Primase from Escherichia coli primes single-stranded templates in the absence of single-stranded DNA-binding protein or other auxiliary proteins. Template sequence requirements based on the bacteriophage G4 complementary strand origin and Okazaki fragment initiation sites.

We find that neither the hairpins nor bound single-stranded DNA-binding protein (SSB) are required for in vitro primase activity at the G4 complementary strand origin. Primase has the ability to prime synthetic DNA templates in the absence of any auxiliary proteins, including SSB, and in the absence of any DNA secondary structure. primase does, however, initiate primer synthesis starting at a specific nucleotide sequence present in both the G4 origin system and at sites of Okazaki fragment initiation. We tested a series of single-stranded DNA templates that consisted of various portions of the G4 complementary strand origin for the ability to support primer synthesis. Primer synthesis activity was determined using an assay in which primer length and quantity are monitored by the incorporation of [gamma-32P]ATP once per primer and the products analyzed on a denaturing polyacrylamide gel. We find that primase requires only a short DNA template, 5'-(AC)7CTG CAA AGC-3', to synthesize a 17-nucleotide primer starting at the thymidine residue. We also report primase's ability to incorporate dideoxyribonucleotide triphosphates into primers, which has allowed us to determine directly both length and sequence of primers synthesized.