AFM Studies of DNA Complexes with Intrinsically Disordered Proteins

关于HIV整合酶，对复合体的成像将阐明当HIV整合酶与代表HIV双链DNA的线性DNA的活性末端结合时形成的复合体的结构。我们使用原子力显微镜作为主要的成像工具，并非常清楚地看到了复杂的形状。我们使用了一系列DNA长度(90、150、350、500和1000bps)来形成复合体。复合体的形成效率取决于DNA长度，这意味着蛋白质-DNA相互作用位于活性IN结合位点远端的DNA位点。有趣的是，这些结构中的主要产物是两个DNA分子与蛋白质四聚体结合，这些复合体通过结合蛋白质低聚物而聚集，形成蜘蛛样的结构。我们量化了双DNA/蛋白质复合体中结合粒子的体积，并确认它们与结合的四聚体是一致的。我们还研究了与DNA的整合过程，观察到稳定的突触复合体以及完全整合的DNA。In似乎也影响DNA分子在远离其结合端的构象状态，因为我们观察到两个不同的这样的线性分子形成的上油状结构。我们正在澄清这一现象，这将有助于揭示整合复杂的核运输的详细机制，并将解释IN结合亲和力与DNA长度的依赖关系，尽管IN似乎只在其活性末端与DNA结合。此外，我们正在与“宿主”DNA形成整合产物，以研究半部位整合产物的拓扑结构，这是实际病毒DNA插入宿主DNA的第一步。分析该复合体的大量图像也可以深入了解DNA相对于蛋白质的拓扑排列，从而进一步阐明IN的结构和功能。 对于RAG1和RAG2蛋白对VDJ基因片段的处理，我们在不同的生理条件下形成了复合体，并用原子力显微镜进行成像。蛋白质RAG1和RAG2与VDJ基因片段上的重组信号序列(RSS)结合，在重组发生之前导致双链断裂。对该复合体成像，我们建立了结合蛋白复合体的化学计量学，我们正在澄清DNA相对于蛋白质复合体的拓扑排列。此外，每个RAG1和RAG2都包含一个麦芽糖结合结构域(MBP)，我们正在使用针对这些结构域的特异性抗体来进一步阐明功能复合体的组成成分的排列。

With respect to the HIV Integrase, imaging of the complex will elucidate the structure of the complex formed when HIV-integrase binds the active end of linear DNA which represents the HIV double strand DNA. We used the AFM as the primary imaging tool and have seen the complex form very clearly. We used a range of DNA lengths (90, 150, 350, 500 and 1000 bps) in forming the complexes. The complex formation efficiency depends on the DNA length which implies protein-DNA interactions at DNA sites distal to the active IN binding site. It is interesting that the primary product in these constructs is that two DNA molecules bind to a protein tetramer and these complexes go on to aggregate by binding among the protein oligomers, forming spider-like structures. We quantified the volumes of the bound particles in two-DNA/protein complexes and confirmed them to be consistent with bound tetramers. We also examined the integration process to "host" DNA and observe stable synaptic complexes as well as fully integrated DNA. IN also appears to affect the conformational state of DNA molecules away from their binding ends, as we observe supecoil-like structures formed by two distinct such linear molecules. We are in the process of clarifying this phenomenon which would shed light into the detailed mechanics of integration complex nuclear transport and would explain the dependence of IN binding affinity to DNA length even though IN appears to bind DNA only at its active ends. In addition we are forming integration products with "host" DNA to study the topology of half-site integration products, the first step in the actual viral DNA insertion into the host DNA. Analyzing large numbers of images of the complex may also give insights into the topological arrangement of the DNA relative to the protein thus clarifying the structure-function of IN further. With respect to the VDJ gene fragment processing by RAG1 and RAG2 proteins we form the complex under different physiological conditions and use the AFM for imaging. Proteins RAG1 and RAG2 bind the recombination signal sequences (RSS) on the VDJ gene segments and cause a double strand break before recombination can take place. Imaging the complex we established the stoichiometry of the bound protein complex and we are in the process of clarifying the topological arrangement of the DNA relative to the protein complex. In addition, each both RAG1 and RAG2 contain a maltose binding domain (MBP) and we are using specific antibodies to these domains to further clarify the arrangement of the constituent components of the functional complex.