The major aim of the proposed project is to get a deeper knowledge of the protein-primed mechanism of initiation of replication. We will study: 1) The template requirements for phi29 DNA replication. 2) The interaction of the terminal protein and DNA polymerase with the phi29 DNA ends. 3) The critical amino acids in the terminal protein involved in the catalysis of the initiation reaction and in the interaction with the DNA polymerase and DNA, by using site-directed mutants. 4) The functional domains in the phi29 DNA polymerase involved in initiation, polymerization, pyrophosphorolysis and 3' > 5' exonuclease activities and, more specifically, critical amino acids involved in processivity, single-and double-stranded DNA binding, interaction with the terminal protein and dNTPs; these studies will be carried out using site-directed mutants and protease treatment. 5) The contribution of nucleotide selection and 3' > 5' exonucleolytic correction to the fidelity of DNA synthesis by the phi29 DNA polymerase. 6) A system based on the phi29 DNA replication origins to amplify DNA will be constructed. 7) The structural and functional properties of the protein p5-ssDNA complex and the critical amino acids in protein p5 involved in ssdna binding. 8) The mechanism of activation of the initiation reaction by protein p6, the structural properties of the p6-DNA complex 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 protein in phi29 DNA replication. 10) The replication genes mutagenized in vitro will be studied in vivo. 11) The transcriptional activation in phi29 DNA replication and functional domains in protein p4 for DNA binding, DNA bending and interaction with the RNA polymerase. Health-related viruses such as adeno, polio, encephalomyocarditis, hepatitis A and B and viruses of socio-economic importance such as foot and mouth and several plant viruses, have a protein covalently linked to the 5' end (s) of the nucleic acid. Evidence for a protein-primed mechanism of replication has been obtained for phi29, adenovirus and hepatitis B. The long term objective of the project is to find specific ways to interfere with this initiation reaction.
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