DnaA-dependent assembly of the ABC primosome at the A site, a single-stranded DNA hairpin containing a dnaA box.

Abstract

The ABC primosome is assembled from DnaA, DnaB and DnaC proteins at a stem-and-loop structure containing a dnaA box within its stem (A site), and catalyses primer RNA synthesis for DNA chain elongation. The DnaA protein can bind to the A site and the A-site-DnaA-protein complex can be isolated by gel-filtration chromatography in the absence of nucleotides. Mutations within the dnaA box completely abolish the binding of DnaA protein. Point mutations within the stem region outside the dnaA box also severely reduce the affinity of DnaA protein for the A site. These results indicate that not only the dnaA box but also other nucleotides and/or secondary structure features of the stem are important for proper recognition of the A site by DnaA protein. The preprimosome, which is able to synthesize RNA primers upon addition of primase, can be isolated by gel-filtration chromatography in the presence of ATP or adenosine 5'-[gamma-thio]triphosphate, a non-hydrolyzable analogue of ATP. The preprimosome can translocate along Escherichia coli single-stranded-DNA-binding protein-coated single-stranded DNA, utilizing the energy released by hydrolysis of ATP, as indicated by its helicase activity. dATP, as well as dCTP, can support the helicase activity of the preprimosome to some extent, while they are inert in helicase assays with DnaB protein in the absence of E. coli single-stranded DNA-binding protein. In keeping with this result, the isolated preprimosome, which appears to contain DnaA and DnaB proteins, is capable of hydrolyzing dATP as well as ATP and GTP. In a reconstituted replication assay, addition of excess dATP restores replication activities which have been inhibited by addition of adenosine 5'-[gamma-thio]triphosphate. The ability of dATP to support helicase and replicative activities of the ABC primosome indicates that the formation of the complex somehow modulates the structures of its component(s) so that they can utilize otherwise inert nucleotides. On the basis of these results, a scheme for the assembly of the ABC primosome at the A site is presented.