Bischerour Julien, Chalmers Ronald
University of Oxford, Department of Biochemistry, South Parks Road, Oxford, OX1 3QU, UK.
Nucleic Acids Res. 2007;35(8):2584-95. doi: 10.1093/nar/gkm186. Epub 2007 Apr 4.
Many enzymes that repair or modify bases in double-stranded DNA gain access to their substrates by base flipping. Although crystal structures provide stunning snap shots, biochemical approaches addressing the dynamics have proven difficult, particularly in complicated multi-step reactions. Here, we use protein-DNA crosslinking and potassium permanganate reactivity to explore the base-flipping step in Tn5 transposition. We present a model to suggest that base flipping is driven by a combination of factors including DNA bending and the intrusion of a probe residue. The forces are postulated to act early in the reaction to create a state of tension, relieved by base flipping after cleavage of the first strand of DNA at the transposon end. Elimination of the probe residue retards the kinetics of nicking and reduces base flipping by 50%. Unexpectedly, the probe residue is even more important during the hairpin resolution step. Overall, base flipping is pivotal to the hairpin processing reaction because it performs two opposite but closely related functions. On one hand it disrupts the double helix, providing the necessary strand separation and steric freedom. While on the other, transposase appears to position the second DNA strand in the active site for cleavage using the flipped base as a handle.
许多修复或修饰双链DNA中碱基的酶通过碱基翻转来接触其底物。尽管晶体结构提供了惊人的瞬间图像，但解决动力学问题的生化方法已被证明很困难，尤其是在复杂的多步反应中。在这里，我们使用蛋白质-DNA交联和高锰酸钾反应性来探索Tn5转座中的碱基翻转步骤。我们提出了一个模型，表明碱基翻转是由多种因素共同驱动的，包括DNA弯曲和一个探测残基的侵入。这些力被假定在反应早期起作用，以产生一种张力状态，在转座子末端的第一条DNA链切割后通过碱基翻转得以缓解。去除探测残基会延迟切口动力学，并使碱基翻转减少50%。出乎意料的是，探测残基在发夹结构解析步骤中更为重要。总体而言，碱基翻转对于发夹处理反应至关重要，因为它执行了两个相反但密切相关的功能。一方面，它破坏双螺旋，提供必要的链分离和空间自由度。另一方面，转座酶似乎利用翻转的碱基作为手柄，将第二条DNA链定位在活性位点进行切割。
