Rumora Amy E, Kolodziejczak Katarzyna M, Malhowski Wagner Anne, Núñez Megan E
Department of Chemistry, Mount Holyoke College, South Hadley, Massachusetts 01075.
Biochemistry. 2008 Dec 9;47(49):13026-35. doi: 10.1021/bi801417u.
Despite significant progress in the past decade, questions still remain about the complete structural, dynamic, and thermodynamic effect of the cis-syn cyclobutane pyrimidine dimer lesion (hereafter called the thymine dimer) on double-stranded genomic DNA. We examined a 19-mer oligodeoxynucleotide duplex containing a thymine dimer lesion using several small, base-selective reactive chemical probes. These molecules probe whether the presence of the dimer causes the base pairs to be more accessible to the solution, either globally or adjacent to the dimer. Though all of the probes confirm that the overall structure of the dimer-containing duplex is conserved compared to that of the undamaged parent duplex, reactions with both diethyl pyrocarbonate and Rh(bpy)(2)(chrysi)(3+) indicate that the duplex is locally destabilized near the lesion. Reactions with potassium permanganate and DEPC hint that the dimer-containing duplex may also be globally more accessible to the solution through a subtle shift in the double-stranded DNA ↔ single-stranded DNA equilibrium. To begin to distinguish between kinetic and thermodynamic effects, we determined the helix melting thermodynamic parameters for the dimer-containing and undamaged parent duplexes by microcalorimetry and UV melting. The presence of the thymine dimer causes this DNA duplex to be slightly less stable enthalpically but slightly less unstable entropically at 298 K, causing the overall free energy of duplex melting to remain unchanged by the dimer lesion within the error of the experiment. Here we consider these results in the context of what has been learned about the thymine dimer lesion from NMR, X-ray crystallographic, and molecular biological methods.
尽管在过去十年中取得了重大进展，但关于顺式-顺式环丁烷嘧啶二聚体损伤（以下简称胸腺嘧啶二聚体）对双链基因组DNA的完整结构、动力学和热力学影响，仍然存在一些问题。我们使用几种小的、碱基选择性反应性化学探针，研究了一个含有胸腺嘧啶二聚体损伤的19聚体寡脱氧核苷酸双链体。这些分子探测二聚体的存在是否会使碱基对在整体上或与二聚体相邻处更容易与溶液接触。尽管所有探针都证实，与未受损的亲本双链体相比，含二聚体的双链体的整体结构是保守的，但焦碳酸二乙酯和Rh(bpy)(2)(chrysi)(3+)的反应表明，双链体在损伤附近局部不稳定。高锰酸钾和DEPC的反应暗示，含二聚体的双链体也可能通过双链DNA↔单链DNA平衡的微妙变化，在整体上更容易与溶液接触。为了开始区分动力学和热力学效应，我们通过微量热法和紫外熔解法，测定了含二聚体和未受损亲本双链体的螺旋熔解热力学参数。胸腺嘧啶二聚体的存在导致该DNA双链体在298 K时焓稳定性略有降低，但熵不稳定性略有降低，导致双链体熔解的总自由能在实验误差范围内因二聚体损伤而保持不变。在这里，我们结合从核磁共振、X射线晶体学和分子生物学方法中了解到的关于胸腺嘧啶二聚体损伤的知识，来考虑这些结果。
