DNA strand arrangement within the SfiI-DNA complex: Atomic force microscopy analysis
DNA strand arrangement within the SfiI-DNA complex: Atomic force microscopy analysis
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DOI：
10.1021/bi051767c
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发表时间：
2006-01-10
期刊：
影响因子：
2.9
通讯作者：
Lyubchenko, YL
中科院分区：
文献类型：
--
作者：
Lushnikov, AY;Potaman, VN;Lyubchenko, YL
SfiI限制酶与DNA结合为四聚体，在切割四条DNA链之前将两个通常相距较远的DNA识别位点结合在一起。为了阐明SfiI-DNA复合物的结构特性，在非切割条件下（反应缓冲液中的Ca 2+而不是Mg 2+）进行了复合物的原子力显微镜（AFM）成像。分析了由一个DNA分子中的两个结合位点之间的蛋白质相互作用（顺式相互作用）以及由不同分子中的两个位点的相互作用（反式相互作用）形成的分子内复合物。通过在DNA交叉点处存在一个高的球形斑点来明确地鉴定复合物。为了表征复合物内DNA的路径，系统地分析了复合物附近的DNA螺旋之间的角度。所有的数据都显示出以对应于60度和120度的峰值为中心的明确的双峰分布。为了明确区分复合物内DNA方向的交叉和弯曲模型，设计了SfiI结合位点侧翼具有不同臂长的DNA分子。对这种类型的复合物的AFM图像的分析得出结论，复合物内的DNA识别位点是交叉的。DNA螺旋之间的60度或120度角对应于其中一个螺旋相对于另一个螺旋的取向翻转的复合体。还分析了由具有不同中心碱基对的五种不同识别序列（5 '-GGCNNNNNGGCC-3'）形成的复合物。我们的结果表明，含有两种可能的螺旋取向的复合物几乎相等地形成。这表明复合物内的DNA同源位点没有优先取向，表明DNA结合位点的中心部分不与蛋白质形成强的序列特异性接触。
The SfiI restriction enzyme binds to DNA as a tetramer holding two usually distant DNA recognition sites together before cleavage of the four DNA strands. To elucidate structural properties of the SfiI-DNA complex, atomic force microscopy (AFM) imaging of the complexes under noncleaving conditions (Ca2+ instead of Mg2+ in the reaction buffer) was performed. Intramolecular complexes formed by protein interaction between two binding sites in one DNA molecule (cis interaction) as well as complexes formed by the interaction of two sites in different molecules (trans interaction) were analyzed. Complexes were identified unambiguously by the presence of a tall spherical blob at the DNA intersections. To characterize the path of DNA within the complex, the angles between the DNA helices in the proximity of the complex were systematically analyzed. All the data show clear-cut bimodal distributions centered around peak values corresponding to 60 degrees and 120 degrees. To unambiguously distinguish between the crossed and bent models for the DNA orientation within the complex, DNA molecules with different arm lengths flanking the SfiI binding site were designed. The analysis of the AFM images for complexes of this type led to the conclusion that the DNA recognition sites within the complex are crossed. The angles of 60 degrees or 120 degrees between the DNA helices correspond to a complex in which one of the helices is flipped with respect to the orientation of the other. Complexes formed by five different recognition sequences (5'-GGCCNNNNNGGCC-3'), with different central base pairs, were also analyzed. Our results showed that complexes containing the two possible orientations of the helices were formed almost equally. This suggests no preferential orientation of the DNA cognate site within the complex, suggesting that the central part of the DNA binding site does not form strong sequence specific contacts with the protein.