Structure of the Tertiary Complex of the RepA Hexameric Helicase of Plasmid RSF1010 with the ssDNA and Nucleotide Cofactors in Solution

Abstract

The structure of the complex of the hexameric replicative helicase RepA protein of plasmid RSF1010 with ssDNA has been examined using the fluorescence energy transfer and analytical ultracentrifugation methods. We utilized the fact that the RepA monomer contains a single, natural cysteine residue. The cysteine residue has been modified with a fluorescent marker, which serves as the donor to the acceptor placed in different locations on the DNA. Using the two independent fluorescence donor-acceptor pairs and different DNA oligomers, we provide direct evidence that, in the complex with the enzyme, the ssDNA passes through the inner channel of the RepA hexamer. In the stationary complex, the RepA hexamer assumes a strictly single orientation with respect to the polarity of the sugar-phosphate backbone of the nucleic acid, with the large domain of protomers facing the 3' end of the bound DNA. Interactions with the helicase induce profound changes in the structure of the bound DNA, and these changes are predominantly localized in the proper DNA-binding site. The heterogeneity of the structure of the bound DNA reflects the heterogeneous structure of the total RepA helicase DNA-binding site. This is in excellent agreement with the thermodynamic data. The structure of the RepA hexamer, in solution, differs considerably from the crystal structure of the enzyme. Both fluorescence energy transfer and analytical ultracentrifugation data indicate a significant conformational flexibility of the RepA hexamer. Implications of these results for the mechanism of interactions of the hexameric helicase with the DNA are discussed.

Full-length Dengue Virus RNA-dependent RNA Polymerase-RNA/DNA Complexes

“…The concentration of the protein is 1.89 ϫ 10 Ϫ6 M (monomer). Inspection of the profiles clearly shows that there is a single moving boundary, indicating the presence of a single molecular species (41,42,44). To obtain the sedimentation coefficient of the protein, s 20,w , the sedimentation velocity scans have been analyzed using the time derivative approach (45, 46).…”

Interactions between the Dengue Virus Polymerase NS5 and Stem-Loop A

“…The size and shape of SLA 80 were analyzed by analytical ultracentrifugation using an Optima XL-A analytical ultracentrifuge (Beckman Inc., Palo Alto, CA), as we described previously (29,40,41). Sedimentation equilibrium scans at different MgCl 2 concentrations (0, 1, and 10 mM) were collected at the absorption band of SLA 80 (260 nm).…”

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

“…The obtained values of the apparent fluorescence energy transfer efficiencies are E D = 0.26 ± 0.03 and E A = 0.025 ± 0.005, and the Förster FRET efficiency is E = 0.033 ± 0.005 (Materials and Methods). [28][29][30] Because the limiting anisotropies of all labeled components of the system examined are below ∼ 0.23 (data not shown), the orientation factor κ 2 does not affect the FRET measurements discussed. [28][29][30] The value of E indicates that the average distance between CPM on PriA and the Fl moiety at the 5′ end of the PAS is R = 91 ± 5 Å.…”

“…[26][27][28][29][30] The fluorescence donor [coumarin derivative, 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM)] has been placed on the PriA protein. Labeling of the PriA protein with CPM has been performed according to the protocol previously reported.…”

“…Labeling of the PriA protein with CPM has been performed according to the protocol previously reported. 28,29 The protein has 11 cysteine residues. 31 However, the degree of labeling is only q = 0.36 ± 0.1.…”