation between the mutagenic and carcinogenic effect of agents that damage DNA. Experiments on base selection will involve: (i) a more complete study of the specificity of nucleotide insertion; (ii) a more refined kinetic analysis of the reaction pathway for incorporation of correct and incorrect nucleotides. Work on editing will address: (i) the intrinsic exonuclease specificity of the editing subunit of polymerase III; (ii) contribution of the polymerase subunit to editing specificity, especially by kinetics of chain elongation from a mispaired base. For the SOS work, the experiments will seek to reconstitute the mutagenic pathway with purified proteins and to explore the mechanism of the nonmutagenic pathway termed replication-restart. Experiments will fall into four groups: (i) use of a simple replication system with currently available proteins; (ii) study of possible additional protein components; (iii) analysis of protein-protein interactions; (iv) investigation of more complex double strand replication systems.
