Y-family DNA polymerases, characterized by low fidelity and processivity, are able to bypass different classes of DNA lesions in vivo. By monitoring time-dependent fluorescence resonance energy transfer (FRET) signal changes at multiple sites in each domain and DNA during catalysis, we show a real-time picture of the global conformational transitions of Sulfolobus solfataricus Dpo4, a model Y-family enzyme. Our results provide evidence for a hypothetical DNA translocation event followed by a rapid protein conformational change prior to catalysis and a subsequent slow, post-chemistry protein conformational change. Surprisingly, the DNA translocation step was induced by the binding of a correct nucleotide. Moreover, we have determined the directions, rates, and activation energy barriers of the protein conformational transitions, which indicated that the four domains of Dpo4 moved in a synchronized manner. These results showed conclusively that a pre-chemistry conformational change associated with domain movements was too fast to be the rate-limiting step.

Global conformational dynamics of a Y-Family DNA polymerase during catalysis<i> </i>

242nd National Meeting (2011)

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