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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM054627-02
Application #
2459702
Study Section
Biochemistry Study Section (BIO)
Project Start
1996-08-03
Project End
2000-07-31
Budget Start
1997-08-01
Budget End
1998-07-31
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Petrov, Vasiliy M; Nolan, James M; Bertrand, Claire et al. (2006) Plasticity of the gene functions for DNA replication in the T4-like phages. J Mol Biol 361:46-68
Wang, C X; Zakharova, E; Li, J et al. (2004) Pre-steady-state kinetics of RB69 DNA polymerase and its exo domain mutants: effect of pH and thiophosphoryl linkages on 3'-5' exonuclease activity. Biochemistry 43:3853-61
Zakharova, E; Wang, J; Konigsberg, W (2004) The activity of selected RB69 DNA polymerase mutants can be restored by manganese ions: the existence of alternative metal ion ligands used during the polymerization cycle. Biochemistry 43:6587-95
Borjac-Natour, Jamilah M; Petrov, Vasiliy M; Karam, Jim D (2004) Divergence of the mRNA targets for the Ssb proteins of bacteriophages T4 and RB69. Virol J 1:4
Petrov, V M; Karam, J D (2004) Diversity of structure and function of DNA polymerase (gp43) of T4-related bacteriophages. Biochemistry (Mosc) 69:1213-8
Petrov, Vasiliy M; Karam, Jim D (2002) RNA determinants of translational operator recognition by the DNA polymerases of bacteriophages T4 and RB69. Nucleic Acids Res 30:3341-8
Petrov, Vasiliy M; Ng, San-San; Karam, Jim D (2002) Protein determinants of RNA binding by DNA polymerase of the T4-related bacteriophage RB69. J Biol Chem 277:33041-8
Bebenek, A; Dressman, H K; Carver, G T et al. (2001) Interacting fidelity defects in the replicative DNA polymerase of bacteriophage RB69. J Biol Chem 276:10387-97
Lin, T C; Wang, C X; Joyce, C M et al. (2001) 3'-5' Exonucleolytic activity of DNA polymerases: structural features that allow kinetic discrimination between ribo- and deoxyribonucleotide residues. Biochemistry 40:8749-55
Pavlov, A R; Karam, J D (2000) Nucleotide-sequence-specific and non-specific interactions of T4 DNA polymerase with its own mRNA. Nucleic Acids Res 28:4657-64

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