Greenwood Sharon N, Dispensa Alexis N, Wang Matthew, Bauer Justin R, Vaden Timothy D, Liu Zhiwei, Weiser Brian P
Department of Molecular Biology, Rowan-Virtua School of Osteopathic Medicine, Rowan University, Stratford, New Jersey 08084, United States.
Department of Molecular Biology, Rowan-Virtua School of Translational Biomedical Engineering & Sciences, Rowan University, Stratford, New Jersey 08084, United States.
Biochemistry. 2025 May 20;64(10):2332-2344. doi: 10.1021/acs.biochem.5c00067. Epub 2025 May 7.
Because of their ubiquitous presence, ions interact with numerous macromolecules in the cell and affect critical biological processes. Here, we discuss how cations including Mg alter the enzymatic activity of a DNA glycosylase by tuning its affinity for DNA. The response of uracil DNA glycosylase (UNG2) to Mg ions in solution is biphasic and paradoxical, where low concentrations of the ion stimulate the enzyme, but high concentrations inhibit the enzyme. We analyzed this phenomenon by modeling experimental data with a statistical framework that we empirically derived to understand molecular systems that display biphasic behaviors. Parameters from our statistical model indicate that DNA substrates are nearly saturated with cations under ideal conditions for UNG2 activity. However, the enzyme slows rather abruptly when the ionic content becomes too low or too high due to changes in the electrostatic environment that alter protein affinity for DNA. We discuss how ion occupancy on DNA is dependent on DNA length; thus, the sensitivity of UNG2 to cations is also dependent on DNA length. Finally, we found that Mg-induced changes in DNA base stacking and dynamics have minimal effects on UNG2, as these outcomes occur at ion concentrations that are much lower than is required for efficient enzyme activity. Altogether, our work demonstrates how cation-DNA interactions, which are likely common in the nucleus, are a key determinant of uracil base excision repair mediated by UNG2.
由于离子无处不在，它们在细胞内与众多大分子相互作用，并影响关键的生物过程。在此，我们讨论包括镁离子在内的阳离子如何通过调节对DNA的亲和力来改变DNA糖基化酶的酶活性。尿嘧啶DNA糖基化酶（UNG2）在溶液中对镁离子的反应是双相且矛盾的，低浓度的该离子会刺激酶活性，但高浓度则会抑制酶活性。我们通过用一个根据经验推导得出的统计框架对实验数据进行建模来分析这一现象，该框架用于理解呈现双相行为的分子系统。我们统计模型的参数表明，在UNG2活性的理想条件下，DNA底物几乎被阳离子饱和。然而，由于改变蛋白质对DNA亲和力的静电环境变化，当离子含量变得过低或过高时，酶的活性会相当突然地减慢。我们讨论了DNA上离子占据情况如何取决于DNA长度；因此，UNG2对阳离子的敏感性也取决于DNA长度。最后，我们发现镁离子诱导的DNA碱基堆积和动力学变化对UNG2的影响极小，因为这些变化发生时的离子浓度远低于高效酶活性所需的浓度。总之，我们的工作证明了阳离子与DNA的相互作用（这在细胞核中可能很常见）是UNG2介导的尿嘧啶碱基切除修复的关键决定因素。
