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

Abstract

Functional interactions of the E. coli PriA helicase 181N-terminal domain with the DNA and nucleotide cofactors have been quantitatively examined. The isolated 181N-terminal domain forms a stable dimer in solution, most probably reflecting the involvement of the domain in specific cooperative interactions of the intact PriA protein - dsDNA complex. Only one monomer of the domain dimer binds the DNA, i.e., the dimer has one effective DNA-binding site. Although the total site-size of the dimer - ssDNA complex is ~13 nucleotides, the DNA-binding subsite engages in direct interactions ~5 nucleotides. A small number of interacting nucleotides indicates that the DNA-binding subsites of the PriA helicase, i.e., the strong subsite on the helicase domain and the weak subsite on the N-terminal domain, are spatially separated in the intact enzyme. Contrary to current views, the subsite has only a slight preference for the 3′-end OH group of the ssDNA and lacks any significant base specificity, although it has a significant dsDNA affinity. Unlike the intact helicase, the DNA-binding subsite of the isolated domain is in an open conformation, indicating the presence of the direct helicase domain - N-terminal domain interactions. The discovery that the 181N-terminal domain possesses a nucleotide-binding site places the allosteric, weak nucleotide-binding site of the intact PriA on the N-terminal domain. The specific ADP effect on the domain DNA-binding subsite indicates that in the intact helicase, the bound ADP not only opens the DNA-binding subsite but also increases its intrinsic DNA affinity.

The Escherichia coli Primosomal DnaT Protein Exists in Solution as a Monomer–Trimer Equilibrium System

“…All steady-state fluorescence titrations were performed using the ISS (Urbana, IL) PC-1 spectrofluorometer, as previously described by us. − The state of the DnaT protein oligomerization has been analyzed using the fluorescence anisotropy, r , of the protein tryptophan residues defined as − r = I VV − G I VH I VV + 2 G I VH where I VV and I VH are fluorescence intensities where the first and second subscripts refer to vertical (V) polarization of the excitation and vertical (V) or horizontal (H) polarization of the emitted light, respectively. − The factor ( G = I HV / I HH ) corrects for the different sensitivity of the emission monochromator for vertically and horizontally polarized light. − The sample was excited at 296 nm (protein tryptophan absorption band) and the emission recorded at 347 nm (protein tryptophan emission band).…”

Structural Insight into the DNA-Binding Mode of the Primosomal Proteins PriA, PriB, and DnaT

“…The native PriA is a monomer with a molecular mass of ~82 kDa. The tertiary structure of the monomer contains two functional domains, namely, the helicase domain (HD), which encompasses ~540 amino acid residues from the C-terminus, and the DNA-binding domain, which comprises ~181 amino acid residues from the N-terminus [ 31 – 33 ]. PriA is a DEXH-type helicase that unwinds DNA with a 3′ to 5′ polarity [ 34 ].…”

“…EcPriA has a highly electropositive ssDNA-binding region (amino acid residues 1–198) containing 8 Lys and 14 Arg residues in DBD; thus, the basic DBD in EcPriA may be involved in complex with EcPriB [ 73 ]. The DBD of EcPriA alone in solution forms a dimer and not a monomer as EcPriA [ 31 ], suggesting that another unknown stabilization factor is needed. The DBD of PriA and one of the monomers of PriB may bind to ssDNA cooperatively to decrease the dissociation rate of PriA from the DNA during helix unwinding [ 73 ].…”