Nagaich A K, Bhattacharyya D, Brahmachari S K, Bansal M
Molecular Biophysics Unit, Indian Institute of Science, Bangalore.
J Biol Chem. 1994 Mar 11;269(10):7824-33.
An analysis of the base pair doublet geometries in available crystal structures indicates that the often reported intrinsic curvature of DNA containing oligo-(d(A).d(T)) tracts may also depend on the nature of the flanking sequences. The presence of CA/TG doublet in particular at the 5' end of these tracts is expected to enhance their intrinsic bending property. To test this proposition, three oligonucleotides, d(GAAAAAC-CCCCC), d(CCCCCCAAAAAG), d(GAAAAATTTTTC), and their complementary sequences were synthesized to study the effect of various flanking sequences, at the 5' and 3' ends of the A-tracts, on the curvature of DNA in solution. An analysis of the polyacrylamide gel electrophoretic mobilities of these sequences under different conditions of salts and temperatures (below their melting points) clearly showed that the oligomer with CA/TG sequence in the center was always more retarded than the oligomer with AC/GT sequence, as well as the oligomer with AT/AT sequence. Hydroxyl radical probing of the sequences with AC/GT and CA/TG doublet junctions gives a similar cutting pattern in the A-tracts, which is quite different from that in the C-tracts, indicating that the oligo(A)-tracts have similar structures in the two oligomers. KMnO4 probing shows that the oligomer with a CA/TG doublet junction forms a kink that is responsible for its inherent curvature and unusual electrophoretic mobility. UV melting shows a reduced thermal stability of the duplex with CA/TG doublet junction, and circular dichroism (CD) studies indicate that a premelting transition occurs in the oligomer with CA/TG doublet step before global melting but not in the oligomer with AC/GT doublet step, which may correspond to thermally induced unbending of the oligomer. These observations indicate that the CA/TG doublet junction at the 5' end of the oligo(A)-tract has a crucial role in modulating the overall curvature in DNA.
对现有晶体结构中碱基对双峰几何结构的分析表明，常报道的含有寡聚（d(A).d(T)）片段的DNA的固有曲率可能也取决于侧翼序列的性质。特别是在这些片段的5'端存在CA/TG双峰，预计会增强其固有弯曲特性。为了验证这一观点，合成了三种寡核苷酸，d(GAAAAAC-CCCCC)、d(CCCCCCAAAAAG)、d(GAAAAATTTTTC)及其互补序列，以研究A片段5'和3'端各种侧翼序列对溶液中DNA曲率的影响。对这些序列在不同盐浓度和温度条件下（低于其熔点）的聚丙烯酰胺凝胶电泳迁移率分析清楚地表明，中心含有CA/TG序列的寡聚物总是比含有AC/GT序列的寡聚物以及含有AT/AT序列的寡聚物迁移更慢。对具有AC/GT和CA/TG双峰连接的序列进行羟基自由基探测，在A片段中得到了相似的切割模式，这与C片段中的模式有很大不同，表明这两种寡聚物中的寡聚（A）片段具有相似的结构。高锰酸钾探测表明，具有CA/TG双峰连接的寡聚物形成了一个扭结，这是其固有曲率和异常电泳迁移率的原因。紫外熔解显示含有CA/TG双峰连接的双链体热稳定性降低，圆二色性（CD）研究表明，含有CA/TG双峰步的寡聚物在整体熔解之前会发生预熔解转变，而含有AC/GT双峰步的寡聚物则不会，这可能对应于寡聚物的热诱导解弯曲。这些观察结果表明，寡聚（A）片段5'端的CA/TG双峰连接在调节DNA的整体曲率方面起着关键作用。
