PMID:340457
| Citation |
Rowen, L and Kornberg, A (1978) Primase, the dnaG protein of Escherichia coli. An enzyme which starts DNA chains. J. Biol. Chem. 253:758-64 |
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| Abstract |
Conversion of the viral DNA of phage G4 to the duplex form provided an opportunity to isolate and determine the function of the dnaG protein, the product of a gene known to be essential for replication of the Escherichia coli chromosome. This stage of G4 DNA replication requires action of three proteins: the E. coli DNA-binding protein, the dnaG protein, and the DNA polymerase III holoenzyme. The dnaG protein has been purified approximately 25,000-fold to near-homogeneity. The native protein contains a single polypeptide of 60,000 daltons. It has been assayed for its activity on G4 DNA in three ways: (a) RNA synthesis, (b) complementation for replication of an extract of a temperature-sensitive dnaG mutant, and (c) priming of DNA replication by DNA polymerase III holoenzyme. The dnaG protein is highly specific for G4 DNA and synthesizes a unique 29-residue RNA primer to be described in the suceeding paper. Other single-stranded and duplex DNA templates are inactive. RNA primer synthesis by the dnaG protein has an apparent Km for ribonucleoside triphosphates near 10 micrometer, and a narrow optimum for Mg2+. The sharp specificity of the dnaG protein in choice of template and the utilization of either deoxyribonucleotides or ribonucleotides to produce a hybrid piece only a few residues long (as described in a succeeding paper) suggests that the dnaG protein previously named RNA polymerase by renamed primase. |
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| Keywords |
Coliphages; DNA Replication; Escherichia coli/enzymology; Escherichia coli/genetics; Genes; RNA Nucleotidyltransferases/isolation & purification; RNA Nucleotidyltransferases/metabolism; Templates, Genetic |
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- Describes the purification of DnaG
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