The Study Of DNA Polymerase Binding With DNA By SPR Biosensor

The kinetics of biomolecular interaction can be studied by surface plasmon resonance (SPR) biosensor. SPR biosensor provides an excellent tool to overcome the various limitations of traditional techniques on the kinetics study. It can measure the the biospecific interaction between an analyte in solution and a ligand immobilized on the sensor chip surface in real time, without labeling any of the interactants. In this dissertation, SPR biosensor is used to study the DNA polymerase binding with DNA, including the effects of base mismatch on the DNA polymerase binding with DNA and on the formation of enzyme#DNA#dNMP ternary complex, the effects of inhibitors on DNA polymerase binding with DNA. The dissertation also introduces the setup of a SPR imaing system (SPM) which can be used to measure dozens of biomolecular interactions simultaneously and in real time.The followings are the results of the project.1. The affinity of Taq pol binding with DNA T-P decreased when the number of mismatched bases increased. 'Correct' dNMP induced the conformation change of enzyme-DNA complex and enhanced the binding tightness. In the presence of'correct' or 'incorrect' dNMP, the affinity of pol β binding with DNA template-primer (DNA T-P) increased greatly.2. The two fatty acid, linoleic acid and nervonic acid, could bind the DNA binding site of the 8kDa domain of human pol β and inhibit competitively the enzyme binding with DNA. The nervonic acid inhibited the binding much more than linoleic acid. Pamoic acid also inhibited the 8kDa domain, but its inhibition was weaker than fatty acid. Quercetin was verified as an inhibitor of human pol β and the 31kDa domain and 8kDa domain of the enzyme involved in the inhibition.