Kinetics and thermodynamics of BI-BII interconversion altered by T:G mismatches in DNA.
Kinetics and thermodynamics of BI-BII interconversion altered by T:G mismatches in DNA.
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DNA 中 T:G 错配改变的 BI-BII 互变的动力学和热力学。
DOI：
10.1016/j.bpj.2022.03.031
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发表时间：
2022
影响因子：
3.4
通讯作者：
Meints,GaryA
中科院分区：
文献类型：
--
作者：
Westwood,MN;Johnson,CC;Oyler,NathanA;Meints,GaryA
T：G错配在人类中主要是由甲基化的CpG位点去氨基造成的。它们被冗余的系统修复，如胸腺嘧啶DNA糖基酶(TDG)和甲基结合域酶(MBD4)，这些位点的维持与表观遗传过程有关。这些酶在不正确的碱基配对环境中识别典型DNA碱基的过程仍然是一个谜。然而，修复酶与DNA骨架的保守接触表明，蛋白质-磷酸相互作用在识别和修复过程中发挥了作用。我们用31P核磁共振研究了DNA骨架BI-BII相互转换的能量学，为此我们重点研究了相互转换的激活势垒的变化以及与经典DNA相比错配的影响。我们发现T：G基对与U：G基对相互转化的ΔG的变化非常相似，其形式是ΔG的步长比未修饰的DNA1-2千卡/摩尔的步长大1-2千卡/摩尔，这表明这一结果对TDG底物具有普遍性。同样，我们看到BI-BII相互转换的自由能(∼1千卡/摩尔)和激活热焓(2-5千卡/摩尔)的扰动局限于失配两侧的磷酸盐。总体而言，我们的结果有力地表明T：G碱基对中受扰动的骨架能量学在DNA修复酶的识别过程中发挥着重要作用。
T:G mismatches in DNA result in humans primarily from deamination of methylated CpG sites. They are repaired by redundant systems, such as thymine DNA glycosylase (TDG) and methyl-binding domain enzyme (MBD4), and maintenance of these sites has been implicated in epigenetic processes. The process by which these enzymes identify a canonical DNA base in the incorrect basepairing context remains a mystery. However, the conserved contacts of the repair enzymes with the DNA backbone suggests a role for protein-phosphate interaction in the recognition and repair processes. We have used31P NMR to investigate the energetics of DNA backbone BI-BII interconversion, and for this work have focused on alterations to the activation barriers to interconversion and the effect of a mismatch compared with canonical DNA. We have found that alterations to the ΔG of interconversion for T:G basepairs are remarkably similar to U:G basepairs in the form of stepwise differences in ΔG of 1–2 kcal/mol greater than equivalent steps in unmodified DNA, suggesting a universality of this result for TDG substrates. Likewise, we see perturbations to the free energy (∼1 kcal/mol) and enthalpy (2–5 kcal/mol) of activation for the BI-BII interconversion localized to the phosphates flanking the mismatch. Overall our results strongly suggest that the perturbed backbone energetics in T:G basepairs play a significant role in the recognition process of DNA repair enzymes.