HIV_Integrase complexes with DNA

HIV_Integrase 与 DNA 的复合物

• 批准号: 8177743
• 负责人: Richard Leapman
• 金额: $ 3.05万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8177743
• 关键词: Affect; Affinity; Binding; Complex; DNA; DNA Binding; Dependence; Goals; HIV; HIV Integrase; Imaging Device; Length; Light; Measures; Mechanics; Molecular Conformation; Pathway interactions; Positioning Attribute; Process; Proteins; Reaction; Site; Spiders; Structure; Synapses; Time; ds-DNA; insight; interest; particle; protein complex; protein oligomer; viral DNA

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.

我们使用 AFM 作为主要成像工具，并且非常清楚地看到了复杂的形状。我们使用末端序列与 HIV DNA 末端序列相同的各种 DNA 长度（90、150、350、500 和 1000 bp）作为底物。复合物的形成效率取决于 DNA 长度，尽管不是太强。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继续聚集，形成蜘蛛状结构。 我们定量了双 DNA/蛋白质复合物中结合颗粒的体积，并确认它们与结合四聚体一致。 我们还检查了“宿主”DNA 的整合过程，并观察了稳定的突触复合体以及完全整合的 DNA。 IN 似乎还会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此类线性分子形成的超螺旋状结构。 我们正在澄清这一现象，这将揭示整合的详细机制，并解释 IN 结合亲和力对 DNA 长度的依赖性，尽管 IN 似乎仅在其活性末端结合 DNA。复合物形成的时间过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。