Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 610 Taylor Rd, Piscataway, NJ08854, USA.
The Rutgers Cancer Institute of New Jersey, New Brunswick, NJ08901, USA.
Q Rev Biophys. 2021 Dec 6;55:e1. doi: 10.1017/S0033583521000093.
We demonstrate that reshaping of the dynamic, bulged-loop energy landscape of DNA triplet repeat ensembles by the presence of an abasic site alters repair outcomes by the APE1 enzyme. This phenomenon depends on the structural context of the lesion, despite the abasic site always having the same neighbors in sequence space. We employ this lesion-induced redistribution of DNA states and a kinetic trap to monitor different occupancies of the DNA bulge loop states. We show how such dynamic redistribution and associated differential occupancies of DNA states impact APE1 repair outcomes and APE1 induced interconversions. We correlate the differential biophysical properties of the dynamic, DNA ensemble states, with their ability to be recognized and processed as substrates by the APE1 DNA repair enzyme. Enzymatic digestions and biophysical characterizations reveal that APE1 cuts a fraction (10-12%) of the dynamic, rollameric substrates within the initial kinetic distribution. APE1 interactions also 'induce' rollamer redistribution from a kinetically trapped distribution to an equilibrium distribution, the latter not containing viable APE1 substrates. We distinguish between kinetically controlled ensemble (re)distributions of potential DNA substrates, versus thermodynamically controlled ensemble (re)distribution; features of importance to DNA regulation. We conclude that APE1 activity catalyzes/induces ensembles that represent the thermodynamically optimal loop distribution, yet which also are nonviable substrate states for abasic site cleavage by APE1. We propose that by inducing substrate redistributions in a dynamic energy landscape, the enzyme actually reduces the available substrate competent species for it to process, reflective of a regulatory mechanism for enzymatic self-repression. If this is a general phenomenon, such a consequence would have a profound impact on slowing down and/or misdirecting DNA repair within dynamic energy landscapes, as exemplified here within triplet repeat domains. In short, APE1-instigated redistribution of potential substrates induces a preferred pathway to an equilibrium ensemble of enzymatically incompetent states.
我们证明了碱基切除修复酶 APE1 通过改变存在无碱基位点的 DNA 三核苷酸重复序列的动态膨出环能量景观的形状来改变修复结果。尽管无碱基位点在序列空间中始终具有相同的邻居，但这种现象取决于损伤的结构背景。我们利用这种损伤诱导的 DNA 状态再分布和动力学陷阱来监测 DNA 凸起环状态的不同占据。我们展示了这种动态再分布和相关的 DNA 状态的差异占据如何影响 APE1 修复结果和 APE1 诱导的相互转化。我们将动态，DNA 整体状态的差异生物物理特性与它们作为 APE1 DNA 修复酶的底物被识别和处理的能力相关联。酶消化和生物物理特性表明，APE1 在初始动力学分布中切割动态，滚环底物的一部分（10-12％）。APE1 相互作用还通过将滚环从动力学捕获分布诱导到平衡分布来“诱导”滚环再分布，后者不包含可行的 APE1 底物。我们区分了潜在 DNA 底物的动力学控制的整体（再）分布与热力学控制的整体（再）分布; 这些特征对 DNA 调节很重要。我们得出的结论是，APE1 活性催化/诱导了代表热力学最优环分布的整体，然而，这些整体也是 APE1 切割无碱基位点的不可行的底物状态。我们提出，通过在动态能量景观中诱导底物再分布，酶实际上减少了其可处理的可用底物有活性的物质，反映了酶自身抑制的调节机制。如果这是一种普遍现象，那么这种后果将对在动态能量景观中减缓和/或误导 DNA 修复产生深远的影响，正如这里在三核苷酸重复域中所体现的那样。简而言之，APE1 引发的潜在底物的再分配诱导了一种向酶无能状态的平衡整体的首选途径。
