Tsoi Pui Yan, Zhang Xiaomeng, Sui Sen-Fang, Yang Mengsu
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, China.
Analyst. 2003 Sep;128(9):1169-74. doi: 10.1039/b305474h.
In this study, surface plasmon resonance (SPR) biosensor techniques were used to obtain quantitative information on the kinetics of the DNA and polymerase I (Klenow fragment) interaction. DNA duplexes containing different base compositions at the binding site were immobilized on the SPR sensor surface via biotin-streptavidin chemistry and the subsequent binding of the polymerase was measured in real time. Various kinetic models were tested and a translocation model was shown to provide the best fit for the binding and dissociation profiles. The results revealed that the enzyme binds to DNA at both the polymerase and the exonuclease domains with different association and dissociation rates as well as affinity constants, depending on the presence of mismatches near the primer 3'-end. Introduction of unpaired bases increases the DNA binding affinity towards the exonuclease domain and promotes the translocation of DNA from the polymerase site to the exonuclease site. The results also demonstrated that SPR biosensors may be used as a sensitive technique for studying molecular recognition events such as single-base discrimination involved in protein-DNA interaction.
在本研究中，表面等离子体共振（SPR）生物传感器技术被用于获取有关DNA与聚合酶I（克列诺片段）相互作用动力学的定量信息。在结合位点含有不同碱基组成的DNA双链通过生物素-链霉亲和素化学法固定在SPR传感器表面，并实时测量聚合酶随后的结合情况。测试了各种动力学模型，结果表明转位模型最能拟合结合和解离曲线。结果显示，根据引物3'端附近错配的存在情况，该酶以不同的缔合和解离速率以及亲和常数与DNA的聚合酶结构域和外切核酸酶结构域结合。引入不成对碱基会增加DNA对外切核酸酶结构域的结合亲和力，并促进DNA从聚合酶位点向核酸外切酶位点的转位。结果还表明，SPR生物传感器可作为一种灵敏的技术，用于研究分子识别事件，如蛋白质-DNA相互作用中涉及的单碱基识别。
