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PDBsum entry 2isp
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Transferase/DNA
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PDB id
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2isp
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References listed in PDB file
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Key reference
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Title
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Modifying the beta,Gamma leaving-Group bridging oxygen alters nucleotide incorporation efficiency, Fidelity, And the catalytic mechanism of DNA polymerase beta.
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Authors
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C.A.Sucato,
T.G.Upton,
B.A.Kashemirov,
V.K.Batra,
V.Martínek,
Y.Xiang,
W.A.Beard,
L.C.Pedersen,
S.H.Wilson,
C.E.Mckenna,
J.Florián,
A.Warshel,
M.F.Goodman.
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Ref.
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Biochemistry, 2007,
46,
461-471.
[DOI no: ]
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PubMed id
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Abstract
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DNA polymerase catalysis and fidelity studies typically compare incorporation of
"right" versus "wrong" nucleotide bases where the leaving group is
pyrophosphate. Here we use dGTP analogues replacing the beta,gamma-bridging O
with CH2, CHF, CF2, or CCl2 to explore leaving-group effects on the nucleotidyl
transfer mechanism and fidelity of DNA polymerase (pol) beta. T.G mismatches
occur with fidelities similar to dGTP with the exception of the CH2 analogue,
which is incorporated with 5-fold higher fidelity. All analogues are observed to
bind opposite template C with Kds between 1 and 4 microM, and structural
evidence suggests that the analogues bind in essentially the native
conformation, making them suitable substrates for probing linear free energy
relationships (LFERs) in transient-kinetics experiments. Importantly, Brnsted
correlations of log(kpol) versus leaving-group pKa for both right and wrong base
incorporation reveal similar sensitivities (betalg approximately -0.8) followed
by departures from linearity, suggesting that a chemical step rather than enzyme
conformational change is rate-limiting for either process. The location of the
breaks relative to pKas of CF2, O, and the sterically bulky CCl2-bridging
compounds suggests a modification-induced change in the mechanism by
stabilization of leaving-group elimination. The results are addressed
theoretically in terms of the energetics of successive primer 3'-O addition
(bond forming) and pyrophosphate analogue elimination (bond breaking) reaction
energy barriers.
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