Escherichia coli Replicative Helicase PriA Protein-Single-stranded DNA Complex

Abstract

SUMMARYAnalyses of interactions of the E. coli replicative helicase, PriA protein, with a singlestranded DNA have been performed, using the quantitative fluorescence titration technique.The stoichiometry of the PriA helicase -ssDNA complex has been examined in binding experiments with a series of ssDNA oligomers. The total site-size of the PriAssDNA complex, i.e., the maximum number of nucleotide residues occluded by the PriA helicase in the complex is 20 ± 3 residues per protein monomer. However, the protein can efficiently form a complex with a minimum of 8 nucleotides. Thus, the enzyme has a strong ssDNA-binding site that engages in direct interactions with a significantly smaller number of nucleotides than the total site-size. The ssDNA-binding site is located in the center of the enzyme molecule, with the protein matrix protruding over a distance of ~6 nucleotides on both sides of the binding site.

Size-Independent and Noncooperative Recognition of dsRNA by the Rice Stripe Virus RNA Silencing Suppressor NS3

“…An early approach modified the site size to n − 2b in the case of oneligand binding and to n − b in the case of twoligand binding. 39,41 Such a correction cannot be readily applied to lattices that can accommodate more than two ligands. In the present work, we took a different approach to correct the ligand border effect by extending the nominal length of the lattice at each end by b sites while keeping the ligand site size constant along the whole lattice (Fig.…”

“…As an example of ligand with border region, E. coli helicase PriA also has an occluded site size larger than its minimal binding size on ssDNA. 39,41 When such a ligand binds at the end of a lattice, the border region protrudes out, reducing the effective site size. For the purpose of quantitative analysis, we assumed that the border regions of NS3 dimer are evenly partitioned around its central lattice-interacting region, with a size of b on each side (Fig.…”

“…30 As the short dsRNAs used in our experiments could not be treated as infinite lattices, we employed the exact combinatorial expression for finite lattices to analyze the binding isotherms of dsRNAs that accommodate two NS3 dimers. [37][38][39] This type of ligand-lattice interacting system is characterized by the lattice length M, the total site size of the ligand n, the interaction site size c, the intrinsic binding constant K int , and the cooperativity parameter ω. Each base pair of dsRNA is regarded as a repeating unit of the lattice.…”

“…3-6 can be derived to relate fractions of the free RNA, one-ligand complex, and two-ligand complex to the concentration of free ligand L and binding parameters M, n, K int , and ω (see Materials and Methods). 37,39 We applied these equations to globally fit the binding data of 21-, 22-, and 30-bp dsRNAs with overhangs to derive the parameters K int and ω. The lattice length M was corrected for the ligand border effect and the dsRNA overhang effect, as discussed above.…”

The N-Terminal Domain of the Escherichia coli PriA Helicase Contains Both the DNA- and Nucleotide-Binding Sites. Energetics of Domain–DNA Interactions and Allosteric Effect of the Nucleotide Cofactors

“…The commencement of the primosome assembly occurs through the recognition of the damaged DNA site structure/sequence by the PriA protein, although the nature of this recognition reaction is not yet understood (4,5,9–11,13). The native PriA protein is a monomer with a molecular mass of 81.7-kDa (1,2,5,15–17). The tertiary structure of the monomer contains two functional domains, the helicase domain encompassing ~540 amino acid residues from the C-terminus and the N-terminal domain, which comprises the ~181 amino acid residues (181N terminal domain) from the N-terminus (14,18).…”