Komazin-Meredith Gloria, Mirchev Rossen, Golan David E, van Oijen Antoine M, Coen Donald M
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10721-6. doi: 10.1073/pnas.0802676105. Epub 2008 Jul 25.
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.
许多与DNA相互作用的蛋白质在DNA上扩散以执行其生化功能。持续性因子在DNA上扩散，以使与其相关的DNA聚合酶能够不受阻碍地进行延伸，但关于它们的扩散速率和机制却知之甚少。单纯疱疹病毒DNA聚合酶的持续性因子UL42，与通常在DNA周围形成环的“滑动夹”持续性因子不同，它作为单体直接且紧密地结合DNA，但仍能在DNA上扩散。为了研究UL42在DNA上的扩散机制，我们研究了盐浓度对扩散系数的影响。利用相对较短的DNA进行电泳迁移率变动分析的整体研究表明，在较高而非较低盐浓度下，UL42从DNA上的解离速率取决于DNA长度，这与扩散系数依赖于盐的情况一致。对单个荧光标记的UL42分子沿DNA运动的直接测定表明，在较高盐浓度下扩散增加。值得注意的是，在这些测定中观察到的扩散系数比从整体实验计算出的扩散系数大约高10^4倍。单分子实验中观察到UL42以盐依赖的方式从DNA末端相对缓慢地释放，从而解决了单分子结果与整体结果之间的差异。结果表明，UL42是“跳跃”而非“滑动”，也就是说，它在扩散时从微观上与DNA解离并重新结合，而不是与DNA紧密结合到磷酸骨架上的阳离子凝聚不影响其运动的程度。这些发现可能与其他持续性因子和DNA结合蛋白的机制有关。
