Interactions of Escherichia coli Replicative Helicase PriA Protein with Single-Stranded DNA

Abstract

Quantitative analyses of the interactions of the Escherichia coli replicative helicase PriA protein with a single-stranded DNA have been performed, using the thermodynamically rigorous fluorescence titration technique. The analysis of the PriA helicase interactions with nonfluorescent, unmodified nucleic acids has been performed, using the macromolecular competition titration (MCT) method. Thermodynamic studies of the PriA helicase binding to ssDNA oligomers, as well as competition studies, show that independently of the type of nucleic acid base, as well as the salt concentration, the type of salt in solution, and nucleotide cofactors, the PriA helicase binds the ssDNA as a monomer. The enzyme binds the ssDNA with significant affinity in the absence of any nucleotide cofactors. Moreover, the presence of AMP-PNP diminishes the intrinsic affinity of the PriA protein for the ssDNA by a factor approximately 4, while ADP has no detectable effect. Analyses of the PriA interactions with different ssDNA oligomers, over a large range of nucleic acid concentrations, indicates that the enzyme has a single, strong ssDNA-binding site. The intrinsic affinities are salt-dependent. The formation of the helicase-ssDNA complexes is accompanied by a net release of 3-4 ions. The experiments have been performed with ssDNA oligomers encompassing the total site size of the helicase-ssDNA complex and with oligomers long enough to encompass only the ssDNA-binding site of the enzyme. The obtained results indicate that salt dependence of the intrinsic affinity results predominantly, if not exclusively, from the interactions of the ssDNA-binding site of the helicase with the nucleic acid. There is an anion effect on the studied interactions, which suggests that released ions originate from both the protein and the nucleic acid. Contrary to the intrinsic affinities, cooperative interactions between bound PriA molecules are accompanied by a net uptake of approximately 3 ions. The PriA protein shows preferential intrinsic affinity for pyrimidine ssDNA oligomers. In our standard conditions (pH 7.0, 10 degrees C, 100 mM NaCl), the intrinsic binding constant for the pyrimidine oligomers is approximately 1 order of magnitude higher than the intrinsic binding constant for the purine oligomers. The significance of these results for the mechanism of action of the PriA helicase is discussed.

The Escherichia coli PriA Helicase Specifically Recognizes Gapped DNA Substrates

“…Although there are no cooperative interactions between the PriA helicase bound in the gap complex and to the dsDNA of the gapped DNA substrates, the increase of [NaCl] (20,21). In the case of the gap complex, low ADP or ATP␥S concentrations only slightly affect ⌬F 1 , ⌬F 2 , and ⌬F 3 , as compared with the same parameters determined in the absence of the cofactors, indicating similar structures of the formed complexes (Tables 1 and 2).…”

“…The slope of the log-log plot, ∂logK G / ∂log[NaCl] ϭ Ϫ6.1 Ϯ 0.7, whereas the analogous slopes, obtained for the ssDNA 10-mer and 18-mer, respectively, engaging only the strong DNA-binding subsite, are ∂logK 10 / ∂log[NaCl] ϭ Ϫ2.5 Ϯ 0.4 and ∂logK 18 /∂log[NaCl] ϭ Ϫ3.0 Ϯ 0.5, respectively (21). Clearly, the net number of ions released, ϳ6, in the formation of the gap complex is even larger than the number of the nucleotides in the ssDNA gap.…”

“…In the case of the ssDNA, the increase of the salt concentration in solution strongly increases the weak positive cooperative interactions, which at high [NaCl] become an important part of the free energy of binding (21). Although there are no cooperative interactions between the PriA helicase bound in the gap complex and to the dsDNA of the gapped DNA substrates, the increase of [NaCl] (20,21).…”

Binding of Two PriA–PriB Complexes to the Primosome Assembly Site Initiates Primosome Formation

“…In this context, the PriA protein has very different stoichiometries and affinities for single-stranded DNA (ssDNA) and dsDNA conformations. [19][20][21][22][23][24] While the site size of the protein on ssDNA is ∼ 20 nucleotides, the site size of the PriA-dsDNA complex is only ∼5 bp. Moreover, the affinity for dsDNA is ∼1-2 orders of magnitude higher than the affinity for ssDNA.…”

Functional Characterization of the Multidomain F Plasmid TraI Relaxase-Helicase

“…The presence of different conformational states during each NTPase cycle has been observed in most established helicases, although the detailed mechanism can vary. For example, the helicase DnaB exhibits 210-fold greater affinity for ssDNA upon NTP binding (51), whereas nucleotide binding reduced the affinity of PriA for ssDNA about 5-fold (43). Structural studies of SF1 helicases have shown that nucleotide binding induces the close up of the cleft between 1A and 2A domains (52)(53)(54).…”