HU binding to DNA: Evidence for multiple complex formation and DNA bending
HU binding to DNA: Evidence for multiple complex formation and DNA bending
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DOI：
10.1021/bi002382r
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发表时间：
2001-02-27
期刊：
影响因子：
2.9
通讯作者：
Mukerji, I
中科院分区：
文献类型：
--
作者：
Wojtuszewski, K;Hawkins, ME;Mukerji, I
HU是一种非特异性组蛋白样DNA结合蛋白，作为辅助蛋白参与许多基因组事件，并与DNA形成多种复合物。HU-DNA结合相互作用的特征在于荧光，产生与鸟苷类似物3-甲基-8-（2-脱氧-β-D-呋喃核糖基）异黄蝶呤（3-MI）直接纳入DNA双链体。HU与13和34 bp的DNA双链体之间形成的复合物的化学计量和平衡结合常数的测定使用荧光各向异性和分析超离心。这些测量结果表明，三个HU分子结合到34 bp的双链体，而两个HU分子结合到13 bp的双链体。通过独立结合位点模型很好地描述了数据，并且两个双链体的第一个结合事件的缔合常数相似（类似于1 × 10（6）M-1），表明HU结合亲和力与双链体长度无关。根据非特异性结合模型对结合曲线的进一步分析表明，HU与DNA的结合表现出很少或没有协同性。荧光强度在HU结合后也增加，这与3-MI荧光探针的碱基堆积减少和溶剂暴露增加一致。这些结果提示DNA的局部弯曲或解旋。在这些结果的基础上，我们提出了一个模型，其中弯曲的DNA伴随着HU绑定。在HU与34 bp双链体结合的凝胶迁移率变动测定中观察到多达5个复合物条带。我们认为，蛋白质诱导的DNA弯曲导致观察到的复合物在凝胶中，具有相同的分子量，但不同的相对迁移率。
HU, a nonspecific histone-like DNA binding protein, participates in a number of genomic events as an accessory protein and forms multiple complexes with DNA. The HU-DNA binding interaction was characterized by fluorescence, generated with the guanosine analogue 3-methyl-8-(2-deoxy-beta -D-ribofuranosyl)isoxanthopterin (3-MI) directly incorporated into DNA duplexes. The stoichiometry and equilibrium binding constants of complexes formed between HU and 13 and 34 bp DNA duplexes were determined using fluorescence anisotropy and analytical ultracentrifugation. These measurements reveal that three HU molecules bind to the 34 bp duplexes, while two HU molecules bind to the 13 bp duplex. The data are well described by an independent binding site model, and the association constants for the first binding event for both duplexes are similar(similar to1 x 10(6) M-1), indicating that HU binding affinity is independent of duplex length. Further analysis of the binding curves in terms of a nonspecific binding model is indicative that HU binding to DNA exhibits little to no cooperativity. The fluorescence intensity also increases upon HU binding, consistent with decreased base stacking and increased solvent exposure of the 3-MI fluorescence probe. These results are suggestive of a local bending or unwinding of the DNA. On the basis of these results we propose a model in which bending of DNA accompanies HU binding. Up to five complex bands are observed in gel mobility shift assays of HU binding to the 34 bp duplexes. We suggest that protein-induced bending of the DNA leads to the observation of complexes in the gel, which have the same molecular weight but different relative mobilities.