Jezewska M J, Rajendran S, Bujalowski W
Department of Human Biological Chemistry and Genetics, the University of Texas Medical Branch, Galveston, Texas 77555-1053, USA.
J Biol Chem. 2000 Sep 8;275(36):27865-73. doi: 10.1074/jbc.M004104200.
Analyses of interactions of the Escherichia coli replicative helicase, PriA protein, with a single-stranded (ss) DNA have been performed, using the quantitative fluorescence titration technique. The stoichiometry of the PriA helicase.ssDNA complex has been examined in binding experiments with a series of ssDNA oligomers. The total site-size of the PriA.ssDNA complex, i.e. the maximum number of nucleotide residues occluded by the PriA helicase in the complex, is 20 +/- 3 residues per protein monomer. However, the protein can efficiently form a complex with a minimum of 8 nucleotides. Thus, the enzyme has a strong ssDNA-binding site that engages in direct interactions with a significantly smaller number of nucleotides than the total site-size. The ssDNA-binding site is located in the center of the enzyme molecule, with the protein matrix protruding over a distance of approximately 6 nucleotides on both sides of the binding site. The analysis of the binding of two PriA molecules to long oligomers was performed using statistical thermodynamic models that take into account the overlap of potential binding sites, cooperative interactions, and the protein.ssDNA complexes with different stoichiometries. The intrinsic affinity depends little upon the length of the ssDNA. Moreover, the binding is accompanied by weak cooperative interactions.
利用定量荧光滴定技术，对大肠杆菌复制解旋酶PriA蛋白与单链（ss）DNA的相互作用进行了分析。在与一系列ssDNA寡聚物的结合实验中，检测了PriA解旋酶-ssDNA复合物的化学计量。PriA-ssDNA复合物的总位点大小，即复合物中PriA解旋酶所占据的核苷酸残基的最大数量，为每个蛋白质单体20±3个残基。然而，该蛋白质能够与最少8个核苷酸有效形成复合物。因此，该酶具有一个强大的ssDNA结合位点，它与核苷酸的直接相互作用数量明显少于总位点大小。ssDNA结合位点位于酶分子的中心，蛋白质基质在结合位点两侧突出约6个核苷酸的距离。使用统计热力学模型对两个PriA分子与长寡聚物的结合进行了分析，该模型考虑了潜在结合位点的重叠、协同相互作用以及不同化学计量的蛋白质-ssDNA复合物。内在亲和力几乎不依赖于ssDNA的长度。此外，结合伴随着较弱的协同相互作用。
