Modulating the DNA polymerase ß reaction equilibrium to dissect the reverse reaction.
Nat Chem Biol
; 13(10): 1074-1080, 2017 Oct.
Article
en En
| MEDLINE
| ID: mdl-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 ß 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 ß- and γ-phosphates of the incoming nucleotide in reaction chemistry, enzyme conformational changes, and overall reaction equilibrium.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Termodinámica
/
ADN Polimerasa beta
Límite:
Humans
Idioma:
En
Revista:
Nat Chem Biol
Asunto de la revista:
BIOLOGIA
/
QUIMICA
Año:
2017
Tipo del documento:
Article
País de afiliación:
Estados Unidos