Eriksson S, Nordén B, Takahashi M
Department of Physical Chemistry, Chalmers University of Technology, Göteborg, Sweden.
J Biol Chem. 1993 Jan 25;268(3):1805-10.
The binding of single- as well as double-stranded DNA to RecA, in the presence of the cofactor analog ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)), leads to about 20% quenching of the tyrosine fluorescence of the protein but to no essential change of the tryptophan fluorescence. The excitation spectrum of the fluorescent DNA analog poly(d epsilon A), complexed with RecA, shows no sign of energy transfer from the tyrosine residues of RecA to the etheno-modified adenine bases of the polynucleotide. From this observation we reject stacking interaction between tyrosine residues and DNA bases. The RecA filament may bind up to three molecules of single-stranded DNA; however, the observed fluorescence change occurs only upon the binding of the first DNA strand, indicating that the binding mode of this first strand is different from those of the others. The fluorescence change is interpreted in terms of a conformational change of the RecA protein promoted by cooperative binding to DNA. A larger quenching (40%) upon the binding of single-stranded DNA is observed in the absence of cofactor. At high salt condition, which induces ATPase activity in RecA just as DNA binding does, the tyrosine fluorescence is more pronounced than at low salt conditions, indicating that the effect induced by high salt is different from the conformational change induced by DNA binding.
在辅因子类似物三磷酸腺苷γ-S（腺苷5'-O-（3-硫代三磷酸））存在的情况下，单链和双链DNA与RecA的结合会导致该蛋白质酪氨酸荧光约20%的淬灭，但色氨酸荧光没有本质变化。与RecA复合的荧光DNA类似物聚（dεA）的激发光谱没有显示出从RecA的酪氨酸残基到多核苷酸的乙烯修饰腺嘌呤碱基的能量转移迹象。基于这一观察结果，我们排除了酪氨酸残基与DNA碱基之间的堆积相互作用。RecA丝最多可结合三个单链DNA分子；然而，观察到的荧光变化仅在第一条DNA链结合时发生，这表明第一条链的结合模式与其他链不同。荧光变化被解释为RecA蛋白通过与DNA协同结合而促进的构象变化。在没有辅因子的情况下，观察到单链DNA结合时淬灭程度更大（40%）。在高盐条件下，RecA会像结合DNA一样诱导ATP酶活性，此时酪氨酸荧光比低盐条件下更明显，这表明高盐诱导的效应与DNA结合诱导的构象变化不同。
