Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch at Galveston, Galveston, TX 77555-1053, USA.
J Mol Biol. 2010 Sep 17;402(2):344-62. doi: 10.1016/j.jmb.2010.07.008. Epub 2010 Jul 17.
The Escherichia coli PriA helicase complex with the double-stranded DNA (dsDNA), the location of the strong DNA-binding subsite, and the effect of the nucleotide cofactors, bound to the strong and weak nucleotide-binding site of the enzyme on the dsDNA affinity, have been analyzed using the fluorescence titration, analytical ultracentrifugation, and photo-cross-linking techniques. The total site size of the PriA-dsDNA complex is only 5±1 bp, that is, dramatically lower than 20±3 nucleotides occluded in the enzyme-single-stranded DNA (ssDNA) complex. The helicase associates with the dsDNA using its strong ssDNA-binding subsite in an orientation very different from the complex with the ssDNA. The strong DNA-binding subsite of the enzyme is located on the helicase domain of the PriA protein. The dsDNA intrinsic affinity is considerably higher than the ssDNA affinity and the binding process is accompanied by a significant positive cooperativity. Association of cofactors with strong and weak nucleotide-binding sites of the protein profoundly affects the intrinsic affinity and the cooperativity, without affecting the stoichiometry. ATP analog binding to either site diminishes the intrinsic affinity but preserves the cooperativity. ADP binding to the strong site leads to a dramatic increase of the cooperativity and only slightly affects the affinity, while saturation of both sites with ADP strongly increases the affinity and eliminates the cooperativity. Thus, the coordinated action of both nucleotide-binding sites on the PriA-dsDNA interactions depends on the structure of the phosphate group. The significance of these results for the enzyme activities in recognizing primosome assembly sites or the ssDNA gaps is discussed.
已使用荧光滴定法、分析超速离心法和光交联技术分析了大肠杆菌 PriA 解旋酶与双链 DNA（dsDNA）、强 DNA 结合亚基位置以及核苷酸辅助因子的效应复合物在 dsDNA 亲和力上的结合，该酶的强和弱核苷酸结合位点， PriA-dsDNA 复合物的总结合位大小仅为 5±1 bp，即明显低于酶-单链 DNA（ssDNA）复合物中封闭的 20±3 个核苷酸。解旋酶使用其强 ssDNA 结合亚基以与 ssDNA 复合物非常不同的方向与 dsDNA 结合。酶的强 DNA 结合亚基位于 PriA 蛋白的解旋酶结构域上。dsDNA 的固有亲和力明显高于 ssDNA 亲和力，并且结合过程伴随着显著的正协同作用。辅助因子与蛋白质的强和弱核苷酸结合位点的结合会深刻影响固有亲和力和协同作用，而不影响配位数。ATP 类似物结合任一部位都会降低固有亲和力，但保持协同作用。ADP 结合到强位点会导致协同作用急剧增加，而仅略微影响亲和力，而用 ADP 饱和两个位点会强烈增加亲和力并消除协同作用。因此，两个核苷酸结合位点对 PriA-dsDNA 相互作用的协调作用取决于磷酸基团的结构。这些结果对酶识别引发酶组装位点或 ssDNA 缺口的活性的意义进行了讨论。
