Single-molecule FRET analysis of DNA binding and bending by yeast HMGB protein Nhp6A
Single-molecule FRET analysis of DNA binding and bending by yeast HMGB protein Nhp6A
复制标题
DOI：
10.1093/nar/gks1208
复制
发表时间：
2013-01-01
影响因子：
14.9
通讯作者：
Rasnik, Ivan
中科院分区：
文献类型：
--
作者：
Coats, Julie E.;Lin, Yuyen;Rasnik, Ivan
高迁移率族B（HMGB）蛋白结合双链体DNA，无序列特异性，通过引入DNA扭结增加DNA的表观柔性，促进紧凑核蛋白结构的形成。目前尚不清楚HMGB结合和DNA扭结是否同时发生，以及诱导的扭结是刚性（静态）还是柔性。HMG诱导的DNA“软化”的详细分子机制，探索在这里通过单分子荧光共振能量转移研究的单个酵母Nhp6A（yNhp6A）蛋白结合短DNA双链体。我们表明，yNhp6A蛋白结合DNA的局部效果是一致的，形成一个单一的静态扭结是短暂的（几秒钟的寿命）在生理缓冲液条件下。在我们实验的时间分辨率内，这种静态扭结发生在蛋白质与DNA结合的瞬间，而DNA在蛋白质与DNA分离的瞬间变直。我们的观察结果支持一个模型，其中HMGB蛋白软化DNA通过随机动态结合和解离，伴随着DNA扭结和拉直，分别。
High-mobility group B (HMGB) proteins bind duplex DNA without sequence specificity, facilitating the formation of compact nucleoprotein structures by increasing the apparent flexibility of DNA through the introduction of DNA kinks. It has remained unclear whether HMGB binding and DNA kinking are simultaneous and whether the induced kink is rigid (static) or flexible. The detailed molecular mechanism of HMGB-induced DNA 'softening' is explored here by single-molecule fluorescence resonance energy transfer studies of single yeast Nhp6A (yNhp6A) proteins binding to short DNA duplexes. We show that the local effect of yNhp6A protein binding to DNA is consistent with formation of a single static kink that is short lived (lifetimes of a few seconds) under physiological buffer conditions. Within the time resolution of our experiments, this static kink occurs at the instant the protein binds to the DNA, and the DNA straightens at the instant the protein dissociates from the DNA. Our observations support a model in which HMGB proteins soften DNA through random dynamic binding and dissociation, accompanied by DNA kinking and straightening, respectively.