Biochemical investigation of primase and helicase in bacteria

The initiation recognition sequences for primase (DnaG) in the species Escherichia coli, Aquifex aeolicus, Staphylococcus aureus and Geobacillus stearothermophilus have been determined. All are composed of three nucleotides and initiation takes place from the central nucleotide. However, the initiation specificities appear to differ among bacterial phyla. The hypothesis of this study is that bacteria from the same phyla have the same initiation specificity. To test this hypothesis, the primases for Pseudomonas aeruginosa and Yersinia pestis (Phylum=Proteobacteria) were compared to those of S. aureus and Bacillus anthracis (Phylum = Firmicutes). Primases were first screened for activity using single-stranded deoxyribonucleic acid (ssDNA) templates that contained all possible trinucleotides. These experiments were followed by DnaG assays using specific trinucleotide sequences in ssDNA templates. The effect of replicative helicase (DnaB) on template specificity was also determined. Primase specificity was furthered studied by mutagenesis studies of amino acids positioned in the zinc-binding domain (ZBD). Point mutations were done on S. aureus DnaG to resemble gamma proteobacteria and on E. coli DnaG to resemble Y. pestis primase. Primer synthesis was analyzed using the WAVERTM High Performance Liquid Chromatography Nucleic Acid Fragment Analysis System under denaturing conditions. All novel DnaGs belonging to Y. pestis, P. aeruginosa and B. anthracis recognized a broad spectrum of trinucleotides and were found to be equally processive. The specificity of E. coli DnaG was found to be broad and with activity considerably lower than Y. pestis and P. aeruginosa primases. The DnaB of Y. pestis and P. aeruginosa stimulated their corresponding DnaG to synthesize more of the same primers. E. coli DnaB stimulated the synthesis of longer primers and restored the specificity of DnaG for the trinucleotide CTG. Several S. aureus DnaG mutants produced enzymes with the ability to recognize CTG. However, the mutations did not improve the processivity of their corresponding enzyme. The E. coli DnaG mutants produced enzymes that had the same relative activity as the wild-type enzyme and did produce full length primers with CTG, suggesting the ZBD did not contribute to the ability of the enzyme to make longer primers. This work suggests that primase trinucleotide specificity can be predicted by phyla with 80% certainty. Future studies will focus on determining the amino acids in the RNA polymerase domain that contribute to enhanced processivity and activity in primases.