AFM Studies of DNA Complexes with Intrinsically Disordered Proteins

对于HIV整合酶，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成像工具，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）来形成复合物。复合物的形成效率取决于DNA的长度，这意味着蛋白质-DNA相互作用的DNA位点远低于活性IN结合位点。有趣的是，这些结构的主要产物是两个DNA分子与一个蛋白质四聚体结合，这些复合物通过蛋白质寡聚体之间的结合继续聚集，形成蜘蛛状结构。我们量化了两个dna /蛋白质复合物中结合颗粒的体积，并确认它们与结合的四聚体一致。我们还研究了与“宿主”DNA的整合过程，并观察到稳定的突触复合物以及完全整合的DNA。IN似乎也影响DNA分子的构象状态，远离它们的结合端，因为我们观察到由两个不同的线性分子形成的超线圈状结构。我们正在澄清这一现象，这将阐明整合复杂核运输的详细机制，并将解释in结合亲和力对DNA长度的依赖，即使in似乎只在其活性端结合DNA。此外，我们正在与“宿主”DNA形成整合产物，以研究半位点整合产物的拓扑结构，这是实际将病毒DNA插入宿主DNA的第一步。分析该复合物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的结构功能。

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.