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.
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