TOPOGRAPHICAL CHARACTERIZATION OF THE DNA-POLYMERASE FROM THERMUS-AQUATICUS - DEFINING GROUPS OF INHIBITOR MABS BY EPITOPE MAPPING AND FUNCTIONAL-ANALYSIS USING SURFACE-PLASMON RESONANCE

Among 24 unique monoclonal antibodies (mAb) generated against Tag poly merase (TaqPol) 13 are potent inhibitors of polymerase activity. These antibodies have been sorted into groups defined by their topographica l or functional properties using surface plasmon resonance-based metho ds to examine three different types of interactions. An epitope map of all the pairwise interactions among all 24 antiTaqPol antibodies reve aled the surface of TaqPol as a complex space populated by isolated an tigenic domains with no evident relationship to each other. 11 discret e epitopes or epitope clusters were defined and potent inhibitors boun d to sites within seven of them. The second method examined the abilit y of antiTaqPol mAbs to bind to recombinant forms of the constituent f unctional domains of TaqPol, the N-terminal 5'-nuclease domain and the C-terminal polymerase domain. Most of the antibodies demonstrated a c lear specificity for one domain or the other. The third method measure d the ability of each mAb to block the interaction of TaqPol with a pr eformed, immobilized primer:template complex (PTC). Some antibodies ha d no effect on this interaction while others effectively blocked it. T ogether these latter two methods resolved many of the antibodies into five distinct groups. In addition, antibodies that bound to overlappin g epitopes in the pairwise interaction analysis were members of the sa me group by their interaction with the polymerase fragment and PTC. Th ree groups of polymerase inhibitors were clearly resolved by these ana lyses: (1) those that recognize an epitope on the 5'-nuclease domain a nd have no effect on the interaction of TaqPol with PTC; (2) those tha t recognize an epitope on the polymerase domain and block the interact ion of TaqPol and PTC; and (3) those that recognize an epitope on the polymerase domain, but have no effect on the interaction of TaqPol wit h PTC. The surface of TaqPol bears at least three antigenic regions th at are topographically and functionally distinct and may correspond to sites for inhibition of different steps in the enzymatic activity of TaqPol.