Structural studies on an inhibitory antibody against Thermus aquaticus DNA polymerase suggest mode of inhibition |
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Authors: | Murali R; Helmer-Citterich M; Sharkey DJ; Scalice ER; Daiss JL; Sullivan MA; Krishna Murthy HM |
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Affiliation: | Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia 19104, USA. |
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Abstract: | TP7, an antibody against Thermus aquaticus DNA polymerase I (TaqP), is used
as a thermolabile switch in 'hot start' variations of PCR to minimize
non-specific amplification events. Earlier studies have established that
TP7 binds to the polymerase domain of TaqP, competes with primer template
complex for binding and is a potent inhibitor of the polymerase activity of
TaqP. We report crystallographic determination of the structure of an Fab
fragment of TP7 and computational docking of the structure with the known
three-dimensional structure of the enzyme. Our observations strongly
suggest that the origin of inhibitory ability of TP7 is its binding to
enzyme residues involved in DNA binding and polymerization mechanism.
Although criteria unbiased by extant biochemical data have been used in
identification of a putative solution, the resulting complex offers an
eminently plausible structural explanation of biochemical observations. The
results presented are of general significance for interpretation of docking
experiments and in design of small molecular inhibitors of TaqP, that are
not structurally similar to substrates, for use in PCR. Structural and
functional similarities noted among DNA polymerases, and the fact that
several DNA polymerases are pharmacological targets, make discovery of
non-substrate based inhibitors of additional importance.
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