The major aim of the proposed project is to study the function in replication of protein p3, covalently linked to the 5' end of Bacillus subtilis phage 029 DNA. We will study: (a) The mechanism of the synthesis of the 029 DNA protein p3 complex. We will purify native protein p3 and study whether p3 itself has the enzymatic activity to catalyze the formation of the covalent bond with dAMP or some other phage-induced or bacterial activity is needed. The requirements in the template DNA and protein moieties will be also determined. (b) 029 DNA conformation required for replication. To investigate if circularization of 029 DNA in vivo has any role in replication we will repare DNA in which the protein at either one of the ends has been removed. After determining that this DNA is unable to circularize in vitro we will use it to transfect competent B. subtilis. (c) Possible role of transcription in DNA replication. The requirement for RNA polymerase activity or for the early promoters located close to the left and right DNA ends in the in vitro system of initiation of replication will be studied. (d) Cloning of 029 DNA fragments containing gene 3 in plasmids to overproduce protein p3. Bacteria transformed by these plasmids will be used as a starting material for the purification of protein p3. (e) Study of 029-induced enzymatic activities related to DNA replication and characterization of the genes coding for these activities by using sus and ts mutants in the genes involved in viral DNA synthesis. (f) Location of the serine residue in the protein involved in the linkage with the DNA, by identification and sequence determination of peptides containing the surrounding amino acids. (g) The role of protein p3 in DNA encapsulation and possible enzymatic activities in p3 (gyrase, ATPase) relevant to DNA packaging will be determined. Several health-related viruses such as adeno, hepatitis B, polio and encephalomyocarditis have a protein covalently linked to the 5' ends of the nucleic acid and a mechanism for the initiation of replication of these viruses similar to that of 029 has been proposed. The long-term objective of the project will be to find a specific way to interfere with this new initiation reaction once we understand its mechanism.
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