THERMODYNAMIC PROPERTIES OF EXOCYCLIC DNA ADDUCTS

在目前的资助期内，我们已经应用光谱和量热技术来表征诱变损伤对DNA双链的稳定性、构象偏好、温度依赖性转变和熔化协同性的热（δ Go、δ Ho、δ So、δ Cp）和热力学外影响。在所要求的供资期内，我们的方案分为两个部分，将并行进行。在第一部分中，我们将通过评估来建立包含病变的DNA双链的胚胎数据库：(I)通过DNA主干和/或堆叠碱基传播病变效应；（ii）损伤对DNA长度和柔韧性的影响；（iii）一种新的基于荧光的方法，用于快速、可靠地测定DNA双链稳定性；（iv）附加损伤对DNA双链物理化性质的影响，特别是链间交联、FapydG（鸟嘌呤的主要丙烯醛加合物）、dA的碳环类似物、8-氧合g和碱性位点。序列背景（跨链伴侣碱基和侧翼碱基）对这些损伤的热力学和额外热力学影响的影响将被评估，并着眼于进一步评估结构相似DNA结构域的“能量辨别/识别”的作用。在我们计划的第二部分中，我们将使用量热法(i)定义大肠杆菌聚合酶i的外切酶缺陷Klenow片段的模板定向DNA合成的能量景观和（II）表征修复蛋白识别DNA损伤的热力学。这些努力旨在定义损伤诱导的物理化学差异，这些差异可能为修复机制选择性识别受损DNA位点提供基础。我们的最终目标是利用我们的光谱和量热结果，以及核磁共振研究，来定义结构、能量学和生物活性之间的关系。这种相关性应该有助于我们阐明化学和辐射诱导的诱变、病变识别和修复的机制。

DESCRIPTION: (Applicant's Description) During the current funding period, we have applied spectroscopic and calorimetric techniques to characterize the thermal (delta Go, delta Ho, delta So, delta Cp) and extra-thermodynamic impacts of mutagenic lesions on the stabilities, conformational preferences, temperature-dependent transitions, and melting cooperativities of DNA duplexes. Our program for the requested funding period is divided into two parts, to be pursued in parallel. In Part I, we will build on our embryonic database for lesion-containing DNA duplexes by evaluating: (i) the communication of lesion effects through the DNA backbone and/or stacked bases; (ii) the impact of lesions on the length and flexibility of DNA; (iii) a new fluorescence-based methodology for rapid and reliable determination of DNA duplex stabilities; and (iv) the impact of additional lesions on the physicochemical properties of DNA duplexes, particularly interstrand crosslinks, FapydG, the major acrolein adduct of guanine, and the carbocyclic analogs of dA, 8-oxodG and the abasic site. The effects of sequence context (cross-strand partner bases and flanking bases) on the thermodynamic and extra-thermodynamic impacts of these lesions will be assessed with an eye towards further evaluating the role of "energetic discrimination/recognition" of structurally similar DNA domains. In Part II of our program, we will use calorimetric methods (i) to define the energetic landscape of template-directed DNA synthesis by an exonuclease deficient Klenow fragment of E. coli polymerase I and (ii) to characterize the thermodynamics of repair protein recognition of damage in DNA. These efforts are designed to define lesion-induced physicochemical differences that may provide the basis for selective recognition of damaged DNA sites by the machinery of repair. Our ultimate goal is to use our spectroscopic and calorimetric results, together with NMR studies, to define relationships between structure, energetics, and biological activity. Such correlations should help us elucidate mechanisms of chemical and radiation induced mutagenesis, lesion recognition, and repair.