THERMODYNAMIC PROPERTIES OF EXOCYCLIC DNA ADDUCTS

产品说明：（申请人描述）在当前资助期间，我们应用光谱和量热技术来表征诱变损伤对DNA双链体的稳定性、构象偏好、温度依赖性转变和熔融协同性的热（Δ Go、Δ Ho、Δ So、Δ Cp）和超热力学影响。我们在申请资助期间的方案分为两个部分，并行进行。 在第一部分中，我们将建立在我们的胚胎数据库的病变包含DNA双链体的评估：（i）通过DNA骨架和/或堆叠的碱基的病变效应的通信;（ii）病变的长度和灵活性的DNA的影响;（iii）一个新的荧光为基础的方法，快速和可靠的测定DNA双链体的稳定性;和（iv）额外的损伤对DNA双链体的物理化学性质的影响，特别是链间交联、FaplidG（鸟嘌呤的主要丙烯醛加合物）和dA的碳环类似物、8-oxodG和脱碱基位点。 序列上下文（交叉链伴侣碱基和侧翼碱基）对这些病变的热力学和超热力学影响的影响将进行评估，着眼于进一步评估结构相似的DNA结构域的“能量歧视/识别”的作用。在我们计划的第二部分中，我们将使用量热方法（i）定义由E.大肠杆菌聚合酶I和（ii）表征修复蛋白识别DNA损伤的热力学。 这些努力的目的是确定病变引起的物理化学差异，可能提供的基础上，通过修复机制的损伤DNA位点的选择性识别。我们的最终目标是使用我们的光谱和量热结果，与NMR研究一起，来定义结构，能量学和生物活性之间的关系。 这种相关性将有助于我们阐明化学和辐射诱导的诱变，损伤识别和修复的机制。

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.