Molecular basis and manipulation of mariner/Tc1 transposon DNA integration

转座子是DNA片段，可以在宿主基因组中从一个位置移动到另一个位置。这些DNA片段在基因组周围的移动是由转座子本身编码的转座酶蛋白协调的。这种酶将DNA从原来的位置切割出来，并粘贴到一个新的位置。这种剪切和粘贴特性正在被利用来开发用于科学研究和医疗应用的生物技术工具。更多地了解转座酶是如何移动DNA的，将使科学家能够了解如何更好地控制这些酶，从而加快转座子工具的开发。Mariner/Tc1转座子家族特别广泛，在昆虫、植物和动物(包括人类)中都有发现，有证据表明，随着进化时间的推移，它们可以从一个宿主物种转移到另一个宿主物种。这个家族的不同寻常之处在于，所有的元素都被粘贴到特定的、但非常短的DNA序列中，这些DNA序列只有两个DNA碱基。这项提议旨在发现这个家族的转座酶如何选择这个序列，以及它们如何将转座子DNA插入到他们的靶标中。为了做到这一点，我们将获得在转座过程的DNA整合步骤中起作用的Mos1转座酶的分子快照。这将涉及解决与转座子DNA和靶DNA结合的酶的X射线晶体结构。我们已经在转座过程的早期阶段解决了酶和转座子DNA的结构。生化实验的结果将支持和验证我们的发现。在对转座机制有了详细的分子理解后，我们建议对转座酶进行改造，以便它可以被引导到需要的地方插入转座子DNA。基于对其三维结构及其化学机制的透彻了解，控制该酶的能力应该对开发这类转座子家族的基因治疗应用具有巨大的好处，而精确控制将是必不可少的。

Transposons are segments of DNA that can move from one place to another in their host's genome. The movement of these DNA segments around genomes is orchestrated by a transposase protein encoded in the transposon itself. This enzyme cuts the DNA out of its original location and pastes it into a new site. This cut-and-paste property is being exploited to develop biotechnology tools for scientific research and medical applications. Learning more about how transposases move DNA will enable scientists to understand how to better control these enzymes and thereby speed the development of transposon tools. The mariner/Tc1 family of transposons is particularly widespread, being found in insects, plants, and animals including humans, and there is evidence that they can move from one host species to another over evolutionary time. This family is unusual in that all the elements are pasted into specific, but very short sequences of DNA comprising just two DNA bases. This proposal is aimed at discovering how transposases from this family choose this sequence and how they insert the transposon DNA into their target. To do this we will obtain a molecular snapshot of the Mos1 transposase in action at the DNA integration step of the transposition process. This will involve solving an X-ray crystal structure of the enzyme bound to both transposon DNA and target DNA. We have already solved structures of the enzyme and transposon DNA at earlier stages in the transposition process. The results of biochemical experiments will support and validate our findings. Having gained this detailed molecular understanding of the transposition machinery, we propose to engineer the transposase so that it can be directed to insert transposon DNA precisely where it is required. The ability to control the enzyme, based on a thorough knowledge of its three-dimensional structure as well as its chemical mechanism, should be of enormous benefit to developing gene therapy applications of this family of transposons, where precise control will be essential.