SPECIFICITY OF DNA REPAIR FOR OXIDIZED ABASIC SITES

氧化碱基位点 DNA 修复的特异性

• 批准号: 6633207
• 负责人: Bruce F. Demple
• 金额: $ 38.12万
• 依托单位: HARVARD SCHOOL OF PUBLIC HEALTH
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-25 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6633207
• 关键词: DNA damage; DNA repair; Escherichia coli; aldehydes; bacterial DNA; bacterial proteins; bacteriophage M13; bleomycin; endonuclease; enzyme activity; esters; exonuclease; gamma radiation; glycolates; high performance liquid chromatography; hydrogen peroxide; ketones; lactones; mutagens; mutant; neocarzinostatin; nitric oxide; oxidation; oxidizing agents; yeasts

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.

对DNA的自由基损害与癌变有关，正常的衰老过程和神经变性会产生多种病变，包括各种氧化的无碱性位点（OAS）。这些包括1'-氧化，4'-氧化或碎片的脱氧核糖残基，诸如H2O2，电离辐射和博来霉素的已知产物。这种损害具有细胞毒性和诱变潜力，可能是上述年龄和疾病影响的基础，但是OA很难通过现有方法进行具体，敏感和定量的特定测量。该项目将利用其最初的成功，以继续开发并应用新方法进行此类分析，并着重于使用DNA修复酶来解放特定的损害，以通过质谱的先进技术来检测。这些方法将用于探索体内单个OAS的形成和修复。 abasic（AP）核酸内切酶与这些损害的修复有关，但在体内尚未证明这一点。我们将检查AP内核酸酶缺陷型细菌，酵母和（如果有的话）哺乳动物细胞中OA的修复缺陷。特定OA的体外和诱变潜力的可修复性将使用合成的，光敏的1'-T-丁基酮导数评估，以靶向特定序列的损伤。这项工作对于了解自由基损害在衰老，癌症和神经变性中的作用的未来工作将非常重要，并且它将大大改善对这一重要的氧化DNA损伤的分子分析方法。