The major aim of the project will be to get further insight on the mechanism of replication by protein priming. The PI will study: 1. The interaction of the phi29 terminal protein (TP)/DNA polymerase heterodimer with the phi29 ends. 2. Transition from TP-primed initiation to DNA-primed elongation during phi29 TP-DNA replication using mutant replication origin and DNA polymerases. 3. Critical amino acids in TP involved in the interaction with the DNA polymerase, with the parental TP and with specific phi29 DNA terminal sequences. 4. Critical amino acids in the DNA polymerase involved in processivity, strand displacement, insertion fidelity and interaction with TP. 5. Amplification vectors based in the phi29 replication origins and engineering of phi29 DNA polymerase for DNA sequencing. 6. The role of the reiterated bases at the phi29 DNA ends and that of the TP on the termination of phi29 DNA replication. 7. Residues in the phi29 SSB critical for protein-protein and protein-DNA interaction, as well as the in vivo role of phi29 SSB. 8. Amino acids in p6 involved in dimer and oligomer formation, as well as DNA determinants for p6 binding. 9. Interaction of p1, p17 and p16.7 with replication proteins, characterization of other viral proteins and possible role of cellular proteins in phi29 DNA replication. 10. Function of SpoOJ and SpoOA binding sites present in the phi29 genome. 11. Amino acids in p4 critical for interaction with DNA, for dimerization and for interaction with the alpha-CTD of B. subtilis RNA polymerase at promoters A3 and A2c, the switch from early to late transcription in phage GA-1, and the mechanism of p6 repression at promoters C2 and A2c.
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