The overall goal of this project is to understand the enzymatic mechanisms of DNA metabolism in Escherichia coli and in phage infected E. coli. Major emphasis will be on the replication of the linear, duplex DNA of phage T7. Additional phage and host mutants defective in T7 replication will be isolated. One such E. coli mutant that cannot support the replication of T7 gene 1.1/1.2 mutants will be characterized, and the host protein responsible for this phenotype will be purified. The initiation of T7 DNA replication will be investigated by (1) identifying, cloning and sequencing the origin of replication, (2) establishing an in vitro initiation system, (3) further characterizing a host protein that makes possible site-specific initiation of T7 DNA, and (4) identifying additional phage and host proteins of initiation. The gene 1.1/1.2 protein, a protein required for T7 DNA replication in a host mutant, will be purified and studied. Studies on the DNA-dependent nucleoside 5'-triphosphatase activity and the oligoribonucleotide synthesizing activity of the gene 4 protein will be continued. T7 DNA polymerase will be examined with regard to the role of its subunit, thioredoxin, in replication and the mechanism by which the polymerase can use short RNA primers. The metabolism of concatemers will be studied, including the roles of the gene 2 protein (an inhibitor of E. coli RNA polymerase) and gene 20 protein in this process. Two separate studies are: (1) the mechanism by which DNA containing hydroxymethylcytosine is restricted by E.coli, and (2) the in vivo role of the exonuclease activities of DNA polymerase III of E. coli.
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