title: E2-mediated small ubiquitin-like modifier (SUMO) modification of thymine DNA glycosylase is efficient but not selective for the enzyme-product complex.
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 E2∼SUMO-1 thioester. The hyperbolic dependence of kobs on TDG concentration gives kmax = 1.6 min(-1) and K1/2 = 0.55 μM, suggesting that E2∼SUMO-1 has higher affinity for TDG than for the SUMO targets RanGAP1 and p53 (peptide). Whereas sumoylation substantially weakens TDG binding to DNA, TDG∼SUMO-1 still binds relatively tightly to AP-DNA (Kd ∼50 nM). Although E2∼SUMO-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.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201 and.
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Coey
authors.affiliation: From the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201 and.
authors.forename: Megan E
authors.initials: ME
authors.lastname: Fitzgerald
authors.affiliation: From the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201 and.
authors.forename: Atanu
authors.initials: A
authors.lastname: Maiti
authors.affiliation: the Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland 21205.
authors.forename: Katherine H
authors.initials: KH
authors.lastname: Reiter
authors.affiliation: the Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland 21205.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Guzzo
authors.affiliation: the Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland 21205.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Matunis
authors.affiliation: From the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201 and adrohat@som.umaryland.edu.
authors.forename: Alexander C
authors.initials: AC
authors.lastname: Drohat
mesh_terms: D019175:DNA Methylation / Q000502:physiology; D004260:DNA Repair / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D011499:Protein Processing, Post-Translational / Q000502:physiology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D058207:Sumoylation / Q000502:physiology; D044502:Thymine DNA Glycosylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
chemical_list: D004268:DNA-Binding Proteins; D020690:GTPase-Activating Proteins; C092317:RANGAP1 protein, human; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D044763:Ubiquitin-Conjugating Enzymes; D044502:Thymine DNA Glycosylase; D000098843:Ubiquitin-Conjugating Enzyme UBC9
keywords: Base Excision Repair (BER); DNA Methylation; DNA Repair; Enzyme Turnover; Post-translational Modification (PTM); Small Ubiquitin-like Modifier (SUMO); Sumoylation; Ubiquitin-conjugating Enzyme (E2 Enzyme)
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