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EFFECTS OF BASE SUBSTITUTIONS ON THE BINDING OF A DNA-BENDING PROTEIN

机译:碱基取代对DNA弯曲蛋白结合的影响

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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. (C) 1995 Academic Press Limited [References: 28]
机译:为了研究位于B型螺旋小沟中的鸟嘌呤的2-氨基是否可以直接影响蛋白质的DNA柔韧性和大沟识别,我们研究了含肌苷和/或DNA分子的特性2,6-二氨基嘌呤(DAP)残基。通过聚合酶链反应制备适当取代的tyrT(A93)DNA片段。它们在非变性凝胶中的流动性受到影响,这与各向异性柔性的变化相一致,这导致由于G-> I取代而导致曲率增加,以及由于用DAP替代腺嘌呤而导致曲率降低。 FIS蛋白结合的带移分析显示与含肌苷的DNA相互作用更容易,与DAP含DNA的结合明显减少,这归因于可弯曲性的改变。 DNase足迹实验证实,在给定的蛋白质浓度下,较少的位点会结合含DAP的DNA中的FIS,而含肌苷的分子则会发生更高的结合水平。因此,影响嘌呤2-氨基在小沟中的位置和存在的碱基取代可以影响DNA的固有曲率和可弯曲性。 (C)1995 Academic Press Limited [参考号:28]

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