Department of Physics, University of Illinois at Chicago, Chicago, IL 60607, USA.
Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, Rochester, MN 55905, USA.
Nucleic Acids Res. 2019 Apr 8;47(6):2871-2883. doi: 10.1093/nar/gkz022.
The yeast Nhp6A protein (yNhp6A) is a member of the eukaryotic HMGB family of chromatin factors that enhance apparent DNA flexibility. yNhp6A binds DNA nonspecifically with nM affinity, sharply bending DNA by >60°. It is not known whether the protein binds to unbent DNA and then deforms it, or if bent DNA conformations are 'captured' by protein binding. The former mechanism would be supported by discovery of conditions where unbent DNA is bound by yNhp6A. Here, we employed an array of conformational probes (FRET, fluorescence anisotropy, and circular dichroism) to reveal solution conditions in which an 18-base-pair DNA oligomer indeed remains bound to yNhp6A while unbent. In 100 mM NaCl, yNhp6A-bound DNA unbends as the temperature is raised, with no significant dissociation of the complex detected up to ∼45°C. In 200 mM NaCl, DNA unbending in the intact yNhp6A complex is again detected up to ∼35°C. Microseconds-resolved laser temperature-jump perturbation of the yNhp6a-DNA complex revealed relaxation kinetics that yielded unimolecular DNA bending/unbending rates on timescales of 500 μs-1 ms. These data provide the first direct observation of bending/unbending dynamics of DNA in complex with yNhp6A, suggesting a bind-then-bend mechanism for this protein.
酵母 Nhp6A 蛋白（yNhp6A）是真核生物 HMGB 类染色质因子家族的成员，可增强 DNA 的表观灵活性。yNhp6A 以纳摩尔亲和力非特异性地结合 DNA，使 DNA 强烈弯曲 >60°。目前尚不清楚该蛋白是先结合未弯曲的 DNA 然后使其变形，还是弯曲的 DNA 构象被蛋白结合“捕获”。如果发现未弯曲的 DNA 被 yNhp6A 结合，那么前一种机制将得到支持。在这里，我们使用了一系列构象探针（FRET、荧光各向异性和圆二色性）来揭示溶液条件，在这些条件下，18 个碱基对的 DNA 寡聚体确实在未弯曲的情况下与 yNhp6A 结合。在 100mM NaCl 中，yNhp6A 结合的 DNA 在升温时会变直，在高达约 45°C 时未检测到复合物有明显的解离。在 200mM NaCl 中，完整的 yNhp6A 复合物中的 DNA 变直再次在高达约 35°C 时检测到。yNhp6a-DNA 复合物的微秒分辨激光温度跃变扰动揭示了松弛动力学，在 500μs-1ms 的时间尺度上产生了 DNA 弯曲/变直的单分子反应速率。这些数据提供了 yNhp6A 与 DNA 复合物中 DNA 弯曲/变直动力学的首次直接观察结果，表明该蛋白的结合-然后弯曲机制。
