Our long range goal is to understand the molecular mechanism of DNA replication and the processes which regulate DNA synthesis in both prokaryotic and eukaryotic cells. As a model system we will study DNA replication in bacteriophage T7. Since the basic mechanism of DNA replication is probably similar for both prokaryotes and eukaryotes, much that we learn from our studies on DNA replication in this simple bacteriophage system will help to elucidate these mechanisms in higher cells. This knowledge is fundamental to our understanding of such important questions as the mechanism of viral replication and the growth and differentiation of both normal and malignant cells. 1) During purification, the 3' greater than 5' exonuclease associated with the T7 DNA polymerase can be irreversibly inactivated. We will investigate the molecular basis for this loss of exonuclease activity. We will examine the effect of this nuclease on the fidelity of DNA synthesis and on the ability of the DNA polymerase to move past pyrimidine dimers on a template strand. 2) T7 RNA polymerase stimulates the initiation of DNA synthesis on duplex T7 DNA by T7 DNA polymerase and T7 gene 4 protein. However, DNA syntheses with these three proteins is not initiated at the origin(s) used in vivo. We will examine the effects of ionic strength and Mg++ concentration on the specificity of initiation in vitro. If necessary, we will search for additional protein factors required for specific initiation. We will also attempt to identify and characterize an RNA primer synthesized by the T7 RNA polymerase and used for the initiation of T7 DNA replication in vitro. 3) We will investigate the mechanism by which the E. coli RNA polymerase interferes with T7 DNA replication. We will test the effect of this enzyme on T7 DNA syntheses carried out with our purified T7 replication proteins. We will also study T7 mutants (T7Beta) which are able to grow on the mutant host E. coli tsnB. E. coli tsnB produces an RNA polymerase which is resistant to inhibition by the T7 gene 2 protein. Genetic and biochemical analyses of the T7Beta mutants may provide new insight into the mechanisms by which the bacterial RNA polymerase interferes with phage DNA replication.
| Chung, Y B; Nardone, C; Hinkle, D C (1990) Bacteriophage T7 DNA packaging. III. A ""hairpin"" end formed on T7 concatemers may be an intermediate in the processing reaction. J Mol Biol 216:939-48 |
| Chung, Y B; Hinkle, D C (1990) Bacteriophage T7 DNA packaging. I. Plasmids containing a T7 replication origin and the T7 concatemer junction are packaged into transducing particles during phage infection. J Mol Biol 216:911-26 |
| Chung, Y B; Hinkle, D C (1990) Bacteriophage T7 DNA packaging. II. Analysis of the DNA sequences required for packaging using a plasmid transduction assay. J Mol Biol 216:927-38 |
