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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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Related in: MedlinePlus

A schematic representation of the main results obtained. (A) SUMO-1 covalent conjugation to K330 leads to a change in the C-terminal conformation of TDG. SUMO-1 thereby also interacts with SBM2. TDG-RD is not displaced from the TDG-CAT domain and hence can rest in its "closed" conformation. Sumoylation thereby influences third party interactions with the RD and therefore "locks" the RD. The presence of free SUMO-1, just as covalent SUMO-1 addition to "open" TDG conformers, increases especially G:U turnover rates ("primed"). Note that also SUMO-modified proteins might be recruited to TDG via SBM2 and have similar effects on TDG's turnover rate. SUMO, when bound via SBM2, sterically competes with TDG-RD for the TDG-CAT surface. The TDG-RD hence adopts a partially "open" conformation which leads to increased G:U repair activity. Also, when SUMO is bound to the SBM2 site, the C-terminus of TDG adopts a conformation similar to the one in the sumoylated protein. The enzymatic turnover especially on G:U mismatches is enhanced through the DNA interaction of either SBM2 recruited or covalently attached SUMO-1. Note that the effect in the case of transient SBM2 interaction is likely due to a local concentration effect as it does not require prolonged SBM2 binding by SUMO. (B) SUMO-1 conjugation or binding to the SBM2 might also occur post-repair once TDG has been trapped on its abasic G:- product to salvage TDG activity by overcoming product inhibition. In the case of non-covalently bound SUMO-1 alternatively a third protein carrying the SUMO-1 group might bring SUMO-1 sufficiently close to TDG for the 'salvage' effect.
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Figure 8: A schematic representation of the main results obtained. (A) SUMO-1 covalent conjugation to K330 leads to a change in the C-terminal conformation of TDG. SUMO-1 thereby also interacts with SBM2. TDG-RD is not displaced from the TDG-CAT domain and hence can rest in its "closed" conformation. Sumoylation thereby influences third party interactions with the RD and therefore "locks" the RD. The presence of free SUMO-1, just as covalent SUMO-1 addition to "open" TDG conformers, increases especially G:U turnover rates ("primed"). Note that also SUMO-modified proteins might be recruited to TDG via SBM2 and have similar effects on TDG's turnover rate. SUMO, when bound via SBM2, sterically competes with TDG-RD for the TDG-CAT surface. The TDG-RD hence adopts a partially "open" conformation which leads to increased G:U repair activity. Also, when SUMO is bound to the SBM2 site, the C-terminus of TDG adopts a conformation similar to the one in the sumoylated protein. The enzymatic turnover especially on G:U mismatches is enhanced through the DNA interaction of either SBM2 recruited or covalently attached SUMO-1. Note that the effect in the case of transient SBM2 interaction is likely due to a local concentration effect as it does not require prolonged SBM2 binding by SUMO. (B) SUMO-1 conjugation or binding to the SBM2 might also occur post-repair once TDG has been trapped on its abasic G:- product to salvage TDG activity by overcoming product inhibition. In the case of non-covalently bound SUMO-1 alternatively a third protein carrying the SUMO-1 group might bring SUMO-1 sufficiently close to TDG for the 'salvage' effect.

Mentions: It had previously been shown (i) that SUMO-1 can interact with TDG also in a non-covalent manner through apparently two distinct binding sites (residues 133-137 and 308-311, namely SUMO-binding motif SBM1 and SBM2, respectively) located within TDG-CAT (see red shading in Figure 1, as well as the model in Figure 8) [14,29,34] and (ii) that the interactions of TDG with DNA as well as sumoylation of TDG prevent further SUMO-1 intermolecular interactions [29]. The non-covalent interactions with SUMO-1 could be either implicated in the TDG sumoylation process itself - as intermediate states, or in functional interactions between TDG and other sumoylated proteins [29,30]. Moreover, since SUMO conjugation to TDG was shown to reduce its DNA binding activity, which suggests when seen in context of previous works, a putative modification of the TDG N-terminal conformation [11,18,31], we have investigated the intermolecular interactions between TDG and SUMO-1 by NMR spectroscopy. In direct binding experiments, we have not detected chemical shift perturbations of the resonances of the isolated N-terminal domain (residues 1-111) in the presence of a 3-fold excess of SUMO-1 (data not shown). These data confirm that there are no direct interactions between SUMO-1 and the N-terminal domain of TDG. Moreover, in 15N-labeled full-length TDG, the resonances of the regulatory domain (residues 51 to 111) become partially detectable upon unlabeled SUMO-1 addition (Figure 3A) while no modification was detected for the first fifty N-terminal residues. We indeed show a number of new resonances on the 15N-1H HSQC spectrum of the 15N-labeled TDG protein in the presence of SUMO-1 (Figure 3A) that match very well with those of TGD-RD observed in the context of the isolated TDG N-terminus (Figure 3C, blue spectrum) indicating that SUMO-1 produces a conformational change of TDG-RD upon binding to SBMs. These resonances are of lower intensity as compared with those of the N[1-42]50]-terminal region suggesting a partial effect on TDG-RD conformation. An increase of RD resonances was measured when adding increasing amounts of SUMO-1 over TDG (ranging from an equimolar amount to a 10-fold excess). We were also able to detect a gradual decrease of signal intensities for some resonances of the TDG C-terminus (from A328 to A345) in presence of SUMO-1 (Figures 3A and see Additional file 1, Figure S1) which indicates a modification of the C-terminal dynamics and conformation upon SUMO-1 intermolecular binding to SBMs. Remarkably, the non-covalent interaction of SUMO-1 and the covalent SUMO-1 modification of TDG induce a perturbation of the same TDG C-terminal resonances. This effect is obviously more pronounced for SUMO-1 conjugation than for the non-covalent binding and leads to the only consistent interpretation that cis and trans SUMO-1 target at least one identical region of TDG-CAT: the C-terminal SUMO-binding motif (SBM2, see Figures 1, 8). To confirm this interaction, we have acquired a 15N-1H HSQC spectrum on 15N-labeled SUMO-1 in presence of TDG. Despite we observed some slight signal perturbations upon TDG addition it seems rather to be induced by weak, non-specific interactions (data not shown). However, an overall 2-fold decrease of SUMO-1 signal intensity in the presence of TDG was noticed with exception of its N-terminal residues (K7-G14) that remain unchanged (see Additional file 2, Figure S2). Hence, the SUMO-1 population bound to TDG cannot be detected on the 15N-1H HSQC spectrum of 15N-labeled SUMO-1 as already observed for SUMO-1 conjugated to TDG. Only the remaining free SUMO-1 molecules are detected. Taken together, our data indicate that non-covalent interactions between SUMO-1 and TDG exist, but do not directly involve the TDG N-terminus which is in accordance with previous studies [29,30].


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)

A schematic representation of the main results obtained. (A) SUMO-1 covalent conjugation to K330 leads to a change in the C-terminal conformation of TDG. SUMO-1 thereby also interacts with SBM2. TDG-RD is not displaced from the TDG-CAT domain and hence can rest in its "closed" conformation. Sumoylation thereby influences third party interactions with the RD and therefore "locks" the RD. The presence of free SUMO-1, just as covalent SUMO-1 addition to "open" TDG conformers, increases especially G:U turnover rates ("primed"). Note that also SUMO-modified proteins might be recruited to TDG via SBM2 and have similar effects on TDG's turnover rate. SUMO, when bound via SBM2, sterically competes with TDG-RD for the TDG-CAT surface. The TDG-RD hence adopts a partially "open" conformation which leads to increased G:U repair activity. Also, when SUMO is bound to the SBM2 site, the C-terminus of TDG adopts a conformation similar to the one in the sumoylated protein. The enzymatic turnover especially on G:U mismatches is enhanced through the DNA interaction of either SBM2 recruited or covalently attached SUMO-1. Note that the effect in the case of transient SBM2 interaction is likely due to a local concentration effect as it does not require prolonged SBM2 binding by SUMO. (B) SUMO-1 conjugation or binding to the SBM2 might also occur post-repair once TDG has been trapped on its abasic G:- product to salvage TDG activity by overcoming product inhibition. In the case of non-covalently bound SUMO-1 alternatively a third protein carrying the SUMO-1 group might bring SUMO-1 sufficiently close to TDG for the 'salvage' effect.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: A schematic representation of the main results obtained. (A) SUMO-1 covalent conjugation to K330 leads to a change in the C-terminal conformation of TDG. SUMO-1 thereby also interacts with SBM2. TDG-RD is not displaced from the TDG-CAT domain and hence can rest in its "closed" conformation. Sumoylation thereby influences third party interactions with the RD and therefore "locks" the RD. The presence of free SUMO-1, just as covalent SUMO-1 addition to "open" TDG conformers, increases especially G:U turnover rates ("primed"). Note that also SUMO-modified proteins might be recruited to TDG via SBM2 and have similar effects on TDG's turnover rate. SUMO, when bound via SBM2, sterically competes with TDG-RD for the TDG-CAT surface. The TDG-RD hence adopts a partially "open" conformation which leads to increased G:U repair activity. Also, when SUMO is bound to the SBM2 site, the C-terminus of TDG adopts a conformation similar to the one in the sumoylated protein. The enzymatic turnover especially on G:U mismatches is enhanced through the DNA interaction of either SBM2 recruited or covalently attached SUMO-1. Note that the effect in the case of transient SBM2 interaction is likely due to a local concentration effect as it does not require prolonged SBM2 binding by SUMO. (B) SUMO-1 conjugation or binding to the SBM2 might also occur post-repair once TDG has been trapped on its abasic G:- product to salvage TDG activity by overcoming product inhibition. In the case of non-covalently bound SUMO-1 alternatively a third protein carrying the SUMO-1 group might bring SUMO-1 sufficiently close to TDG for the 'salvage' effect.
Mentions: It had previously been shown (i) that SUMO-1 can interact with TDG also in a non-covalent manner through apparently two distinct binding sites (residues 133-137 and 308-311, namely SUMO-binding motif SBM1 and SBM2, respectively) located within TDG-CAT (see red shading in Figure 1, as well as the model in Figure 8) [14,29,34] and (ii) that the interactions of TDG with DNA as well as sumoylation of TDG prevent further SUMO-1 intermolecular interactions [29]. The non-covalent interactions with SUMO-1 could be either implicated in the TDG sumoylation process itself - as intermediate states, or in functional interactions between TDG and other sumoylated proteins [29,30]. Moreover, since SUMO conjugation to TDG was shown to reduce its DNA binding activity, which suggests when seen in context of previous works, a putative modification of the TDG N-terminal conformation [11,18,31], we have investigated the intermolecular interactions between TDG and SUMO-1 by NMR spectroscopy. In direct binding experiments, we have not detected chemical shift perturbations of the resonances of the isolated N-terminal domain (residues 1-111) in the presence of a 3-fold excess of SUMO-1 (data not shown). These data confirm that there are no direct interactions between SUMO-1 and the N-terminal domain of TDG. Moreover, in 15N-labeled full-length TDG, the resonances of the regulatory domain (residues 51 to 111) become partially detectable upon unlabeled SUMO-1 addition (Figure 3A) while no modification was detected for the first fifty N-terminal residues. We indeed show a number of new resonances on the 15N-1H HSQC spectrum of the 15N-labeled TDG protein in the presence of SUMO-1 (Figure 3A) that match very well with those of TGD-RD observed in the context of the isolated TDG N-terminus (Figure 3C, blue spectrum) indicating that SUMO-1 produces a conformational change of TDG-RD upon binding to SBMs. These resonances are of lower intensity as compared with those of the N[1-42]50]-terminal region suggesting a partial effect on TDG-RD conformation. An increase of RD resonances was measured when adding increasing amounts of SUMO-1 over TDG (ranging from an equimolar amount to a 10-fold excess). We were also able to detect a gradual decrease of signal intensities for some resonances of the TDG C-terminus (from A328 to A345) in presence of SUMO-1 (Figures 3A and see Additional file 1, Figure S1) which indicates a modification of the C-terminal dynamics and conformation upon SUMO-1 intermolecular binding to SBMs. Remarkably, the non-covalent interaction of SUMO-1 and the covalent SUMO-1 modification of TDG induce a perturbation of the same TDG C-terminal resonances. This effect is obviously more pronounced for SUMO-1 conjugation than for the non-covalent binding and leads to the only consistent interpretation that cis and trans SUMO-1 target at least one identical region of TDG-CAT: the C-terminal SUMO-binding motif (SBM2, see Figures 1, 8). To confirm this interaction, we have acquired a 15N-1H HSQC spectrum on 15N-labeled SUMO-1 in presence of TDG. Despite we observed some slight signal perturbations upon TDG addition it seems rather to be induced by weak, non-specific interactions (data not shown). However, an overall 2-fold decrease of SUMO-1 signal intensity in the presence of TDG was noticed with exception of its N-terminal residues (K7-G14) that remain unchanged (see Additional file 2, Figure S2). Hence, the SUMO-1 population bound to TDG cannot be detected on the 15N-1H HSQC spectrum of 15N-labeled SUMO-1 as already observed for SUMO-1 conjugated to TDG. Only the remaining free SUMO-1 molecules are detected. Taken together, our data indicate that non-covalent interactions between SUMO-1 and TDG exist, but do not directly involve the TDG N-terminus which is in accordance with previous studies [29,30].

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