SPECIFICITY OF DNA REPAIR FOR OXIDIZED ABASIC SITES

自由基对DNA的损伤与癌发生、正常衰老过程和神经退行性变有关，产生各种病变，包括各种氧化脱碱基位点（OAS）。这些包括1 '-氧化的、4'-氧化的或片段化的脱氧核糖残基，已知的试剂如H2 O2、电离辐射和博来霉素的产物。这种损伤具有细胞毒性和致突变潜力，可能是上述年龄和疾病影响的基础，但OAS难以通过现有方法进行特异性、灵敏性和定量测量。该项目将利用其初步成功，继续开发和应用这种分析的新方法，重点是使用DNA修复酶来释放特定的损害，以便通过先进的质谱技术进行检测。这些方法将用于探索个体OAS在体内的形成和修复。非碱性（AP）核酸内切酶参与这些损伤的修复，但这在体内尚未得到具体证实。我们将研究是否有缺陷的修复OAS在AP核酸内切酶缺陷的细菌，酵母，如果有的话，哺乳动物细胞。将使用合成的光敏1 '-叔丁基酮衍生物靶向特定序列中的损伤，评估特定OAS的体外修复性和体内致突变潜力。这项工作将是重要的，为未来的工作，了解自由基损伤在衰老，癌症和神经退行性疾病中的作用，它将大大提高这一重要类别的氧化DNA损伤的分子分析方法。

Free radical damage to DNA, which has been associated with carcinogenesis, the normal aging process, and neurodegeneration, produces a variety of lesions, including various oxidized abasic sites (OAS). These include 1'-oxidized, 4'-oxidized or fragmented deoxyribose residues, known products of agents such as H2O2, ionizing radiation, and bleomycin. Such damage have cytotoxic and mutagenic potential that might underlie the age and disease effects cited above, but OAS have been difficult to measure specifically, sensitively and quantitatively by existing methods. This project will capitalize on its initial success to continue development of and to apply new methods for such analysis, with an emphasis on the use of DNA repair enzymes to liberate specific damages for detection by advanced techniques of mass spectrometry. These methods will be used to explore the formation and repair of individual OAS in vivo. Abasic (AP) endonucleases are implicated in the repair of these damages, but this specifically has not been demonstrated in vivo. We will examine whether there are defects in repair of OAS in AP endonuclease-deficient bacteria, yeast and, if available, mammalian cells. The repairability in vitro and mutagenic potential in vivo of a specific OAS will be assessed using a synthetic, photosensitive 1'-t-butyl ketone derivative to target the damage in specific sequences. This work will be important for future work toward understanding the role of free-radical damage in aging, cancer, and neurodegeneration, and it will substantially improve the methodology for molecular analysis of this important class of oxidative DNA damage.