Preparation and biophysical characterization of neutral and cationic DNA lesions, and their role in damage recognition by DNA glycosylases

项目摘要 基因组的完整性不断受到普遍存在的烷化剂的威胁，发现在内源性 外生的来源。不能修复DNA烷基化损伤可能导致诱变或 细胞毒DNA对烷基化损伤敏感，特别是在位于嘌呤和嘌呤上的亲核位点处。 嘧啶核碱基。但是，这些位点并不等价，有些位点的烷基化程度可能更高 对DNA结构、完整性和细胞健康有害。特别是烷基化损伤 沿着DNA的大沟（例如鸟嘌呤和腺嘌呤的N7）可以影响DNA结构， 动力学与沿着位于小沟中的位点的烷基化非常不同（例如鸟嘌呤的N3和 腺嘌呤）。然而，关于小沟与大沟病变的影响的信息很少， 部分是由于它们相对于自发和酶介导的脱嘌呤的固有不稳定性。 此外，DNA热力学“签名”在DNA修复酶识别损伤中的作用， 作为DNA糖基化酶，人们对它知之甚少，这也是因为这种酶催化这些损伤的修复， 排除了在相应的蛋白质/DNA复合物中对它们进行生物化学/结构研究。 我建议制备和掺入甲基化核糖核苷酸3-甲基腺苷（3 mA）和7- 甲基鸟苷（7 mG，代替它们的“正常”脱氧核糖对应物）转化为双链DNA，作为类似物 具有稳定的糖苷键，用于生物物理、生物化学和结构研究。结合使用 生物物理技术，如圆二色性、差示扫描量热法和热熔融 实验中，我将描述甲基的影响-一个常见的DNA烷基损伤-到 双链体DNA的大沟和小沟（其中7 mG和3 mA提供位于大沟和小沟中的甲基， 分别为小沟）。感兴趣的热力学特征包括以下物质的摩尔数： 每摩尔DNA的水合水分子数，相关抗衡离子的摩尔数，B到A形式DNA 转变倾向（在加入三氟乙醇后在相对湿度下测量）和DNA双链体解链 温度这些特征提供了关于如何强溶剂化的DNA双链体含有 小沟或大沟损伤的阳离子烷基嘌呤，静电/离子气氛变化 与这些修饰相关的碱基堆积/配对相互作用和一般的B型构象 稳定然后将这些性质与生物化学数据进行比较，例如与 DNA糖基化酶AAG和AlkD，并将用作结构探针，以获得有关识别的见解 以及这些酶所采用的切除机制。电泳迁移率变化将用于 确定结合常数，X射线晶体学将用于结构研究。

PROJECT SUMMARY Genomic integrity is constantly threatened by ubiquitous alkylating agents found in both endogenous and exogenous sources. Failure to repair DNA alkylation damage may lead to either or both mutagenesis or cytotoxicity. DNA is susceptible to alkylation damage, particularly at nucleophilic sites located on purine and pyrimidine nucleobases. However, these sites are not equivalent, and alkylation of some sites may be more detrimental to DNA structure, integrity, and to the health of the cell than others. Particularly, alkylation damage along the major groove of DNA (such as at N7 of guanine and adenine) may affect DNA structure and dynamics very differently than alkylation along sites located in the minor groove (such as N3 of guanine and adenine). However, very little information regarding the effects of minor versus major groove lesions exist, in part due to their inherent instability with respect to spontaneous and enzyme-mediated depurination. Furthermore, the role of DNA thermodynamic “signatures” in lesion recognition by DNA repair enzymes, such as DNA glycosylases, is poorly understood, again because such enzymes catalyze the repair of these lesions, precluding their biochemical/structural study in corresponding protein/DNA complexes. I propose preparation and incorporation of methylated ribonucleotides 3-methyladenosine (3mA) and 7- methylguanosine (7mG, in place of their “normal”, deoxyribose counterparts) into duplex DNA, as analogues with stabilized glycosidic bonds for biophysical, biochemical, and structural studies. Using a combination of biophysical techniques such as circular dichroism, differential scanning calorimetry, and thermal melting experiments, I will characterize the effects that methyl groups – a common DNA alkyl lesion – have into the major and minor groove of duplex DNA (where 7mG and 3mA provide methyl groups located in the major and minor grooves, respectively). Thermodynamic characteristics of interest include the number of moles of hydrating water molecules per mole of DNA, the number of moles of associated counterions, B-to-A form DNA transition propensity (measured in relative humidity upon addition of trifluoroethanol), and DNA duplex melting temperature. These characteristics give insight regarding how strongly solvated DNA duplexes containing cationic alkylpurines with minor groove or major groove lesions are, electrostatic/ionic atmosphere changes associated with these modifications, base stacking/pairing interactions, and general B-form conformational stability. These properties will then be compared to biochemical data, such as equilibrium binding constants to DNA glycosylases AAG and AlkD, and will be utilized as structural probes to gain insight regarding recognition and excision mechanisms employed by these enzymes. Electrophoretic mobility shifts will be used to determine binding constants, and X-ray crystallography will be used for structural studies.