Margarita Salas - US grants

Our aim is to get a better knowledge of the mechanism of initiation of replication by protein-priming and to study other requirements for 029 DNA replication. We will study: 1) Template requirements for the initiation reaction. The role of the parental terminal protein and of the sequence at the ends of 029 DNA will be determined. 2) Function of the terminal protein p3 in 029 DNA replication. The binding of protein p3 to terminal 029 DNA fragments and its role in elongation will be studied. Mutants of protein p3 will be obtained and the effect of the mutations in the initiation and elongation reactions determined. 3) Function of the 029 DNA polymerase in replication. The DNA polymerase and 3' to 5' exonuclease activities of protein p2 and their role in 029 DNA replication will be studied. The interaction between p2 and p3 will be determined. 4) Function of host and viral proteins in 029 DNA replication. The host factor(s) that stimulates the initiation reaction will be purified and its interaction with terminal fragments of 029 DNA determined. The activity of protein p6 as a single-strand DNA binding protein and its effect on replication will be studied. Other viral proteins will be purified and their effect on 029 DNA replication determined. THe effect on 029 DNA replication of the B. subtilis RNA polymerase and of protein p4, that controls 029 late transcription, will be studied. 5) replication of the displaced parental strand. Replicative intermediates synthesized in vitro will be isolated and analyzed by electron microscopy. 6) role of the inverted terminal repeat in replication. Hybrid 029 DNA molecules containing terminal protein only at one of the ends or lacking nucleotide sequences shorter or longer than the inverted terminal repeat will be used for transfection. 7) The role of the parental terminal protein and of the DNA sequence in encapsidation, and the presence of enzymatic activities in the 029 DNA-protein p3 complex will be determined. Several health-related viruses such as adeno, polio and encephalomyocarditis and viruses of soci-economic importance such as foot and mouth and different plant viruses have a protein covalently linked tothe 5' ends of the nucleic acid. Evidence for a protein-priming mechanism has been shown for 029 and adenovirus and recent results indicate that teplication of poliovirus and encephalomyocarditis is also initiated by a similar mechanism. The long term objective of the project will be to find specific ways to interfere with this new initiation reaction.

DESCRIPTION (adapted from investigator's abstract): The major aim of the project will be to gain a better understanding of the mechanism of replication by protein-priming. Work will primarily focus on the phage phi 29 model and will investigate 1) The interaction of the phi 29 terminal protein (TP) and DNA polymerase with the phi 29 DNA ends. 2) Transition from TP-primed initiation to DNA-primed elongation during phi 29 TP-DNA replication. 3) Critical amino acids in the TP involved in the interaction with the DNA polymerase, with DNA, and with the parental TP, as well as those involved in the transition step. 4) Critical amino acids in the DNA polymerase involved in strand displacement, processivity, insertion fidelity, and ability to interact with TP, ssDNA, dsDNA and template/primer structures. 5) Application of phi 29 DNA polymerase such as amplification vectors based on the phi 29 replication origins and engineering of the enzyme for DNA sequencing. 6) Residues in the phi 29 SSB critical for stability, protein-protein and ssDNA interactions, specificity in phi 29 DNA replication, and interaction with 29 replication proteins. 7) Interaction of protein p6 with TP and DNA polymerase, amino acids critical for dimer or oligomer formation and for interaction with DNA, as well as genome organization by p6. 8) Interactions of proteins p1 and p17 with phi 29 replication proteins, characterization of early viral proteins at the right phi 29 DNA end, and possible role of cellular proteins in phi 29 DNA replication. 9) Critical amino acids in the B. subtilis RNA polymerase alpha subunit for interaction with p4, factors leading to transcription activation or repression, amino acids in p4 involved in DNA binding and dimerization, and mechanism of repression of the A2c promoter by p6. Health-related viruses such as adenovirus or hepatitis B replicate by protein-priming mechanism. The long term objective of the project is to find specific ways to interfere with this initiation reaction.

DESCRIPTION: 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.