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

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 DNA糖基酶区分特定受损碱基和过多的正常碱基的机制仍然是个谜。此外，尽管花了这么多时间询问正常的DNA，这些酶是如何将自己限制在只切割受损碱基的，这一点也不清楚。特别是，识别8-氧鸟嘌呤损伤(OxoG)的壮举令人印象深刻，因为鸟嘌呤和oxoG只有两个原子之差，而且B型DNA中oxoG的存在不会引起显著的结构扰动。为了更好地了解这些问题，我们使用了二硫化物交联技术(DXL)，将人类8-氧鸟嘌呤糖基酶(HOGG1)共价捕获到正常DNA双链中未受损的G：C碱基对。到目前为止，我们已经报道了许多hOGG1/DNA复合体的结构，其中几个具有没有损伤的DNA成分，但所有的结构都被配置为促进目标碱基对的破坏。我们成功地在MutM的情况下获得了螺旋内G：C碱基对，MutM是一种细菌DNA糖基酶，它能识别与hOGG1相同的病变，这是系统地筛选了一些适合引入DXL的位点，然后结晶和求解得到的交联复合体的结构。我们坚信，同样的系统筛选策略将被证明是成功的，以获得hOGG1 DNA询问复合体的结构。通过一个新的交联点以及我们开发的新的二硫键，我们已经得到了有希望的hOGG1/DNA晶体。

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.