Bujalowski Wlodzimierz, Jezewska Maria J
Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch at Galveston, Galveston, Texas, USA.
Methods Enzymol. 2009;466:293-324. doi: 10.1016/S0076-6879(09)66013-4.
Both helicases and polymerases perform their activities when bound to the nucleic acids, that is, the enzymes possess a nucleic acid-binding site. Functional complexity of the helicase or the polymerase action is reflected in the intricate structure of the total nucleic acid-binding site, which allows the enzymes to control and change their nucleic acid affinities during the catalysis. Understanding the fundamental aspects of the functional heterogeneity of the total nucleic acid-binding site of a polymerase or helicase can be achieved through quantitative thermodynamic analysis of the enzyme binding to the nucleic acids oligomers, which differ in their length. Such an analysis allows the experimenter to assess the presence of areas with strong and weak affinity for the nucleic acid, that is, the presence of the strong and the weak nucleic acid-binding subsites, determine the number of the nucleotide occlude by each subsite, and estimate their intrinsic free energies of interactions.
解旋酶和聚合酶在与核酸结合时都会发挥其活性，也就是说，这些酶都拥有一个核酸结合位点。解旋酶或聚合酶作用的功能复杂性体现在整个核酸结合位点的复杂结构中，这使得酶在催化过程中能够控制并改变其对核酸的亲和力。通过对酶与不同长度的核酸寡聚物结合进行定量热力学分析，可以了解聚合酶或解旋酶整个核酸结合位点功能异质性的基本方面。这样的分析能让实验者评估对核酸具有强亲和力和弱亲和力区域的存在情况，即强核酸结合亚位点和弱核酸结合亚位点的存在情况，确定每个亚位点所占据的核苷酸数量，并估算它们相互作用的内在自由能。
