Exploiting Substrate Promiscuity To Develop Activity-Based Probes for Ten-Eleven Translocation Family Enzymes.
J Am Chem Soc
; 140(50): 17329-17332, 2018 12 19.
Article
en En
| MEDLINE
| ID: mdl-30518204
ABSTRACT
Ten-eleven translocation (TET) enzymes catalyze repeated oxidations of 5-methylcytosine in genomic DNA. Because of the challenges of tracking reactivity within a complex DNA substrate, chemical tools to probe TET activity are limited, despite these enzyme's crucial role in epigenetic regulation. Here, building on precedents from related Fe(II)/α-ketoglutarate-dependent dioxygenases, we show that TET enzymes can promiscuously act upon cytosine bases with unnatural 5-position modifications. Oxidation of 5-vinylcytosine (vC) in DNA results in the predominant formation of a 5-formylmethylcytosine product that can be efficiently labeled to provide an end-point read-out for TET activity. The reaction with 5-ethynylcytosine (eyC), moreover, results in the formation of a high-energy ketene intermediate that can selectively trap any active TET isoform as a covalent enzyme-DNA complex, even in the complex milieu of a total cell lysate. Exploiting substrate promiscuity therefore offers a new and needed means to directly track TET activity in vitro or in vivo.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
ADN
/
Sondas de ADN
/
Dioxigenasas
Límite:
Humans
Idioma:
En
Revista:
J Am Chem Soc
Año:
2018
Tipo del documento:
Article