Plumridge Alex, Meisburger Steve P, Andresen Kurt, Pollack Lois
School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.
Department of Chemistry, Princeton University, Princeton, NJ 08544, USA.
Nucleic Acids Res. 2017 Apr 20;45(7):3932-3943. doi: 10.1093/nar/gkx140.
Single-stranded DNA (ssDNA) is notable for its interactions with ssDNA binding proteins (SSBs) during fundamentally important biological processes including DNA repair and replication. Previous work has begun to characterize the conformational and electrostatic properties of ssDNA in association with SSBs. However, the conformational distributions of free ssDNA have been difficult to determine. To capture the vast array of ssDNA conformations in solution, we pair small angle X-ray scattering with novel ensemble fitting methods, obtaining key parameters such as the size, shape and stacking character of strands with different sequences. Complementary ion counting measurements using inductively coupled plasma atomic emission spectroscopy are employed to determine the composition of the ion atmosphere at physiological ionic strength. Applying this combined approach to poly dA and poly dT, we find that the global properties of these sequences are very similar, despite having vastly different propensities for single-stranded helical stacking. These results suggest that a relatively simple mechanism for the binding of ssDNA to non-specific SSBs may be at play, which explains the disparity in binding affinities observed for these systems.
单链DNA（ssDNA）在包括DNA修复和复制在内的重要生物学过程中与单链DNA结合蛋白（SSB）的相互作用值得关注。先前的研究已开始表征与SSB结合的ssDNA的构象和静电性质。然而，游离ssDNA的构象分布一直难以确定。为了捕捉溶液中大量的ssDNA构象，我们将小角X射线散射与新颖的整体拟合方法相结合，获得了不同序列链的大小、形状和堆积特征等关键参数。使用电感耦合等离子体原子发射光谱法进行的补充离子计数测量，用于确定生理离子强度下离子氛围的组成。将这种组合方法应用于聚dA和聚dT，我们发现尽管这些序列在单链螺旋堆积方面有很大不同的倾向，但它们的整体性质非常相似。这些结果表明，ssDNA与非特异性SSB结合的相对简单机制可能在起作用，这解释了这些系统中观察到的结合亲和力差异。
