Evidence against a simple tethering model for enhancement of herpes simplex virus DNA polymerase processivity by accessory protein UL42
Evidence against a simple tethering model for enhancement of herpes simplex virus DNA polymerase processivity by accessory protein UL42
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DOI：
10.1128/jvi.76.20.10270-10281.2002
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发表时间：
2002-10-01
影响因子：
5.4
通讯作者：
Parris, DS
中科院分区：
文献类型：
--
作者：
Chaudhuri, M;Parris, DS
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，由催化亚基（Pol）和加工因子（UL42）组成。HSV-1 UL42不同于大多数DNA聚合酶加工因子，它具有与双链DNA结合的固有能力。有人提出UL42通过将Pol直接连接到引物和模板（P/T）来提高Pol的处理能力。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。尽管Pol的活性被超过50 mM KCl的盐浓度所抑制，但在50至125 mM KCl的离子强度变化下，全酶的活性相对难以改变。我们使用硝化纤维素过滤结合试验和实时生物传感器技术来测量单个亚基和全酶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的短模型P/T。我们发现，从活性观察来看，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改变。在50 mM KCl中，UL42结合P/T的表观亲和力与Pol或Pol/UL42在相同的低离子强度缓冲液中所观察到的差异不超过两倍。然而，离子强度的增加显著降低了UL42对P/T的亲和力，在125 mM KCl中，UL42对P/T的亲和力比Pol/UL42降低了3个数量级以上。实时结合动力学显示，大部分亲和力降低可归因于UL42在高离子强度缓冲液中与P/T的极快解离。尽管UL42在高浓度盐中结合模型P/T具有较低的表观亲和力和较差的稳定性，但模型P/T的全酶的活性、结合亲和力和稳定性对离子强度的增加具有抗性，这表明UL42并不是简单地将Poll绑定到DNA上。相反，UL42与Pol相互作用引起的构象改变可能允许在高离子强度条件下全酶与P/T的高亲和力和高稳定性结合。
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 Poll 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.