Primer synthesis kinetics by Escherichia coli primase on single-stranded DNA templates

Abstract

The kinetics of primer RNA initiation and elongation by Escherichia coli primase were measured. The single-stranded DNA template that was used to develop the system, d(CAGA-(CA)5CTGCAAAGC), contained: (1) the preferred initiating trinucleotide d(CTG); (2) five residues 3' to the d(CTG), the minimum required for efficient primer synthesis; and (3) a single guanine placed near the 5'-end to facilitate study of cytidine triphosphate analog incorporation at a unique site. The assay monitored radiolabeled nucleotide incorporation into the RNA primers. The various primers were separated by length using denaturing polyacrylamide gel electrophoresis. Different types of primers were observed when synthesis was monitored using gamma- versus alpha-radiolabeled nucleotides as the probe. When [gamma-32P]-ATP incorporation was the probe, only primers initiated with ATP from the unique template thymine were observed. The sequences of these primers were complementary to that of the template. No primers shorter than a 12-mer accumulated, demonstrating that formation of the first phosphodiester bond was much slower than that of the next 10 phosphodiester bonds. The longest primer observed when monitoring [gamma-32P]ATP incorporation was 16 nucleotides long, the correct length for a primer completely template-directed and initiated at the unique thymine. Misinsertion of a noncognate nucleotide at the template's guanine indicated very poor nucleotide discrimination by this enzyme. When [alpha-32P]UTP was the probe for primer synthesis, all primers synthesized were observed whether or not they were initiated with ATP. Under these conditions, "overlong" primers and the above-described template length-dependent primers were observed. The template length-dependent primers accumulated faster than the overlong primers, but, at long incubation times, the overlong primers became the dominant species. The overlong primers were not fully related to the template length-dependent primers since they were not initiated complementary to the template d(CTG). Nevertheless, the overlong primers did appear to arise as a consequence of the template length-dependent species since their length was double and they arose in the time course after the length-dependent species.