E2-mediated small ubiquitin-like modifier (SUMO) modification of thymine DNA glycosylase is efficient but not selective for the enzyme-product complex.
J Biol Chem
; 289(22): 15810-9, 2014 May 30.
Article
en En
| MEDLINE
| ID: mdl-24753249
ABSTRACT
Thymine DNA glycosylase (TDG) initiates the repair of G·T mismatches that arise by deamination of 5-methylcytosine (mC), and it excises 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC. TDG functions in active DNA demethylation and is essential for embryonic development. TDG forms a tight enzyme-product complex with abasic DNA, which severely impedes enzymatic turnover. Modification of TDG by small ubiquitin-like modifier (SUMO) proteins weakens its binding to abasic DNA. It was proposed that sumoylation of product-bound TDG regulates product release, with SUMO conjugation and deconjugation needed for each catalytic cycle, but this model remains unsubstantiated. We examined the efficiency and specificity of TDG sumoylation using in vitro assays with purified E1 and E2 enzymes, finding that TDG is modified efficiently by SUMO-1 and SUMO-2. Remarkably, we observed similar modification rates for free TDG and TDG bound to abasic or undamaged DNA. To examine the conjugation step directly, we determined modification rates (kobs) using preformed E2â¼SUMO-1 thioester. The hyperbolic dependence of kobs on TDG concentration gives kmax = 1.6 min(-1) and K1/2 = 0.55 µM, suggesting that E2â¼SUMO-1 has higher affinity for TDG than for the SUMO targets RanGAP1 and p53 (peptide). Whereas sumoylation substantially weakens TDG binding to DNA, TDGâ¼SUMO-1 still binds relatively tightly to AP-DNA (Kd â¼50 nM). Although E2â¼SUMO-1 exhibits no specificity for product-bound TDG, the relatively high conjugation efficiency raises the possibility that E2-mediated sumoylation could stimulate product release in vivo. This and other implications for the biological role and mechanism of TDG sumoylation are discussed.
Palabras clave
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina
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Proteína SUMO-1
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Timina ADN Glicosilasa
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Enzimas Ubiquitina-Conjugadoras
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Reparación del ADN
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Año:
2014
Tipo del documento:
Article