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所在的机构。 该提案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突变体的HincII具有改变的序列特异性，其中突变位点和特异性已被改变的DNA之间的距离是大的。 其次，几乎所有的DNA内切酶，包括那些参与关键的细胞功能，如复制，重组和修复，利用二价阳离子依赖性催化，二价阳离子依赖性的立体化学切割机制的DNA切割HincII将进行研究。最后，存在这样的酶，其在DNA中切割其正常靶序列时具有改变的序列特异性。 将确定与DNA结合的一种这样的酶SgrAI的从头结构。

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.