HIV_Integrase complexes with DNA

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。我们使用不同的DNA长度(90、150、350、500和1000bps)作为底物，其末端序列与HIV DNA末端的序列相同。络合物的形成效率取决于DNA的长度，尽管不是太强烈。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。对复合体形成的时间过程研究强烈地表明了反应途径：蛋白质四聚体首先与单个DNA结合，导致构象变化，从而强烈促进第二个DNA的结合。

We used the AFM as the primary imaging tool and have seen the complex form very clearly. We used various DNA lengths (90, 150, 350, 500 and 1000 bps) whose end sequences are identical to those of HIV DNA ends, as substrates. The complex formation efficiency depends on the DNA length albeit not too strongly. 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 and would explain the dependence of IN binding affinity to DNA length even though IN appears to bind DNA only at its active ends. Time-course studies of complex formation strongly suggests the reactions pathway: A protein tetramer first binds to a single DNA resulting in conformational changes which strongly promote binding of the second DNA.