Mou T C, Gray C W, Terwilliger T C, Gray D M
Department of Molecular and Cell Biology, The University of Texas at Dallas, Box 830688, Richardson, Texas 75083-0688, USA.
Biochemistry. 2001 Feb 20;40(7):2267-75. doi: 10.1021/bi002136f.
The gene 5 protein (g5p) of Ff bacteriophages is a well-studied model ssDNA-binding protein that binds cooperatively to the Ff ssDNA genome and single-stranded polynucleotides. Its affinity, K omega (the intrinsic binding constant times a cooperativity factor), can differ by several orders of magnitude for ssDNAs of different nearest-neighbor base compositions [Mou, T. C., Gray, C. W., and Gray, D. M. (1999) Biophys. J. 76, 1537-1551]. We found that the DNA backbone can also dramatically affect the binding affinity. The K omega for binding phosphorothioate-modified S-d(A)(36) was >300-fold higher than for binding unmodified P-d(A)(36) at 0.2 M NaCl. CD titrations showed that g5p bound phosphorothioate-modified oligomers with the same stoichiometry as unmodified oligomers. The CD spectrum of S-d(A)(36) underwent the same qualitative change upon protein binding as did the spectrum of unmodified DNA, and the phosphorothioate-modified DNA appeared to bind in the normal g5p binding site. Oligomers of d(A)(36) with different proportions of phosphorothioate nucleotides had binding affinities and CD perturbations intermediate to those of the fully modified and unmodified sequences. The influence of phosphorothioation on binding affinity was nearly proportional to the extent of the modification, with a small nearest-neighbor dependence. These and other results using d(ACC)(12) oligomers and mutant proteins indicated that the increased binding affinity of g5p for phosphorothioate DNA was not a polyelectrolyte effect and probably was not an effect due to the altered nucleic acid structure, but was more likely a general effect of the properties of the sulfur in the context of the phosphorothioate group.
丝状噬菌体（Ff bacteriophages）的基因5蛋白（g5p）是一种经过充分研究的单链DNA结合蛋白模型，它能与Ff单链DNA基因组及单链多核苷酸协同结合。对于不同最近邻碱基组成的单链DNA，其亲和力Kω（内在结合常数乘以协同因子）可能相差几个数量级[Mou, T. C., Gray, C. W., and Gray, D. M. (1999) Biophys. J. 76, 1537 - 1551]。我们发现DNA主链也能显著影响结合亲和力。在0.2 M NaCl条件下，g5p与硫代磷酸酯修饰的S-d(A)(36)结合的Kω比与未修饰的P-d(A)(36)结合的Kω高300倍以上。圆二色光谱滴定表明，g5p与硫代磷酸酯修饰的寡聚物结合的化学计量比与未修饰的寡聚物相同。S-d(A)(36)的圆二色光谱在蛋白质结合时发生的定性变化与未修饰DNA的光谱相同，且硫代磷酸酯修饰的DNA似乎在正常的g5p结合位点结合。不同硫代磷酸酯核苷酸比例的d(A)(36)寡聚物的结合亲和力和圆二色光谱扰动介于完全修饰和未修饰序列之间。硫代磷酸化对结合亲和力的影响几乎与修饰程度成正比，且对最近邻碱基有较小的依赖性。使用d(ACC)(12)寡聚物和突变蛋白的这些及其他结果表明，g5p对硫代磷酸酯DNA结合亲和力的增加不是聚电解质效应，也可能不是由于核酸结构改变所致，而更可能是硫代磷酸酯基团中硫的性质的普遍效应。
