HIV_Integrase complexes with DNA

我们使用原子力显微镜作为主要的成像工具，已经非常清楚地看到了复杂的形式。我们使用各种DNA长度（90，150，350，500和1000 bp），其末端序列与HIV DNA末端的序列相同，作为底物。复合物形成效率取决于DNA长度，尽管不是太强烈。有趣的是，这些构建体中的主要产物是两个DNA分子与蛋白质四聚体结合，这些复合物通过蛋白质寡聚体之间的结合而继续聚集，形成蜘蛛状结构。 我们定量了两个DNA/蛋白质复合物中结合颗粒的体积，并证实它们与结合的四聚体一致。 我们还研究了整合过程中的“主机”DNA和观察稳定的突触复合物以及完全整合的DNA。IN也似乎影响远离其结合末端的DNA分子的构象状态，因为我们观察到由两个不同的线性分子形成的supecoil样结构。 我们正在澄清这一现象的过程中，这将揭示到详细的力学整合，并将解释依赖于IN结合亲和力的DNA长度，即使IN似乎只结合DNA在其活性末端。复合物形成的时程研究强烈表明了反应途径：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.