Chaudhuri Murari, Parris Deborah S
Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, Ohio State University, 333W. Tenth Avenue, Columbus, OH 43210, USA.
J Virol. 2002 Oct;76(20):10270-81. doi: 10.1128/jvi.76.20.10270-10281.2002.
The DNA polymerase holoenzyme of herpes simplex virus type 1 (HSV-1) is a stable heterodimer consisting of a catalytic subunit (Pol) and a processivity factor (UL42). HSV-1 UL42 differs from most DNA polymerase processivity factors in possessing an inherent ability to bind to double-stranded DNA. It has been proposed that UL42 increases the processivity of Pol by directly tethering it to the primer and template (P/T). To test this hypothesis, we took advantage of the different sensitivities of Pol and Pol/UL42 activities to ionic strength. Although the activity of Pol is inhibited by salt concentrations in excess of 50 mM KCl, the activity of the holoenzyme is relatively refractory to changes in ionic strength from 50 to 125 mM KCl. We used nitrocellulose filter-binding assays and real-time biosensor technology to measure binding affinities and dissociation rate constants of the individual subunits and holoenzyme for a short model P/T as a function of the ionic strength of the buffer. We found that as observed for activity, the binding affinity and dissociation rate constant of the Pol/UL42 holoenzyme for P/T were not altered substantially in high- versus low-ionic-strength buffer. In 50 mM KCl, the apparent affinity with which UL42 bound the P/T did not differ by more than twofold compared to that observed for Pol or Pol/UL42 in the same low-ionic-strength buffer. However, increasing the ionic strength dramatically decreased the affinity of UL42 for P/T, such that it was reduced more than 3 orders of magnitude from that of Pol/UL42 in 125 mM KCl. Real-time binding kinetics revealed that much of the reduced affinity could be attributable to an extremely rapid dissociation of UL42 from the P/T in high-ionic-strength buffer. The resistance of the activity, binding affinity, and stability of the holoenzyme for the model P/T to increases in ionic strength, despite the low apparent affinity and poor stability with which UL42 binds the model P/T in high concentrations of salt, suggests that UL42 does not simply tether the Pol to DNA. Instead, it is likely that conformational alterations induced by interaction of UL42 with Pol allow for high-affinity and high-stability binding of the holoenzyme to the P/T even under high-ionic-strength conditions.
单纯疱疹病毒1型（HSV-1）的DNA聚合酶全酶是一种稳定的异源二聚体，由一个催化亚基（Pol）和一个持续合成因子（UL42）组成。HSV-1 UL42与大多数DNA聚合酶持续合成因子不同，它具有结合双链DNA的内在能力。有人提出，UL42通过将Pol直接连接到引物和模板（P/T）上，提高了Pol的持续合成能力。为了验证这一假设，我们利用了Pol和Pol/UL42活性对离子强度的不同敏感性。虽然超过50 mM KCl的盐浓度会抑制Pol的活性，但全酶的活性在50至125 mM KCl的离子强度变化中相对不受影响。我们使用硝酸纤维素滤膜结合测定法和实时生物传感器技术，测量各个亚基和全酶对短模型P/T的结合亲和力和解离速率常数，作为缓冲液离子强度的函数。我们发现，正如在活性方面观察到的那样，Pol/UL42全酶对P/T的结合亲和力和解离速率常数在高离子强度缓冲液和低离子强度缓冲液中没有显著变化。在50 mM KCl中，与在相同低离子强度缓冲液中观察到的Pol或Pol/UL42相比，UL42与P/T结合的表观亲和力差异不超过两倍。然而，增加离子强度会显著降低UL42对P/T 的亲和力，使其在125 mM KCl中比Pol/UL42的亲和力降低超过3个数量级。实时结合动力学表明，亲和力降低的很大一部分可归因于在高离子强度缓冲液中UL42从P/T上极其快速的解离。尽管在高盐浓度下UL42与模型P/T结合的表观亲和力较低且稳定性较差，但全酶对模型P/T的活性、结合亲和力和稳定性对离子强度增加具有抗性，这表明UL42并非简单地将Pol连接到DNA上。相反，很可能是UL42与Pol相互作用诱导的构象改变，使得全酶即使在高离子强度条件下也能与P/T进行高亲和力和高稳定性的结合。
