Abstract
TraI, a bifunctional enzyme containing relaxase and helicase activities, initiates and drives the conjugative transfer of the Escherichia coli F plasmid. Here, we examined the structure and function of the TraI helicase. We show that TraI binds to single-stranded DNA (ssDNA) with a site size of ∼25 nucleotides, which is significantly longer than the site size of other known superfamily I helicases. Low cooperativity was observed with the binding of TraI to ssDNA, and a double-stranded DNA-binding site was identified within the N-terminal region of TraI 1-858, outside the core helicase motifs of TraI. We have revealed that the affinity of TraI for DNA is negatively correlated with the ionic strength of the solution. The binding of AMPPNP or ADP results in a 3-fold increase in the affinity of TraI for ssDNA. Moreover, TraI prefers to bind ssDNA oligomers containing a single type of base. Finally, we elucidated the solution structure of TraI using small angle x-ray scattering. TraI exhibits an ellipsoidal shape in solution with four domains aligning along one axis. Taken together, these data result in the assembly of a model for the multidomain helicase activity of TraI.
Highlights
The relaxase and helicase activities necessary for F plasmid transfer are located on a 190-kDa multidomain protein, TraI (6, 7)
To understand the intrinsic DNA-binding property of TraI, we have studied the interaction of TraI with unlabeled DNA oligomers using the Macromolecule Competition Titration (MCT) method as described by Jezewska and Bujalowski (27)
The MCT method enables the construction of a model-independent binding isotherm, which can be used for the determination of thermodynamically rigorous binding parameters for the unlabeled DNA oligomer
Summary
DNA Oligomers—DNA oligomers used in this study were purchased from Integrated DNA Technology (IDT, Coralville, IA). Concentrated protein was loaded on a HiLoadTM 16/60 Superdex 200 column (GE Healthcare) equilibrated with sizing buffer (20 mM TrisHCl, pH 7.5, 100 mM NaCl, 5% glycerol). To perform the direct DNA binding assay, a 6FAM-labeled DNA oligomer was mixed with increasing concentrations of protein in DNA binding buffer in 384-well plates (Corning Glass). To calculate the macroscopic DNA binding constant (KN), normalized data were plotted as average FA versus total protein concentration and fit to Equation 2, FA ϭ FAmin ϩFAmax Ϫ FAmin. A 6FAM-labeled reference DNA oligomer (total concentration, DTR) was mixed with increasing concentrations of protein in DNA binding buffer in 384-well plates in the presence of a competing unlabeled DNA oligomer (total concentration, DTS). The total protein concentration in the presence of a competing DNA oligomer, PT1, was defined as Equation 3,. The IC50, or the concentration of competitor DNA required to displace 50% of the complex formed by protein and reference DNA, was determined by plotting the anisotropy value as a function of competitor DNA concentration and fitting the curve using Equation 11
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