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

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。 这项建议包括旨在通过结构手段了解第二类限制性内切酶类模型酶识别和切割DNA的几个方面的研究。首先，间接读出的作用将通过对具有序列特异性改变的HincII突变体的结构特征进行研究，其中突变位置和特异性发生改变的DNA之间的距离很大。第二，由于几乎所有的DNA内切酶，包括那些参与复制、重组和修复等关键细胞功能的DNA内切酶，都利用二价阳离子依赖的催化，因此将研究HincII切割DNA的二价依赖的立体化学切割机制。最后，当DNA中的正常靶序列被切割时，存在具有改变的序列特异性的酶。其中一种酶SgrAI与DNA结合的De Novo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.