Preliminary Study Of PriC, A Protein Involved In Escherichia Coli DNA Replication

The DNA replication in E. coli is semi-discontinuous, and the processes of replicating of the leading and lagging strands are quite different. Priming of the lagging strand replication is accomplished by primosome. In E. coli, DnaA-dependent primosome and PriA-dependent primosome were identified as two different primosomes. PriA,PriB,PriC,DnaB,DnaC,DnaT and DnaG participate in the assembling of the PriA-dependent primosome. At present, there is little information about the expression and function of PriC. To investigate the biological function and mechanism of PriC, recombination expression plasmid pET21a-PriC was constructed and transformed into E. coli BL21(DE3). PriC purified through Ni chelating sepherose was instable and easily precipitated. According to the prediction of the secondary structure and the result of limited proteolysis experiment, pET21a-PriC1-101 was constructed and transformed into E. coli BL21(DE3). PriC1-101 was purified by Ni chelating sepherose and superdex75. PriC1-101 was stable and identified by Western blot assay. PriC1-101 was basically consisted of a-helix and random coil by CD detection. In order to investigate the interaction between PriC and PriB, pET21a-PriC-notag and pET28at-plus-PriB were transformed into E. coli BL21(DE3) separately, and induced for expression. By co-purification of PriC and PriB, the interaction was detected between PriC and PriB. The stability of PriC was improved. C-terminal amino acids of PriC were identified as the main area interacted with PriB, through the pull-down assays between PriC,PriC1-101 and PriB, separately. This area was also important to the stability of PriC.