Electrostatic properties of complexes along a DNA glycosylase damage search pathway.
Electrostatic properties of complexes along a DNA glycosylase damage search pathway.
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沿DNA糖基酶损伤搜索途径的复合物的静电特性。
DOI：
10.1021/bi501011m
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发表时间：
2014-12-09
期刊：
影响因子：
2.9
通讯作者：
Stivers, James T.
中科院分区：
文献类型：
--
作者：
Cravens, Shannen L.;Hobson, Matthew;Stivers, James T.
人尿嘧啶DNA糖基化酶（hUNG）遵循一个扩展的反应坐标，用于定位基因组DNA中的稀有尿嘧啶碱基。这个过程开始于未受损DNA的扩散控制接合，随后是损伤搜索步骤，其中酶保持与DNA链松散结合（易位），最后是识别步骤，允许酶在遇到尿嘧啶时有效地结合和切除尿嘧啶。在沿着这个坐标的每一步沿着，酶必须形成高度专门化的DNA相互作用，用于快速损伤搜索或催化。在这里，我们做了广泛的测量hUNG活动作为盐浓度的函数，剖析热力学，动力学和静电特性的关键酶状态沿着这个反应坐标。我们发现hUNG与未受损DNA的相互作用是在钾离子生理浓度（ΔGelect = −3.5 ± 0.5 kcal mol-1）下静电驱动的，只有很小的非静电贡献（ΔGnon = −2.0 ± 0.2 kcal mol-1）。相反，与受损DNA的相互作用主要由非静电自由能项（ΔGnon = −7.2 ± 0.1 kcal mol-1）控制，但保留了非特异性静电贡献（ΔGelect = −2.3 ± 0.2 kcal mol-1）。停流动力学实验表明，受损DNA结合的盐敏感性源于kon的减少，而koff弱依赖于盐。从盐依赖性的稳态动力学参数，其中扩散控制的kcat/Km显示类似于kon的盐依赖性，而kcat（由产品释放限制）是弱依赖于盐，得到了类似的结果。最后，在同一DNA链中相隔20 bp的两个尿嘧啶位点之间易位的盐依赖性与kon的盐依赖性是不可区分的。这一结果表明，在此间距的易位的过渡状态类似于从本体溶液中的DNA缔合，并且hUNG在易位过程中逃离了DNA离子云。这些发现为细胞中的离子环境如何影响DNA损伤搜索途径提供了关键见解。
Human uracil DNA glycosylase (hUNG) follows an extended reaction coordinate for locating rare uracil bases in genomic DNA. This process begins with diffusion-controlled engagement of undamaged DNA, followed by a damage search step in which the enzyme remains loosely associated with the DNA chain (translocation), and finally, a recognition step that allows the enzyme to efficiently bind and excise uracil when it is encountered. At each step along this coordinate, the enzyme must form DNA interactions that are highly specialized for either rapid damage searching or catalysis. Here we make extensive measurements of hUNG activity as a function of salt concentration to dissect the thermodynamic, kinetic, and electrostatic properties of key enzyme states along this reaction coordinate. We find that the interaction of hUNG with undamaged DNA is electrostatically driven at a physiological concentration of potassium ions (ΔGelect = −3.5 ± 0.5 kcal mol–1), with only a small nonelectrostatic contribution (ΔGnon = −2.0 ± 0.2 kcal mol–1). In contrast, the interaction with damaged DNA is dominated by the nonelectrostatic free energy term (ΔGnon = −7.2 ± 0.1 kcal mol–1), yet retains the nonspecific electrostatic contribution (ΔGelect = −2.3 ± 0.2 kcal mol–1). Stopped-flow kinetic experiments established that the salt sensitivity of damaged DNA binding originates from a reduction of kon, while koff is weakly dependent on salt. Similar findings were obtained from the salt dependences of the steady-state kinetic parameters, where the diffusion-controlled kcat/Km showed a salt dependence similar to kon, while kcat (limited by product release) was weakly dependent on salt. Finally, the salt dependence of translocation between two uracil sites separated by 20 bp in the same DNA chain was indistinguishable from that of kon. This result suggests that the transition-state for translocation over this spacing resembles that for DNA association from bulk solution and that hUNG escapes the DNA ion cloud during translocation. These findings provide key insights into how the ionic environment in cells influences the DNA damage search pathway.
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影响因子：
14.9
作者：
Dunn AR;Kad NM;Nelson SR;Warshaw DM;Wallace SS
通讯作者：
Wallace SS
影响因子：
16.8
作者：
通讯作者：
--
影响因子：
4.8
作者：
Fitzgerald, Megan E.;Drohat, Alexander C.
通讯作者：
Drohat, Alexander C.
影响因子：
3.3
作者：
Long, H;Kudlay, A;Schatz, GC
通讯作者：
Schatz, GC
DOI：
10.1073/pnas.0509723103
发表时间：
2006-04-11
影响因子：
11.1
作者：
Blainey, PC;van Oijent, AM;Xie, XS
通讯作者：
Xie, XS