A guardian residue hinders insertion of a Fapyâ¢dGTP analog by modulating the open-closed DNA polymerase transition.
Nucleic Acids Res
; 47(6): 3197-3207, 2019 04 08.
Article
en En
| MEDLINE
| ID: mdl-30649431
ABSTRACT
4,6-Diamino-5-formamidopyrimidine (Fapyâ¢dG) is an abundant form of oxidative DNA damage that is mutagenic and contributes to the pathogenesis of human disease. When Fapyâ¢dG is in its nucleotide triphosphate form, Fapyâ¢dGTP, it is inefficiently cleansed from the nucleotide pool by the responsible enzyme in Escherichia coli MutT and its mammalian homolog MTH1. Therefore, under oxidative stress conditions, Fapyâ¢dGTP could become a pro-mutagenic substrate for insertion into the genome by DNA polymerases. Here, we evaluated insertion kinetics and high-resolution ternary complex crystal structures of a configurationally stable Fapyâ¢dGTP analog, ß-C-Fapyâ¢dGTP, with DNA polymerase ß. The crystallographic snapshots and kinetic data indicate that binding of ß-C-Fapyâ¢dGTP impedes enzyme closure, thus hindering insertion. The structures reveal that an active site residue, Asp276, positions ß-C-Fapyâ¢dGTP so that it distorts the geometry of critical catalytic atoms. Removal of this guardian side chain permits enzyme closure and increases the efficiency of ß-C-Fapyâ¢dG insertion opposite dC. These results highlight the stringent requirements necessary to achieve a closed DNA polymerase active site poised for efficient nucleotide incorporation and illustrate how DNA polymerase ß has evolved to hinder Fapyâ¢dGTP insertion.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Conformación Proteica
/
Estrés Oxidativo
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ADN Polimerasa beta
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Nucleótidos de Desoxiguanina
Límite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2019
Tipo del documento:
Article
País de afiliación:
Estados Unidos