Research Project
6920630
DESCRIPTION (provided by applicant): Replicative DNA polymerases are the main determinants of the accuracy of DNA replication and thus of the incidence of heritable birth defects and mutation-primed diseases such as cancer. It is therefore of particular interest to us to understand the mechanism by which the main eukaryotic replicative polymerases achieve high fidelity. Bacteriophage RB69 encodes a replicative DNA polymerase with associated 3"-5" proofreading activity. Like T4 DNA polymerase and many polymerases from archaeons, the RB69 DNA polymerase is a member of the B family which includes the eukaryotic DNA polymerases alpha, delta, and epsilon. The availability of crystallographic structures for this polymerase in both the apo for and the replicating complex makes it an excellent model for structure-function studies. Efficient mutation reporter systems are available both in vivo and in vitro to assess the impact on polymerase accuracy of changes in polymerase residues. To conduct fidelity analyses in vivo, we will use a hybrid system in which T4 DNA replication is driven by a RB69 DNA polymerase. T4 whose DNA polymerase has been mutationally inactivated can be replicated by a cognate RB69 DNA polymerase encoded by a recombinant plasmid in T4-infected Escherichia coil Mutation frequencies are measured using two T4 reporter systems, reversion at the r/I locus, and forward mutation at the rl locus followed by sequencing. These assays provide rapid and sensitive measurements of the mutational specificities of derivatives of RB69 enzyme. To measure fidelity in vitro, we will use the M13mp2 lacZalpha system. This is a gap-filling assay that measures the accuracy of DNA synthesis across a single stranded gap in bacteriophage M13mp2 RF DNA. The gap contains the mutation-reporter lacZalpha as a template. This system detects specific mispairs, and can support measurements of polymerase processivity the role of accessory proteins. In our fidelity studies we will focus first on the highly conserved thumb-domain motif KKRY which contacts primer-template DNA along the minor-groove and is likely to play an essential role in DNA binding and in accurate, processive DNA replication.
