Hopping of a processivity factor on DNA revealed by single-molecule assays of diffusion.

Abstract:

Many DNA-interacting proteins diffuse on DNA to perform their biochemical functions. Processivity factors diffuse on DNA to permit unimpeded elongation by their associated DNA polymerases, but little is known regarding their rates and mechanisms of diffusion. The processivity factor of herpes simplex virus DNA polymerase, UL42, unlike "sliding clamp" processivity factors that normally form rings around DNA, binds DNA directly and tightly as a monomer, but can still diffuse on DNA. To investigate the mechanism of UL42 diffusion on DNA, we examined the effects of salt concentration on diffusion coefficient. Ensemble studies, employing electrophoretic mobility shift assays on relatively short DNAs, showed that off-rates of UL42 from DNA depended on DNA length at higher but not lower salt concentrations, consistent with the diffusion coefficient being salt-dependent. Direct assays of the motion of single fluorescently labeled UL42 molecules along DNA revealed increased diffusion at higher salt concentrations. Remarkably, the diffusion coefficients observed in these assays were approximately 10(4)-fold higher than those calculated from ensemble experiments. Discrepancies between the single-molecule and ensemble results were resolved by the observation, in single-molecule experiments, that UL42 releases relatively slowly from the ends of DNA in a salt-dependent manner. The results indicate that UL42 "hops" rather than "slides," i.e., it microscopically dissociates from and reassociates with DNA as it diffuses rather than remaining so intimately associated with DNA that cation condensation on the phosphate backbone does not affect its motion. These findings may be relevant to mechanisms of other processivity factors and DNA-binding proteins.

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