Research Project
2174902
The major aim of the proposed project will be to better understand the mechanism of replication by protein-priming. We will study: 1) The interaction of the phi29 terminal protein and DNA polymerase with the phi29 DNA ends. 2) The transition from terminal protein-primed initiation to DNA-primed elongation during phi29 DNA replication. 3) By using site- directed mutants, the critical amino acids in the terminal protein involved in the interaction with the DNA polymerase and with DNA, as well as those involved in the transition step. 4) By site-directed and deletion mutagenesis, the critical amino acids in the phi29 DNA polymerase involved in single-stranded DNA binding and strand- displacement activity at the amino-terminal domain, and those at the carboxy-terminal domain involved in the interaction with terminal protein, with double-stranded DNA or template/primer structures, with dNTP, in metal ion effects, or in processivity. 5) The structural and functional mapping of the phi29 DNA polymerase residues involved in insertion fidelity during terminal protein-primed initiation and DNA polymerization. 6) The development of a system for DNA amplification based on the phi29 replication origins and phi29 replication proteins. 7) The structural and functional properties of the phi29 SSB protein p5- ssDNA complex, and the critical amino acids in p5 involved in ssDNA binding. 8) The structural properties of the phi29 protein p6-DNA complex, the mechanism of activation of the initiation reaction by p6, and the functional domains in the protein for DNA binding and dimer formation. 9) The function of the viral proteins p1 and p17 and the possible role of cellular proteins in phi29 DNA replication. 10) The functional domains in the transcriptional activator, protein p4, required for DNA binding, DNA bending, and for dimer formation, and the coupling between transcription and replication in vitro. Health-related viruses such as adeno, polio, encephalomyocarditis, hepatitis A and B, and viruses of socio-economic relevance such as foot and mouth and several plant viruses, have a protein covalently linked at the 5' end (s) of the nucleic acid. Evidence for a protein-primed mechanism of replication has been obtained for phi29, adeno and hepatitis A. The long term objective of the project is to find specific ways to interfere with this initiation reaction.
