SUMO-1 regulates the conformational dynamics of Thymine-DNA Glycosylase regulatory domain and competes with its DNA binding activity

Caroline Smet-Nocca,Hélène Léger,Jean-Michel Wieruszeski,Sebastian Eilebrecht,Arndt Benecke

doi:10.1186/1471-2091-12-4

Abstract

BackgroundThe human thymine-DNA glycosylase (TDG) plays a dual role in base excision repair of G:U/T mismatches and in transcription. Regulation of TDG activity by SUMO-1 conjugation was shown to act on both functions. Furthermore, TDG can interact with SUMO-1 in a non-covalent manner.ResultsUsing NMR spectroscopy we have determined distinct conformational changes in TDG upon either covalent sumoylation on lysine 330 or intermolecular SUMO-1 binding through a unique SUMO-binding motif (SBM) localized in the C-terminal region of TDG. The non-covalent SUMO-1 binding induces a conformational change of the TDG amino-terminal regulatory domain (RD). Such conformational dynamics do not exist with covalent SUMO-1 attachment and could potentially play a broader role in the regulation of TDG functions for instance during transcription. Both covalent and non-covalent processes activate TDG G:U repair similarly. Surprisingly, despite a dissociation of the SBM/SUMO-1 complex in presence of a DNA substrate, SUMO-1 preserves its ability to stimulate TDG activity indicating that the non-covalent interactions are not directly involved in the regulation of TDG activity. SUMO-1 instead acts, as demonstrated here, indirectly by competing with the regulatory domain of TDG for DNA binding.ConclusionsSUMO-1 increases the enzymatic turnover of TDG by overcoming the product-inhibition of TDG on apurinic sites. The mechanism involves a competitive DNA binding activity of SUMO-1 towards the regulatory domain of TDG. This mechanism might be a general feature of SUMO-1 regulation of other DNA-bound factors such as transcription regulatory proteins.

Highlights

The human thymine-DNA glycosylase (TDG) plays a dual role in base excision repair of G:U/T mismatches and in transcription
For small ubiquitinlike modifier (SUMO)-1, only some N-terminal resonances are observable while the major part of SUMO-1 resonances are too broad to be detected, somewhat mimicking the NMR behavior of TDG catalytic domain (TDG-CAT) and TDG regulatory domain (TDG-regulatory domain (RD)) domains (Figure 2)
We have found that the posttranslational modification of TDG by SUMO-1 (i) has no detectable effect on the conformational dynamics of the regulatory domain and rather acts on the TDG-CAT [14] and TDG C-terminal conformations (Figure 2) and (ii) stimulates both G:T and G:U glycosylase activities with a more pronounced effect on G:U substrates (Figure 5)

Summary

Introduction

The human thymine-DNA glycosylase (TDG) plays a dual role in base excision repair of G:U/T mismatches and in transcription. Regulation of TDG activity by SUMO-1 conjugation was shown to act on both functions. The human Thymine-DNA Glycosylase (TDG) is part of the base-excision DNA repair (BER) machinery targeting G:U and G:T mispairs that did not arise due to replication errors. These mismatches frequently occur on double-stranded DNA after spontaneous or catalytically-mediated hydrolysis of cytosine or C5-methylated cytosine leading to uracil and thymine, respectively [1,2,3,4,5]. Functional interactions with the DNA-methyltransferase Dnmt3a were found to regulate the re-methylation of the newly reconstituted G:C canonical pair after TDG-mediated BER [22]. Sumoylation of TDG was found to regulate TDG activity by abolishing interactions with CBP, preventing its CBP-mediated acetylation in vitro, and altering the sub-cellular localization of TDG to the PML oncogenic domains [29]

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Conclusion