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Title
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Structural studies on an inhibitory antibody against Thermus aquaticus DNA polymerase suggest mode of inhibition.
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Authors
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R.Murali,
M.Helmer-Citterich,
D.J.Sharkey,
E.R.Scalice,
J.L.Daiss,
M.A.Sullivan,
H.M.Krishna Murthy.
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Ref.
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Protein Eng, 1998,
11,
79-86.
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PubMed id
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Abstract
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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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