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

Smet-Nocca C, Wieruszeski JM, Léger H, Eilebrecht S, Benecke A - BMC Biochem. (2011)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut de Recherche Interdisciplinaire, Université de Lille1 - Université de Lille2 - CNRS USR3078, Parc de la Haute Borne, 50 avenue de Halley, 59658 Villeneuve d'Ascq, France.

ABSTRACT

Background: 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. Furthermore, TDG can interact with SUMO-1 in a non-covalent manner.

Results: Using 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.

Conclusions: SUMO-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.

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Glycosylase kinetics of TDG and sumoylated TDG proteins in absence and presence of free SUMO-1 or BSA on G:U and G:T repair in NMR buffer. (A, B) G:U repair activity was measured for TDG (A) or sumoylated TDG (B) in the presence or absence of five equimolar amounts of free SUMO-1 or BSA at pH 6.6. (C) G:T repair activity of sumoylated TDG in the presence or absence of five equimolar amounts of free SUMO-1 or BSA at pH 6.6.
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Figure 6: Glycosylase kinetics of TDG and sumoylated TDG proteins in absence and presence of free SUMO-1 or BSA on G:U and G:T repair in NMR buffer. (A, B) G:U repair activity was measured for TDG (A) or sumoylated TDG (B) in the presence or absence of five equimolar amounts of free SUMO-1 or BSA at pH 6.6. (C) G:T repair activity of sumoylated TDG in the presence or absence of five equimolar amounts of free SUMO-1 or BSA at pH 6.6.

Mentions: Although intermolecular SUMO-1 binding did not occur in presence of DNA or with the C-terminal SBM mutation, we have observed a stimulation of the glycosylase activity of wild-type and E310Q mutant TDG proteins. Using a glycosylase assay, we have measured a slight increase of TDG and TDG-E310Q activities and turnover rates upon sumoylation or SUMO-1 addition on the G:T glycosylase reaction (Figure 5A, C). In contrast, the G:U activities and enzymatic turnovers were very sensitive to sumoylation (for wild-type TDG) or SUMO-1 addition in a dose-dependent manner (Figure 5B, D). We have measured a G:U turnover rate increased by a factor of 3.9 for the sumoylated TDG as compared to the non-modified TDG, while a 2.4- and 5.4-fold increase was observed upon addition of 5 and 10 molar equivalents of SUMO-1, respectively (Figure 5C). We have shown in control experiments that the non-covalent SUMO-1 effect is highly specific as same amounts of BSA did not induce such a stimulation of TDG and sumoylated TDG glycosylase activities (Figure 6). Furthermore, indeed, free SUMO-1 can also further increase G:T and G:U processivity of sumoylated TDG unlike BSA (Figure 6). Finally, the increase in activity of TDG that we postulated based on NMR experiments can be shown to take place under the same experimental conditions as the protein-protein and protein-DNA interactions, that is in NMR buffer at pH 6.6 (Figure 6). Note that while TDG's processivity drops by almost an order of magnitude when using acidic buffers, however, the specific stimulation by sumoylation and free SUMO-1 is clearly detectable and comparable to the one detected under standard experimental conditions (Figure 5, Figure 6, and data not shown). Hence SUMO-1, similarly to the sumoylation of TDG, positively acts on the G:U glycosylase activity and also improves albeit weakly the G:T activity. Hence, despite a disruption of SBM2/SUMO-1 interactions in presence of DNA or upon SBM2 mutation, SUMO-1 was still able to activate TDG glycosylase activities on both G:T and G:U substrates in a dose-dependent manner suggesting an indirect mechanism where the TDG/SUMO-1 interaction is not directly responsible for the up-regulation of glycosylase activity (see also Figure 7, and Discussion section).


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

Smet-Nocca C, Wieruszeski JM, Léger H, Eilebrecht S, Benecke A - BMC Biochem. (2011)

Glycosylase kinetics of TDG and sumoylated TDG proteins in absence and presence of free SUMO-1 or BSA on G:U and G:T repair in NMR buffer. (A, B) G:U repair activity was measured for TDG (A) or sumoylated TDG (B) in the presence or absence of five equimolar amounts of free SUMO-1 or BSA at pH 6.6. (C) G:T repair activity of sumoylated TDG in the presence or absence of five equimolar amounts of free SUMO-1 or BSA at pH 6.6.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3040724&req=5

Figure 6: Glycosylase kinetics of TDG and sumoylated TDG proteins in absence and presence of free SUMO-1 or BSA on G:U and G:T repair in NMR buffer. (A, B) G:U repair activity was measured for TDG (A) or sumoylated TDG (B) in the presence or absence of five equimolar amounts of free SUMO-1 or BSA at pH 6.6. (C) G:T repair activity of sumoylated TDG in the presence or absence of five equimolar amounts of free SUMO-1 or BSA at pH 6.6.
Mentions: Although intermolecular SUMO-1 binding did not occur in presence of DNA or with the C-terminal SBM mutation, we have observed a stimulation of the glycosylase activity of wild-type and E310Q mutant TDG proteins. Using a glycosylase assay, we have measured a slight increase of TDG and TDG-E310Q activities and turnover rates upon sumoylation or SUMO-1 addition on the G:T glycosylase reaction (Figure 5A, C). In contrast, the G:U activities and enzymatic turnovers were very sensitive to sumoylation (for wild-type TDG) or SUMO-1 addition in a dose-dependent manner (Figure 5B, D). We have measured a G:U turnover rate increased by a factor of 3.9 for the sumoylated TDG as compared to the non-modified TDG, while a 2.4- and 5.4-fold increase was observed upon addition of 5 and 10 molar equivalents of SUMO-1, respectively (Figure 5C). We have shown in control experiments that the non-covalent SUMO-1 effect is highly specific as same amounts of BSA did not induce such a stimulation of TDG and sumoylated TDG glycosylase activities (Figure 6). Furthermore, indeed, free SUMO-1 can also further increase G:T and G:U processivity of sumoylated TDG unlike BSA (Figure 6). Finally, the increase in activity of TDG that we postulated based on NMR experiments can be shown to take place under the same experimental conditions as the protein-protein and protein-DNA interactions, that is in NMR buffer at pH 6.6 (Figure 6). Note that while TDG's processivity drops by almost an order of magnitude when using acidic buffers, however, the specific stimulation by sumoylation and free SUMO-1 is clearly detectable and comparable to the one detected under standard experimental conditions (Figure 5, Figure 6, and data not shown). Hence SUMO-1, similarly to the sumoylation of TDG, positively acts on the G:U glycosylase activity and also improves albeit weakly the G:T activity. Hence, despite a disruption of SBM2/SUMO-1 interactions in presence of DNA or upon SBM2 mutation, SUMO-1 was still able to activate TDG glycosylase activities on both G:T and G:U substrates in a dose-dependent manner suggesting an indirect mechanism where the TDG/SUMO-1 interaction is not directly responsible for the up-regulation of glycosylase activity (see also Figure 7, and Discussion section).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut de Recherche Interdisciplinaire, Université de Lille1 - Université de Lille2 - CNRS USR3078, Parc de la Haute Borne, 50 avenue de Halley, 59658 Villeneuve d'Ascq, France.

ABSTRACT

Background: 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. Furthermore, TDG can interact with SUMO-1 in a non-covalent manner.

Results: Using 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.

Conclusions: SUMO-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.

Show MeSH
Related in: MedlinePlus