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PDBsum entry 5ugo
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Transferase/DNA
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PDB id
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5ugo
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References listed in PDB file
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Key reference
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Title
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Modulating the DNA polymerase β reaction equilibrium to dissect the reverse reaction.
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Authors
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D.D.Shock,
B.D.Freudenthal,
W.A.Beard,
S.H.Wilson.
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Ref.
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Nat Chem Biol, 2017,
13,
1074-1080.
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PubMed id
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Abstract
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DNA polymerases catalyze efficient and high-fidelity DNA synthesis. While this
reaction favors nucleotide incorporation, polymerases also catalyze a reverse
reaction, pyrophosphorolysis, that removes the DNA primer terminus and generates
deoxynucleoside triphosphates. Because pyrophosphorolysis can influence
polymerase fidelity and sensitivity to chain-terminating nucleosides, we
analyzed pyrophosphorolysis with human DNA polymerase β and found the reaction
to be inefficient. The lack of a thio-elemental effect indicated that this
reaction was limited by a nonchemical step. Use of a pyrophosphate analog, in
which the bridging oxygen is replaced with an imido group (PNP), increased the
rate of the reverse reaction and displayed a large thio-elemental effect,
indicating that chemistry was now rate determining. Time-lapse crystallography
with PNP captured structures consistent with a chemical equilibrium favoring the
reverse reaction. These results highlight the importance of the bridging atom
between the β- and γ-phosphates of the incoming nucleotide in reaction
chemistry, enzyme conformational changes, and overall reaction equilibrium.
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