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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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个oxoguanine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oxoG在B-型DNA中的存在没有引起显著的结构扰动。为了更好地了解这些问题，我们使用了二硫键交联技术，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。该络合物的X射线结构表明，靶鸟嘌呤碱基已经从碱基堆叠中旋转出来，并完全插入到螺旋外的活性中心口袋中。有趣的是，尽管酶具有催化活性，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。即使鸟嘌呤在活性中心完全结合也不会导致碱基裂解，这一事实证明了G和oxoG在催化水平上的区别。

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.