Abstract
Bacteriophage T7 DNA primase (gene-4 protein, 66,000 daltons) enables T7 DNA polymerase to initiate the synthesis of DNA chains on single-stranded templates. An initial step in the process of chain initiation is the formation of an oligoribonucleotide primer by T7 primase. The enzyme, in the presence of natural SS DNA, Mg++ (or Mn++), ATP and CTP (or a mixture of all 4 rNTPs), catalyzes the synthesis of di-, tri-, and tetraribonucleotides all starting at the 5' terminus with pppA. In a subsequent step requiring both T7 DNA polymerase and primase, the short oligoribonucleotides (predominantly pppA-C-C-AOH) are extended by covalent addition of deoxyribonucleotides. With the aid of primase, T7 DNA polymerase can also utilize efficiently a variety of synthetic tri-, tetra-, or pentanucleotides as chain initiators. T7 primase apparently plays an active role in primer extension by stabilizing the short primer segments in a duplex state on the template DNA.
Full text
PDFImages in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Baird J. P., Bourguignon G. J., Sternglanz R. Effect of nalidixic acid on the growth of deoxyribonucleic acid bacteriophages. J Virol. 1972 Jan;9(1):17–21. doi: 10.1128/jvi.9.1.17-21.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bouché J. P., Zechel K., Kornberg A. dnaG gene product, a rifampicin-resistant RNA polymerase, initiates the conversion of a single-stranded coliphage DNA to its duplex replicative form. J Biol Chem. 1975 Aug 10;250(15):5995–6001. [PubMed] [Google Scholar]
- Hausmann R. Bacteriophage T7 genetics. Curr Top Microbiol Immunol. 1976;75:77–110. doi: 10.1007/978-3-642-66530-1_3. [DOI] [PubMed] [Google Scholar]
- Hinkle D. C., Richardson C. C. Bacteriophage T7 deoxyribonucleic acid replication in vitro. Purification and properties of the gene 4 protein of bacteriophage T7. J Biol Chem. 1975 Jul 25;250(14):5523–5529. [PubMed] [Google Scholar]
- Hinkle D. C., Richardson C. C. Bacteriophage T7 deoxyribonucleic acid replication in vitro. Requirements for deoxyribonucleic acid synthesis and characterization of the product. J Biol Chem. 1974 May 10;249(9):2974–2980. [PubMed] [Google Scholar]
- Kolodner R., Richardson C. C. Replication of duplex DNA by bacteriophage T7 DNA polymerase and gene 4 protein is accompanied by hydrolysis of nucleoside 5'-triphosphates. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1525–1529. doi: 10.1073/pnas.74.4.1525. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mark D. F., Richardson C. C. Escherichia coli thioredoxin: a subunit of bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1976 Mar;73(3):780–784. doi: 10.1073/pnas.73.3.780. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Masamune Y., Richardson C. C. Strand displacement during deoxyribonucleic acid synthesis at single strand breaks. J Biol Chem. 1971 Apr 25;246(8):2692–2701. [PubMed] [Google Scholar]
- Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
- Miller J. P., Philipps G. R. Transfer ribonucleic acid nucleotidyltransferase from Escherichia coli. II. Purification, physical properties, and substrate specificity. J Biol Chem. 1971 Mar 10;246(5):1274–1279. [PubMed] [Google Scholar]
- Pongs O., Lanka E. Synthesis of a chemically reactive analog of the initiation codon: its reaction with ribosomes of Escherichia coli. Hoppe Seylers Z Physiol Chem. 1975 Apr;356(4):449–458. doi: 10.1515/bchm2.1975.356.1.449. [DOI] [PubMed] [Google Scholar]
- Reichard P., Eliasson R., Söderman G. Initiator RNA in discontinuous polyoma DNA synthesis. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4901–4905. doi: 10.1073/pnas.71.12.4901. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sanger F., Air G. M., Barrell B. G., Brown N. L., Coulson A. R., Fiddes C. A., Hutchison C. A., Slocombe P. M., Smith M. Nucleotide sequence of bacteriophage phi X174 DNA. Nature. 1977 Feb 24;265(5596):687–695. doi: 10.1038/265687a0. [DOI] [PubMed] [Google Scholar]
- Scherzinger E., Klotz G. Studies on bacteriophage T7 DNA synthesis in vitro. II. Reconstitution of the T7 replication system using purified proteins. Mol Gen Genet. 1975 Dec 1;141(3):233–249. doi: 10.1007/BF00341802. [DOI] [PubMed] [Google Scholar]
- Scherzinger E., Lanka E., Morelli G., Seiffert D., Yuki A. Bacteriophage-T7-induced DNA-priming protein. A novel enzyme involved in DNA replication. Eur J Biochem. 1977 Feb;72(3):543–558. doi: 10.1111/j.1432-1033.1977.tb11278.x. [DOI] [PubMed] [Google Scholar]
- Strätling W., Knippers R. Function and purification of gene 4 protein of phage T7. Nature. 1973 Sep 28;245(5422):195–197. doi: 10.1038/245195a0. [DOI] [PubMed] [Google Scholar]
- Weiner J. H., McMacken R., Kornberg A. Isolation of an intermediate which precedes dnaG RNA polymerase participation in enzymatic replication of bacteriophage phi X174 DNA. Proc Natl Acad Sci U S A. 1976 Mar;73(3):752–756. doi: 10.1073/pnas.73.3.752. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wickner S. DNA or RNA priming of bacteriophage G4 DNA synthesis by Escherichia coli dnaG protein. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2815–2819. doi: 10.1073/pnas.74.7.2815. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zechel K., Bouché J. P., Kornberg A. Replication of phage G4. A novel and simple system for the initiation of deoxyribonucleic acid synthesis. J Biol Chem. 1975 Jun 25;250(12):4684–4689. [PubMed] [Google Scholar]