STRUCTURAL MECHANISMS OF DNA RECOGNITION AND CLEAVAGE BY SEQUENCE SPECIFIC DNA B

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。该建议包括旨在通过结构手段了解II型限制性内切酶类模型酶的DNA识别和切割的几个方面的研究。首先，间接读出的作用将通过结构表征具有改变序列特异性的HincII突变体，其中突变位点与特异性被改变的DNA之间的距离很大来研究。其次，由于几乎所有的DNA内切酶，包括那些参与关键细胞功能的酶，如复制、重组和修复，都利用二价阳离子依赖的催化作用，因此将研究HincII切割DNA的二价阳离子依赖的立体切割机制。最后，存在一些酶，它们在DNA中正常靶序列被切割后具有改变的序列特异性。其中一种与DNA结合的酶SgrAI的从头结构将被确定。

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. This proposal includes studies aimed at understanding, via structural means, of several aspects of DNA recognition and cleavage by model enzymes of the type II restriction endonuclease class. First, the role of indirect readout will be investigated by structurally characterizing mutants of HincII possessing altered sequence specificity, where the distance between the site of mutation and the DNA where specificity has been altered is large. Second, as nearly all DNA endonucleases, including those involved in critical cellular functions such as replication, recombination, and repair, utilize divalent cation dependent catalysis, the divalent cation dependent stereochemical cleavage mechanism of DNA cleavage by HincII will be investigated. Finally, enzymes exist which possess altered sequence specificity upon cleavage of their normal target sequence in DNA. The de Novo structure of one such enzyme, SgrAI, bound to DNA will be determined.