The overall objective of this project is to understand the structure and dynamics of DNA replicases. The applicants are using the replication apparatus of phage T4 and its phylogenetic relatives as their model system. The major focus of the proposed work is the single-chain multi-functional DNA replication polymerase of these viruses. This DNA binding enzyme is also an RNA-binding autogenous translational repressor. They have recently succeeded in isolating and crystallizing the DNA polymerase of phage RB69, a distant relative of T4. Early results from X-ray diffraction studies provide many clues regarding the structural elements used by this replication enzyme to recognize RNA ligands, DNA substrates and other proteins of the DNA replication complex. Extending this study will allow them to explore structure/function relationships particularly with respect to POL and EXO activities as well as binding to their specific operator RNAs. In the proposed work, they will test the biological implications of the enzyme's structure by the use of biochemical, genetic and phylogenetic tools. Five structural domains (N/RNA, EXO, PALM, FINGER and THUMB) will be probed for their roles in catalysis of POL and EXO activities, sequence-specific RNA binding, sequence-independent DNA binding, and recognition of the polymerase accessory proteins as well as other replicase components. They will localize the nucleotide and amino acid determinants of recognition of the mRNA targets for the T4 and RB69 DNA polymerases by the use of UV-cross-linking and genetic studies, and relate these to DNA-binding determinants. Also T4/RB69 DNA polymerase chimeras will be constructed and used to map the protein sites that distinguish the RNA specificities of these two phylogenetic relatives from each other. In additional applications of the phylogenetic tool, they will identify the determinants of protein-protein interactions in the replicase and attempt to dissect the replicase assembly pathway. A special emphasis will be placed on determining the roles of protein subunits of the """"""""clamp loader"""""""" complex and coordination of leading and lagging strand replicase assemblies using genetic, biophysical and biochemical assays.
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