Bailly C, Waring M J, Travers A A
Department of Pharmacology, University of Cambridge, UK.
J Mol Biol. 1995 Oct 13;253(1):1-7. doi: 10.1006/jmbi.1995.0530.
In order to investigate whether the 2-amino group of guanine, which lies in the minor groove of the B-form helix, can directly influence DNA flexibility and major groove recognition by proteins we have examined the properties of DNA molecules containing inosine and/or 2,6-diaminopurine (DAP) residues. Appropriately substituted tyrT(A93) DNA fragments were prepared by the polymerase chain reaction. Their mobility in non-denaturing gels was affected, consistent with changed anisotropic flexibility leading to increased curvature due to G-->I substitution and decreased curvature due to replacement of adenine with DAP. Band-shift assays of FIS protein binding revealed facilitated interaction with inosine-containing DNA and markedly reduced binding to DAP-containing DNA, attributable to altered bendability. DNase footprinting experiments confirmed that fewer sites would bind FIS in DAP-containing DNA at a given protein concentration, whereas higher levels of binding occurred with inosine-containing molecules. Thus base substitutions which affect the placement and presence of the purine 2-amino group in the minor groove can affect both the intrinsic curvature and the bendability of DNA.
为了研究位于B型螺旋小沟中的鸟嘌呤2-氨基基团是否能直接影响DNA的柔韧性以及蛋白质对大沟的识别，我们检测了含有次黄嘌呤和/或2,6-二氨基嘌呤（DAP）残基的DNA分子的特性。通过聚合酶链反应制备了适当取代的tyrT(A93) DNA片段。它们在非变性凝胶中的迁移率受到影响，这与各向异性柔韧性的改变一致，即由于G→I取代导致曲率增加，而由于用DAP取代腺嘌呤导致曲率降低。FIS蛋白结合的凝胶迁移率变动分析显示，与含次黄嘌呤的DNA的相互作用增强，而与含DAP的DNA的结合显著减少，这归因于弯曲性的改变。DNA酶足迹实验证实，在给定的蛋白质浓度下，含DAP的DNA中与FIS结合的位点较少，而含次黄嘌呤的分子则有较高水平的结合。因此，影响嘌呤2-氨基基团在小沟中位置和存在的碱基取代会影响DNA的固有曲率和弯曲性。
