Hopping of a processivity factor on DNA revealed by single-molecule assays of diffusion - PubMed (original) (raw)
Comparative Study
. 2008 Aug 5;105(31):10721-6.
doi: 10.1073/pnas.0802676105. Epub 2008 Jul 25.
Affiliations
- PMID: 18658237
- PMCID: PMC2504831
- DOI: 10.1073/pnas.0802676105
Comparative Study
Hopping of a processivity factor on DNA revealed by single-molecule assays of diffusion
Gloria Komazin-Meredith et al. Proc Natl Acad Sci U S A. 2008.
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.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Effect of salt concentration on DNA length dependence of UL42 dissociation from DNA. Half-lives of UL42-DNA complexes were measured by EMSAs at 10 mM (A), 25 mM (B), and 50 mM (C) NaCl. Data were fitted using the equation t_½ = ln 2/((12_D/_b_2) + _k_off(internal)), relating the length of DNA, b, and the half-life of the protein on DNA, _t_½, to permit calculation of the diffusion coefficient (D) and _k_off(internal) (7) (continuous line). In A and C, data for krelease(ends) (Table 1) from single-molecule experiments at 10 mM (A) and 50 mM (C) NaCl were fitted using the equation _t_½ = ln2·(2_k_release(ends)/b + _k_off(internal))−1, where the _k_off(internal) values were derived from either single-molecule studies (dotted line) or EMSA studies (dashed line).
Fig. 2.
UL42 diffuses bidirectionally on DNA. Histogram showing net displacement of UL42 molecules on lambda DNA measured by single-molecule assays in the presence of buffer flow. Curve represents Gaussian fit.
Fig. 3.
Effect of salt concentration on UL42 binding lifetimes and diffusion coefficients measured by single-molecule experiments. (A) Distribution of binding times of UL42 molecules on lambda DNA at 10 mM, 50 mM, and 100 mM NaCl. Data were fitted to single-exponential decay curves. (B) Mean binding lifetimes of UL42 on lambda DNA determined from curve fit in A (open circle, left axis) and mean diffusion coefficients (D) of UL42 on lambda DNA (open square, right axis) at three different salt concentrations. The error bar for the highest D value indicates standard deviation, which was ≈10% of the mean for each of the three D values. (C) Histograms showing distributions of diffusion coefficients of UL42 on lambda DNA at 10 mM, 50 mM, and 100 mM NaCl. Lines represent smoothed envelope curves. The asterisk in Lower indicates that the frequency shown actually represents the frequency for all D values greater than or equal to the indicated D.
Fig. 4.
Release of UL42 from the ends of DNA. (A and B) Images of UL42 proteins conjugated to fluorescent quantum dots and bound to lambda DNA in the absence (A) or presence (B) of buffer flow. (Scale bar: 5 μm.) (C) Change in fluorescence intensity at the end of lambda DNA over time at 20 mM NaCl. Flow was started at t = 10 s. Data were fitted to a single-exponential decay curve.
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