Structural understanding of Mu transposition

Mu转置的结构理解

• 批准号: 8459942
• 负责人: PHOEBE A RICE
• 金额: $ 29.6万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8459942
• 关键词: Address; Antibiotic Resistance; Architecture; Bacteriophages; Binding; Binding Sites; Biochemical; Biological Assay; Biotechnology; C-terminal; Catalytic Domain; Chimera organism; Collaborations; Complex; Crystallization; Crystallography; DNA; DNA Binding; DNA Binding Domain; DNA Structure; DNA Transposons; DNA-Protein Interaction; Data Set; Dissection; Drops; Elements; Engineering; Enhancers; Enzymes; Evolution; Family; Family member; Fluorescence Resonance Energy Transfer; HIV; In Vitro; Integrase; Investigation; Left; Measures; Mobile Genetic Elements; Modeling; Nature; Pathway interactions; Peptide Hydrolases; Physiological; Play; Protein Engineering; Proteins; Protomer; Reaction; Regulation; Resolution; Rest; Retroviridae; Role; Solutions; Staging; Structure; System; Testing; Translating; Transposase; VDJ Recombinases; Variant; Work; base; chemical reaction; complex R; fluorophore; improved; in vitro Assay; interest; luminescence resonance energy transfer; mu transposase; mutant; prevent; protein protein interaction; public health relevance; rapid technique; recombinase; reconstruction; research study; screening; single molecule; single-molecule FRET; tool; transposon/insertion element; unfoldase; vector

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.

描述（由申请人提供）：这项工作将结合晶体学和生化研究，以了解经典DNA转座酶的组织和调节，该酶现在是一种生物技术工具，即噬菌体MU。这是最彻底的，高度多样化的转座酶和逆转录病毒整合酶家族的特征，对MU的研究将继续照亮转化为家族其他成员的范式。初步结果包括活性MU转座酶-DNA复合物的最近确定的结构，该结构含有240 kDa的蛋白质和135bp的DNA。这是迄今为止任何转座酶-DNA结构中最大，最复杂的结构，并且只有第二个DDE重组酶可以用靶DNA结晶。该结构是进一步研究的跳板。 AIM 1是完成当前的结构并获得最高的分辨率晶体形式。当前的结构指导了具有不同的DNA结合结构域的活性但更紧凑的嵌合式转座酶，该酶可能有助于结晶，将促进我们实验室和其他人的体外实验，并可能有助于设计系统更有用，使其更有用。 目标2是了解构象变化是稳定性和CLPX识别的基础。我们的结构提出了一个模型，用于转座酶如何使用产品结合能来推动原本同工同的化学反应，以及如何优先识别出ATP依赖性的Infordase CLPX，如何优先识别出极为稳定的最终复合物。此目的使用散装和单分子货物和LRET，以及一些晶体学和溶液测定法。 AIM 3是了解以全左右噬菌体末端组装组装的结构基础。尽管一个活跃的复合物仅需要4个转座酶的拷贝（这是我们已经结晶的），但噬菌体的右端和左端都包含3个转座酶结合位点的不同阵列。我们的结构为左端为何与右端不同以及如何将其纳入最终复合物的方式提供了可检验的假设的基础。这项工作将涉及具有针对不同结合位点的精心选择的突变体的复杂形成和活动测定。对该系统的详细理解将为理解为什么许多其他移动DNA元素具有不同的左右两端，并具有看似脱颖而出的重组酶结合位点。