Engineering New Nicking Endonucleases

限制性内切酶是基因工程、克隆和DNA诊断中必不可少的工具。N.BstNBI是一种天然存在的核酸内切酶，它只破坏双链DNA的一条链，是唯一一种市售的核酸内切酶。拟建的研究是将ii型内切酶工程化为切口酶。在I期研究中，我们克隆并鉴定了切口内切酶N.BstNBI和两个相关的ii型内切酶PleI和MlyI。我们的研究结果表明，ii型内切酶PleI和MlyI进行双链切割的过程分为两步，其中DNA首先被切口，然后被另一种酶分子通过二聚化作用在第二链上进一步切割。在N.BstNBI的情况下，二聚化功能可能受到突变的影响，导致一种独特的切DNA的内切酶。我们已经成功地通过破坏其二聚化功能将MlyI转化为一个切口内切酶。因此，我们已经证明了通过突变将ii型内切酶转化为缺口酶是可能的。我们将应用第一阶段的技术以及其他策略将更多的ii型内切酶转化为新的切口酶。潜在的商业应用：可能导致ii型限制性内切酶转化为切口酶，在DNA扩增技术（SDA， RCA）和研究（DNA复制，修复等）中有很大的应用。

Restriction endonucleases are essential tools in genetic engineering, cloning and DNA diagnostics. N.BstNBI is a natural occurring nicking endonuclease which only breaks one strand of double-stranded DNA, and it is the only nicking enzyme that is commercially available. The proposed research is about engineering Type IIs endonucleases into nicking enzymes. In Phase I research, we have cloned and characterized the nicking endonuclease N.BstNBI and two related type IIs endonucleases, PleI and MlyI. Our results showed that type IIs endonucleases PleI and MlyI carry out double-stranded cleavage in a two-step process, in which the DNA is first nicked and then further cleaved on second strand by another enzyme molecule via dimerization. In the case of N.BstNBI, it is the dimerization function that was probably affected by mutation, resulting in a unique endonuclease which nicks DNA. We have successfully converted MlyI into a nicking endonuclease by disrupting its dimerization function. Thus, we have demonstrated that it is possible to convert Type IIs endonucleases into nicking enzymes by mutations. We will apply the techniques used in Phase I as well as additional strategies to convert more Type IIs endonucleases into new nicking enzymes. PROPOSED COMMERCIAL APPLICATIONS: Potentially can lead to the conversion of type IIs restriction endonucleases into nicking enzymes which have great applications in DNA amplification technology (SDA, RCA) and research (DNA replication, repair, etc.).