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: 8361617
• 负责人: GREGORY Lawrence VERDINE
• 金额: $ 0.29万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361617
• 关键词: B-DNA; Bacterial DNA; Base Pairing; Cleaved cell; Complex; DNA; DNA glycosylase; Disulfides; Enzymes; Funding; Grant; Guanine; Human; Lesion; National Center for Research Resources; Principal Investigator; Reporting; Research; Research Infrastructure; Resources; Screening procedure; Site; Source; Structure; Technology; Time; United States National Institutes of Health; base; cost; crosslink; structural biology; success

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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 (DXL) to covalently trap a human 8-oxoguanine glycosylase (hOGG1) to an undamaged G:C base pair in a normal duplex of DNA. To date, we have reported numerous structures of hOGG1/DNA complexes, several of which have borne DNA components having no lesion, but all have been configured so as to promote disruption of the target base-pair. Our success in obtaining the intrahelical G:C base pair in the case of MutM, a bacterial DNA glycosylase that recognizes the same lesion as hOGG1, came about systematically screening a number of suitable sites for introduction of a DXL, then crystallizing and solving structures of the resulting crosslinked complexes. We strongly believe that this same systematic screening strategy will prove successful in obtaining a structure of the hOGG1 DNA interrogation complex. With a new cross-linking site as well as a new disulfide tether that we have developed, we have already obtained promising hOGG1/DNA crystals.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的首席研究员可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 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 (DXL) 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。迄今为止，我们已经报道了许多 hOGG1/DNA 复合物的结构，其中一些带有没有损伤的 DNA 成分，但所有结构都经过配置以促进靶碱基对的破坏。我们成功地获得了 MutM 的螺旋内 G:C 碱基对，MutM 是一种细菌 DNA 糖基化酶，可识别与 hOGG1 相同的病变，系统地筛选了许多适合引入 DXL 的位点，然后结晶并解析了所得交联复合物。我们坚信，同样的系统筛选策略将成功获得 hOGG1 DNA 询问复合物的结构。通过我们开发的新的交联位点以及新的二硫键，我们已经获得了有前途的 hOGG1/DNA 晶体。