title: Characterizing Requirements for Small Ubiquitin-like Modifier (SUMO) Modification and Binding on Base Excision Repair Activity of Thymine-DNA Glycosylase in Vivo.
abstract: Thymine-DNA glycosylase (TDG) plays critical roles in DNA base excision repair and DNA demethylation. It has been proposed, based on structural studies and in vitro biochemistry, that sumoylation is required for efficient TDG enzymatic turnover following base excision. However, whether sumoylation is required for TDG activity in vivo has not previously been tested. We have developed an in vivo assay for TDG activity that takes advantage of its recently discovered role in DNA demethylation and selective recognition and repair of 5-carboxylcytosine. Using this assay, we investigated the role of sumoylation in regulating TDG activity through the use of TDG mutants defective for sumoylation and Small Ubiquitin-like Modifier (SUMO) binding and by altering TDG sumoylation through SUMO and SUMO protease overexpression experiments. Our findings indicate that sumoylation and SUMO binding are not essential for TDG-mediated excision and repair of 5-carboxylcytosine bases. Moreover, in vitro assays revealed that apurinic/apyrimidinic nuclease 1 provides nearly maximum stimulation of TDG processing of G·caC substrates. Thus, under our assay conditions, apurinic/apyrimidinic nuclease 1-mediated stimulation or other mechanisms sufficiently alleviate TDG product inhibition and promote its enzymatic turnover in vivo.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205 and.
authors.forename: Dylan
authors.initials: D
authors.lastname: McLaughlin
authors.affiliation: the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201.
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Coey
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205 and.
authors.forename: Wei-Chih
authors.initials: WC
authors.lastname: Yang
authors.affiliation: the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201 adrohat@som.umaryland.edu.
authors.forename: Alexander C
authors.initials: AC
authors.lastname: Drohat
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205 and mmatuni1@jhu.edu.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Matunis
mesh_terms: D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D019175:DNA Methylation / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D058207:Sumoylation / Q000502:physiology*; D044502:Thymine DNA Glycosylase / Q000235:genetics / Q000378:metabolism*
chemical_list: C560974:5-carboxylcytosine; D025842:SUMO-1 Protein; D003596:Cytosine; D044502:Thymine DNA Glycosylase
keywords: SUMO-interacting motif (SIM); base excision repair (BER); enzyme turnover; small ubiquitin-like modifier (SUMO); sumoylation