Dynamics of the ssDNA Recognition by the RepA Hexameric Helicase of Plasmid RSF1010: Analyses Using Fluorescence Stopped-Flow Intensity and Anisotropy Methods

摘要

The kinetic mechanism of the single-stranded DNA (ssDNA) recognition by the RepA hexameric replicative helicase of the plasmid RSF1010 and the nature of formed intermediates, in the presence of the ATP nonhydrolyzable analog, β,γ-imidoadenosine-5′-triphosphate (AMP-PNP), have been examined, using the fluorescence intensity and anisotropy stopped-flow and analytical ultracentrifugation methods. Association of the RepA hexamer with the ssDNA oligomers that engage the total DNA-binding site and exclusively the strong DNA-binding subsite is a minimum four-step mechanism Helicase+ssDNA⇄k−1k1(H−ssDNA)1⇄k−2k2(H−ssDNA)2⇄k−3k3(H−ssDNA)3⇄k−4k4(H−ssDNA)4 Extreme stability of the RepA hexamer precludes any disintegration of its structure, and the sequential character of the mechanism indicates that the enzyme exists in a predominantly single conformation prior to the association with the nucleic acid. Moreover, the hexameric helicase possesses a DNA-binding site located outside its cross channel. The reaction steps have dramatically different dynamics, with rate constants differing by 2–3 orders of magnitude. Such behavior indicates a very diverse nature of the observed transitions, which comprises binding steps and large conformational transitions of the helicase, including local opening of the hexameric structure. Steady-state fluorescence anisotropies of intermediates indicate that the entry of the DNA into the cross channel is initiated from the 5′ end of the bound nucleic acid. The global structure of the tertiary complex RepA–ssDNA–AMP–PNP is very different from the structure of the binary complex RepA–AMP-PNP, indicating that, in equilibrium, the RepA hexamer–ssDNA–AMP-PNP complex exists as a mixture of partially open states.