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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者的研究机构。 DNA糖基化酶区分特定受损碱基和压倒性过量的正常碱基的机制仍然是一个谜。 此外，也不清楚这些酶，尽管花了这么多时间询问正常的DNA，如何限制自己只切割受损的碱基。 特别是，识别8个氧代鸟嘌呤损伤（oxoG）的壮举令人印象深刻，因为鸟嘌呤和oxoG仅相差两个原子，并且在B型DNA中存在oxoG并没有显着的结构扰动。 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（hOGG 1）共价捕获到正常DNA双链体中未受损的G：C碱基对。 该复合物的X射线结构显示，目标鸟嘌呤碱基已从碱基堆中旋出，并完全插入到螺旋外活性位点口袋中。 有趣的是，虽然这种酶具有催化活性，但目标鸟嘌呤仍然没有被切割，这一观察结果得到了生化分析的支持。 事实上，即使完全结合的鸟嘌呤在活性位点不导致碱基裂解证明存在歧视G和oxoG之间的催化水平。

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.