A DNA primase specified by I-like plasmids.

Abstract

An enzyme was isolated from Escherichia coli strains harboring the I-like plasmid R64drd11, which is capable of initiating DNA synthesis on the circular, single-stranded DNA of phages .vphi.X174, fd and G4. In the conversion of these templates to duplex forms in vitro, the enzyme can substitute for the functions of E. coli dnaB-dnaG proteins, E. coli RNA polymerase and E. coli dnaG protein, respectively. The enzyme requires all 4 ribonucleoside triphosphates [rNTP] for optimal activity, although a combination of ATP, CTP and GTP can almost completely satisfy the rNTP requirement. The enzyme appears to cooperate specifically with DNA polymerase III because single-stranded DNA-dependent synthesis takes place in extracts deficient in DNA polymerases I and II but not in extracts from a dnaZ mutant. Highly purified enzyme preparations consist mostly of 2 major polypeptides, MW 140,000 and 180,000, when analyzed by sodium dodecyl sulfate gel electrophoresis. These polypeptides cosediment with the enzyme activity through a glycerol gradient with a sedimentation coefficient of 3.6 S. DNA priming activity in extracts of E. coli strains harboring the mutant plasmids R64dr11 or Colldr1, which are derepressed in functions of conjugational DNA transfer, is several-fold higher than the activity from strains carrying the corresponding wild-type plasmid. This correlation suggests that the enzyme may play a role in conjugational DNA synthesis.