Structural understanding of Mu transposition

描述（申请者提供）：这项工作将结合晶体学和生化研究来了解一种经典的DNA转座酶的组织和调控，这种转座酶现在是一种生物技术工具，即噬菌体Mu的转座酶。这是庞大且高度多样化的“DDE”转座酶和逆转录病毒整合酶家族中最彻底的特征，对Mu的研究将继续阐明转化为该家族其他成员的范例。初步结果包括最近确定的活性Mu转座酶- DNA复合物的结构，该复合物含有240 kDa的蛋白质和135bp的DNA。这是迄今为止所有转座酶DNA结构中最大和最复杂的结构，也是第二个与目标DNA结晶的DDE重组酶。这种结构是进一步研究的跳板。目标1是完成当前的结构，并获得最高分辨率的晶体形式。目前的结构指导了具有不同DNA结合域的活性但更紧凑的嵌合转座酶的构建，这可能有助于结晶，将促进我们实验室和其他实验室的体外实验，并可能有助于工程系统成为更有用的生物技术工具。目的2是了解增加稳定性和ClpX识别的构象变化。我们的结构为转座酶如何利用产物结合能推动其他等能化学反应提供了一个模型，以及极其稳定的最终复合物如何被atp依赖性展开酶ClpX优先识别。这一目标使用体积和单分子FRET和LRET，以及一些晶体学和溶液分析。目的3是了解左、右噬菌体端完整组装的结构基础。虽然一个有活性的复合体只需要4个转座酶的拷贝（这是我们已经结晶的），噬菌体的左右两端分别包含3个转座酶结合位点的不同阵列。我们的结构提供了一个可测试的假设的基础，关于为什么左端不同于右端，以及它是如何被纳入最终的复合体。这项工作将涉及复杂的形成和活性分析，精心选择的突变体专门针对不同的结合位点。对该系统的详细了解将为理解为什么许多其他移动DNA元件具有不同的左端和右端以及似乎额外的重组酶结合位点提供一个翔实的例子。

DESCRIPTION (provided by applicant): This work will combine crystallographic and biochemical studies to understand the organization and regulation of a classic DNA transposase that is now a biotech tool, that of bacteriophage Mu. This is the most thoroughly characterized of the large and highly diverse "DDE" family of transposases and retroviral integrases, and studies of Mu will continue to illuminate paradigms that translate to the rest of the family. Preliminary results include a recently-determined structure of an active Mu transposase - DNA complex, which contains 240 kDa of protein and 135bp of DNA. This is the largest and most complex structure of any transposase-DNA structure to date, and only the 2nd DDE recombinase to be crystallized with target DNA. This structure is serving as the springboard for further studies. AIM 1 is to complete the current structure and to obtain the highest resolution crystal form possible. The current structure guided the construction of an active but more compact chimeric transposase with a different DNA binding domain that may aid crystallization, will facilitate in vitro experiments in our lab and others', and may aid in engineering the system to be more useful as a biotech tool. AIM 2 is to understand the conformational changes that underlie increasing stability and ClpX recognition. Our structure suggests a model for how product binding energy is used by transposases to drive forward an otherwise isoenergetic chemical reaction, and how the extremely stable final complex can be preferentially recognized by the ATP-dependent unfoldase ClpX. This aim uses bulk and single-molecule FRET and LRET, as well as some crystallography and solution assays. AIM 3 is to understand the structural basis for assembly of with full left and right phage ends. Although an active complex requires only 4 copies of the transposase (which is what we have crystallized), the right and left ends of the phage contain different arrays of 3 transposase binding sites each. Our structure provides a basis for testable hypotheses regarding why the left end differs from the right and how it is incorporated into the final complex. This work will involve complex formation and activity assays with carefully chosen mutants specifically targeted to different binding sites. A detailed understanding of this system will provide an informative example for understanding why many other mobile DNA elements have different left and right ends, with seemingly-extra recombinase binding sites.