DISULPHIDE CROSS-LINKED RARE SEARCH INTERMEDIATE OF HOGG1 ON UNDAMAGED DNA

未受损 DNA 上 HOGG1 的二硫键交联稀有搜索中间体

• 批准号: 8169232
• 负责人: GREGORY Lawrence VERDINE
• 金额: $ 0.35万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169232
• 关键词: Active Sites; B-DNA; Base Pairing; Binding; Biochemical; Catalysis; Cleaved cell; Complex; Computer Retrieval of Information on Scientific Projects Database; DNA; DNA glycosylase; Discrimination; Disulfides; Enzymes; Funding; Grant; Guanine; Human; Institution; Lesion; Research; Research Personnel; Resources; Source; Structure; Technology; Time; United States National Institutes of Health; base; crosslink

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The mechanism by which DNA glycosylases differentiate between specific damaged bases and the overwhelming excess of normal bases remains a mystery. Moreover, it is also unclear how these enzymes, despite spending so much time interrogating normal DNA, constrain themselves to cleaving only damaged bases. In particular, the feat of recognizing 8-oxoguanine lesions (oxoG) is impressive, because guanine and oxoG differ by merely two atoms, and the presence of oxoG in B-form DNA elicits no significant structural perturbation. Per our efforts to better understand these issues, we have used disulfide cross-linking technology to covalently trap a human 8-oxoguanine glycosylase (hOGG1) to an undamaged G:C base pair in a normal duplex of DNA. The x-ray structure of this complex reveals that the target guanine base has been swiveled out from the base stack and is fully inserted into the extrahelical active site pocket. Interestingly, though the enzyme is catalytically active, the target guanine remains uncleaved, an observation supported by biochemical analysis. The fact that even complete binding of guanine in the active site does not result in base cleavage demonstrates the existence of discrimination between G and oxoG at the level of catalysis.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 DNA糖基化酶区分特定受损碱基和大量过量的正常碱基的机制仍然是个谜。此外，我们还不清楚这些酶如何限制自己只切割受损的碱基，尽管花费了如此多的时间来研究正常的 DNA。特别是，识别 8-氧代鸟嘌呤损伤 (oxoG) 的成就令人印象深刻，因为鸟嘌呤和 oxoG 仅相差两个原子，并且 B 型 DNA 中 oxoG 的存在不会引起显着的结构扰动。 为了更好地理解这些问题，我们使用二硫键交联技术将人 8-氧代鸟嘌呤糖基化酶 (hOGG1) 共价捕获到正常 DNA 双链体中未受损的 G:C 碱基对上。该复合物的 X 射线结构表明，目标鸟嘌呤碱基已从碱基堆叠中旋转出来，并完全插入螺旋外活性位点口袋中。有趣的是，尽管该酶具有催化活性，但目标鸟嘌呤仍未被切割，这一观察结果得到了生化分析的支持。即使鸟嘌呤在活性位点完全结合也不会导致碱基裂解，这一事实表明 G 和 oxoG 在催化水平上存在区别。