This is a collaborative project between the Konigsberg and Karam laboratories aimed at understanding the structure and dynamics of DNA replicases. The current focus is the replicative DNA polymerase (gp43) of the T4 family of phages. Phylogenetic, biochemical, structural, and genetic tools are being used to analyze the mechanisms by which the multifunctional gp43 controls 3 categories of function: (a) fidelity of replicative DNA synthesis through accurate base selection and effective editing, (b) translational repression of its own mRNA through a highly specific RNA binding function, and (c) recognition of other replication proteins in the phage replicase. In fidelity studies, the investigators will use complementary biological and biochemical (steady-state and presteady-state kinetic) assay systems to identify gp43 residues that play roles in dNTP discrimination prior to nucleotide transfer. They also plan to identify residues that contribute to the conformational changes that precede chemistry in the nucleotidyl transfer reaction. Their studies draw heavily on knowledge of the crystal structure of RB69 gp43 from phage RB69, a phylogenetic relative of T4. The structure of this enzyme was solved during the previous project period, and they have placed a special emphasis on understanding the structure-function relationships between residues that form a binding pocket for the incoming dNTP and that help to align the complementary base in the template strand so that a sterically constrained space can be formed permitting only the """"""""correctly"""""""" matched dNTP's to fit in the proper geometric orientation for the nucleotidyl transfer reaction. The investigators will attempt to crystallize a RB69 gp43 mutant, Y567A, that is relatively permissive for incorporation of mispaired dNTP's, together with a dideoxy terminated primer-template and mispaired dNTP's. The structure of these complexes should provide insight into the structural requirements for fidelity. The results of the proposed work will contribute to a better understanding of the mechanisms of viral infectious diseases, especially those mediated by viruses that mutate rapidly. It will also provide a structural basis for mutagenic mechanisms that compromise the fidelity of replicative and repair polymerases.
Showing the most recent 10 out of 30 publications
