Uracil-DNA glycosylase efficiency is modulated by substrate rigidity.
Uracil-DNA glycosylase efficiency is modulated by substrate rigidity.
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尿素-DNA糖基化酶效率受底物刚度调节。
DOI：
10.1038/s41598-023-30620-0
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发表时间：
2023-03-08
影响因子：
4.6
通讯作者：
Levitus, Marcia
中科院分区：
文献类型：
--
作者：
Orndorff, Paul B.;Poddar, Souvik;Owens, Aerial M.;Kumari, Nikita;Ugaz, Bryan T.;Amin, Samrat;Van Horn, Wade D.;van der Vaart, Arjan;Levitus, Marcia
尿嘧啶DNA糖基化酶（UNG）是一种DNA修复酶，通过碱基翻转机制从DNA中去除高度诱变的尿嘧啶损伤。虽然这种酶已经进化到可以从不同的序列背景中去除尿嘧啶，但UNG的切除效率取决于DNA序列。为了提供合理的UNG底物偏好的分子基础，我们使用时间分辨荧光光谱、核磁共振亚质子交换测量和分子动力学模拟来测量UNG特异性常数（kcat/KM）和含有中心AUT、TUA、AUA和TUT基序的DNA底物的DNA柔韧性。我们的研究表明，UNG的效率是由病变周围固有的可变形性决定的，建立了底物柔韧性模式与UNG效率之间的直接关系，并表明紧邻尿嘧啶的碱基是变构耦合的，对底物柔韧性和UNG活性的影响最大。底物柔韧性控制UNG效率的发现可能对其他修复酶具有重要意义，并对突变热点发生、分子进化和碱基编辑的理解具有重要意义。
Uracil DNA-glycosylase (UNG) is a DNA repair enzyme that removes the highly mutagenic uracil lesion from DNA using a base flipping mechanism. Although this enzyme has evolved to remove uracil from diverse sequence contexts, UNG excision efficiency depends on DNA sequence. To provide the molecular basis for rationalizing UNG substrate preferences, we used time-resolved fluorescence spectroscopy, NMR imino proton exchange measurements, and molecular dynamics simulations to measure UNG specificity constants (kcat/KM) and DNA flexibilities for DNA substrates containing central AUT, TUA, AUA, and TUT motifs. Our study shows that UNG efficiency is dictated by the intrinsic deformability around the lesion, establishes a direct relationship between substrate flexibility modes and UNG efficiency, and shows that bases immediately adjacent to the uracil are allosterically coupled and have the greatest impact on substrate flexibility and UNG activity. The finding that substrate flexibility controls UNG efficiency is likely significant for other repair enzymes and has major implications for the understanding of mutation hotspot genesis, molecular evolution, and base editing.
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