Structure and dynamics of three-way DNA junctions: atomic force microscopy studies
Structure and dynamics of three-way DNA junctions: atomic force microscopy studies
复制标题
DOI：
10.1093/nar/28.18.3472
复制
发表时间：
2000-09-15
影响因子：
14.9
通讯作者：
Lyubchenko, YL
中科院分区：
文献类型：
--
作者：
Shlyakhtenko, LS;Potaman, VN;Lyubchenko, YL
我们用原子力显微镜（AFM）研究了DNA复制和重组的中间产物--DNA三向连接的构象。通过退火两个部分同源的限制性内切酶片段，得到了一个发夹臂（50、27、18和7 bp）和两个相对较长的线性臂的固定的三向连接。含有特定长度反向重复序列的DNA片段在变性后形成发夹结构。将亲本质粒的一条片段与含有反向重复序列的一条片段的一条链退火，从凝胶中纯化，并通过原子力显微镜观察，获得了三向连接，该分子清晰地被视为具有一个短臂和两个柔性长臂的三臂分子。这一结论得到了干燥样品上检测到的臂间角度高度变异性的支持。通过在液体中对样品进行成像（AFM;原位）直接观察到结的移动性。其次，臂之间的角度的测量得出结论，三向结不是平坦的，而是类似于锯齿状的。在AFM数据的分析中应考虑结的非平坦性。
We have used atomic force microscopy (AFM) to study the conformation of three-way DNA junctions, intermediates of DNA replication and recombination, Immobile three-way junctions with one hairpin arm (50, 27, 18 and 7 bp long) and two relatively long linear arms were obtained by annealing two partially homologous restriction fragments. Fragments containing inverted repeats of specific length formed hairpins after denaturation, Three-way junctions were Obtained by annealing one strand of a fragment from:a parental plasmid with one strand of an inverted repeat-containing fragment, purified from gels, and examined by AFM, The molecules are clearly seen as three-armed molecules with one short aim and two flexible long arms, The AFM analysis revealed two important features of three-way DNA junctions, First, three-way junctions are very dynamic structures. This conclusion is supported by a high variability of the inter-arm angle detected on dried samples. The mobility of the junctions was observed directly by imaging the samples in liquid (AFM;in situ), Second, measurements of the angle between the arms led to the conclusion that three-way junctions are not flat, but rather pyramid-like, Non-flatness of the junction should be taken into account in analysis of the AFM data.