SPECIFICITY OF DNA REPAIR FOR OXIDIZED ABASIC SITES

自由基对DNA的损伤与癌症的发生、正常的衰老过程和神经退行性变有关，会产生各种损伤，包括各种氧化碱性部位(OAS)。这些包括1‘-氧化、4’-氧化或碎裂的脱氧核糖残基、已知的双氧水、电离辐射和博莱霉素的产物。这种损伤具有细胞毒性和诱变潜力，这可能是上述年龄和疾病影响的基础，但用现有方法很难具体、敏感和定量地测量OAS。该项目将利用其初步的成功，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伤，以便通过先进的质谱学技术进行检测。这些方法将被用来探索个体OAS的体内形成和修复。碱性(AP)内切酶与这些损伤的修复有关，但这一点还没有在体内得到明确的证明。我们将检查在AP核酸酶缺陷细菌、酵母和哺乳动物细胞(如果有)中是否存在OAS修复缺陷。将使用一种合成的、光敏的1‘-叔丁基酮衍生物来靶向特定序列的损伤，以评估特定OAS的体外可修复性和体内诱变潜力。这项工作对于了解自由基损伤在衰老、癌症和神经变性中的作用的未来工作将是重要的，它将极大地改进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.