SPECIFICITY OF DNA REPAIR FOR OXIDIZED ABASIC SITES

自由基对DNA的损伤与癌变、正常衰老过程和神经退行性变有关，可产生各种损伤，包括各种氧化基位（OAS）。这些包括1'-氧化，4'-氧化或碎片化的脱氧核糖残基，已知的产物，如H2O2，电离辐射和博来霉素。这种损害具有细胞毒性和诱变的潜力，可能是上述年龄和疾病影响的基础，但美洲国家组织难以用现有方法具体、敏感和定量地衡量。该项目将利用其最初的成功继续开发和应用新的分析方法，重点是使用DNA修复酶来释放特定的损伤，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。碱性（AP）内切酶与这些损伤的修复有关，但这还没有在体内得到证实。我们将研究AP内切酶缺陷细菌、酵母和哺乳动物细胞中OAS的修复是否存在缺陷。将使用合成的光敏1′-t-丁基酮衍生物来评估特定OAS在体外的可修复性和体内的致突变潜力，以针对特定序列的损伤。这项工作将对未来了解自由基损伤在衰老、癌症和神经退行性疾病中的作用具有重要意义，并将大大改进这类重要氧化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.