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D C Hinkle

Publications and source records attributed to D C Hinkle.

29 records · Page 2Linked to original sources
PubMed1980

Evidence for direct involvement of T7 RNA polymerase bacteriophage DNA replication.

Experiments in a number of different systems have suggested that the initiation of DNA replication is often dependent upon transcription at the origin of replication. During infection with bacteriophage T7, the T7 genome is transcribed first by the bacterial RNA polymerase and then by a phage-coded enzyme, the product of gene 1. The bacterial enzyme does not appear to be directly involved in the initiation of replication as phage DNA synthesis is not inhibited by rifampin. For testing whether the T7 RNA polymerase plays a role in replication, a T7 gene 1 temperature-sensitive mutant was used, and the RNA polymerase was inactivated at intervals after infection by rapidly raising the temperature of the culture. The experiments indicated that the inactivation of the T7 RNA polymerase caused the cessation of phage DNA synthesis, even at later times during infection when the inhibition of protein synthesis with chloramphenicol had no effect on DNA replication. This suggests that in addition to its role in gene expression, the T7 RNA polymerase plays a direct role in T7 DNA replication.

Chloramphenicol↗
PubMed1979

Involvement of DNA gyrase in replication and transcription of bacteriophage T7 DNA.

Growth of bacteriophage T7 is inhibited by the antibiotic coumermycin A1, an inhibitor of the Escherichia coli DNA gyrase. Since growth of the phage is insensitive to the antibiotic in strains containing a coumermycin-resistant DNA gyrase, this enzyme appears to be required for phage growth. We have investigated the effect of coumermycin on the kinetics of DNA, RNA, and protein synthesis during T7 infection. DNA synthesis is completely inhibited by the antibiotic. In addition, coumermycin significantly inhibits transcription of late but not early genes. Thus, E. coli DNA gyrase may play an important role in transcription as well as in replication of T7 DNA.

Anti-Bacterial Agents↗
PubMed1975

Bacteriophage T7 deoxyribonucleic acid replication in vitro. Purification and properties of the gene 4 protein of bacteriophage T7.

The T7 gene 4 protein, a protein known from genetic analysis to participate in phage DNA replication in vivo, has been purified approximately 500-fold with an in vitro complementation assay. The protein, purified from cells infected with a T7 gene 4 temperature-sensitive mutant, is thermolabile, establishing that the complementation activity is in the protein product of the phage gene 4. The purified protein has no detectable nuclease, DNA polymerase, or RNA polymerase activity. However, in addition to stimulating the rate of DNA replication in crude extracts of T7 gene 4 mutant-infected cells, the gene 4 protein effects a marked stimulation of DNA synthesis by the purified T7 DNA polymerase when duplex T7 DNA is used as template. This effect is not observed when denatured T7 DNA is used as template, or when phage T4 DNA polymerase or Escherichia coli DNA polymerase I, II, OR III is substituted for the T4 enzyme. Analysis of the DNA synthesized by the T7 DNA polymerase in the presence of the gene 4 protein indicates that much of the product is in short DNA chains which are not covalently attached to the template. This result suggests a novel mechanism for the initiation of DNA chains in this reaction.

Bacterial Proteins↗
PubMed1975

Deoxyribonucleic acid polymerase III of Escherichia coli. Purification and properties.

DNA polymerase III has been purified 4,500-fold from the Escherichis coli mutant, HMS83, which lacks DNA polymerases I and II. When subjected to disc gel electrophoresis, the most purified fraction exhibits a single major protein band from which enzymatic activity may be recovered. Polyacrylamide gel electrophoresis under denaturing conditions produces two protein bands with molecular weights of 140,000 and 40,000. The sedimentation coefficient of the enzyme is 7.0 S, and the Stokes radius is 62 A. Taken together these tow parameters indicate a native molecular weight of 180,000. Purified DNA polymerase III catalyzes the polymerization of nucleotides into DNA when provided with both a DNA template and a complementary primer strand. The newly synthesized DNA is covalently attached to the 3' terminus of the primer strand. Because the extent of polymerization is only 10 to 100 nucleotides, the best substrates are native DNA molecules with small single-stranded regions. The most purified enzyme preparation is devoid of endonuclease activities. In addition to the two exonuclease activities described in the accompanying paper, purified polymerase III also catalyzes pyrophosphorolysis and the exchange of pyrophosphate into deoxynucleoside triphosphates. DNA polymerase III has also been isolated from wild type E. coli containing the other two known DNA polymerases. Futhermore, the enzyme purified from three different polC mutants exhibits altered polymerase III activity, confirming that polC is the structural gene for DNA polymerase III (Gefter, M., Hirota, Y., Kornberb, T., Wechsler, J., and Barnoux, C. (1971) Proc. Natl. Acad. Sci. U. S. A. 68, 3150-3153).

Chemical Phenomena↗