PDBsum entry 5ugp

Transferase/DNA

PDB id: 5ugp

Contents

Protein chain

323 a.a.

DNA/RNA

Ligands

8CP

Metals

_MG ×2

Waters ×318

References listed in PDB file

Key reference

• Title: Modulating the DNA polymerase β reaction equilibrium to dissect the reverse reaction.
• Authors: D.D.Shock, B.D.Freudenthal, W.A.Beard, S.H.Wilson.
• Ref.: Nat Chem Biol, 2017, 13, 1074-1080.
• PubMed id: 28759020

Abstract

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.