Tse Edmund C M, Zwang Theodore J, Bedoya Sebastian, Barton Jacqueline K
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, United States.
ACS Cent Sci. 2019 Jan 23;5(1):65-72. doi: 10.1021/acscentsci.8b00566. Epub 2019 Jan 11.
The stacked aromatic base pairs within the DNA double helix facilitate charge transport down its length in the absence of lesions, mismatches, and other stacking perturbations. DNA repair proteins containing [4Fe4S] clusters can take advantage of DNA charge transport (CT) chemistry to scan the genome for mistakes more efficiently. Here we examine the effective length over which charge can be transported along DNA between these repair proteins. We define the effective CT distance as the length of DNA within which two proteins are able to influence their ensemble affinity to the DNA duplex via CT. Endonuclease III, a DNA repair glycosylase containing a [4Fe4S] cluster, was incubated with DNA duplexes of different lengths (1.5-9 kb), and atomic force microscopy was used to quantify the binding of proteins to these duplexes to determine how the relative protein affinity changes with increasing DNA length. A sharp change in binding slope is observed at 3509 base pairs, or about 1.2 μm, that supports the existence of two regimes for protein binding, one within the range for DNA CT, one outside of the range for CT; DNA CT between the redox proteins bound to DNA effectively decreases the ensemble binding affinity of oxidized and reduced proteins to DNA. Utilizing an Endonuclease III mutant Y82A, which is defective in carrying out DNA CT, shows only one regime for protein binding. Decreasing the temperature to 4 °C or including metallointercalators on the duplex, both of which should enhance base stacking and decrease DNA floppiness, leads to extending the effective length for DNA charge transport to ∼5300 bp or 1.8 μm. These results thus support DNA charge transport between repair proteins over kilobase distances. The results furthermore highlight the ability of DNA repair proteins to search the genome quickly and efficiently using DNA charge transport chemistry.
在没有损伤、错配和其他堆积扰动的情况下，DNA双螺旋内堆叠的芳香碱基对有利于沿其长度方向的电荷传输。含有[4Fe4S]簇的DNA修复蛋白可以利用DNA电荷传输（CT）化学来更有效地扫描基因组中的错误。在这里，我们研究了电荷在这些修复蛋白之间沿DNA传输的有效长度。我们将有效CT距离定义为DNA的长度，在这个长度范围内，两种蛋白质能够通过CT影响它们对DNA双链体的整体亲和力。核酸内切酶III是一种含有[4Fe4S]簇的DNA修复糖基化酶，它与不同长度（1.5 - 9 kb）的DNA双链体一起孵育，然后使用原子力显微镜来量化蛋白质与这些双链体的结合，以确定相对蛋白质亲和力如何随DNA长度增加而变化。在3509个碱基对（约1.2μm）处观察到结合斜率的急剧变化，这支持了蛋白质结合存在两种状态，一种在DNA CT范围内，一种在CT范围之外；与DNA结合的氧化还原蛋白之间的DNA CT有效地降低了氧化和还原蛋白对DNA的整体结合亲和力。利用在进行DNA CT方面有缺陷的核酸内切酶III突变体Y82A，只显示出一种蛋白质结合状态。将温度降至4°C或在双链体上加入金属嵌入剂，这两者都应该增强碱基堆积并减少DNA的柔韧性，导致DNA电荷传输的有效长度延长至约5300 bp或1.8μm。因此，这些结果支持了修复蛋白之间在千碱基距离上的DNA电荷传输。这些结果还突出了DNA修复蛋白利用DNA电荷传输化学快速有效地搜索基因组的能力。
