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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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15N-1H HSQC spectra of TDG (black) and TDG conjugated to SUMO-1 (red). Resonances of the extreme N-terminus of TDG (residues 1 to 50) are annotated between brackets and indicated by arrows, resonances of TDG-RD and TDG C-terminus are in bold and in italic, respectively, and those of SUMO-1 in red.
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Figure 2: 15N-1H HSQC spectra of TDG (black) and TDG conjugated to SUMO-1 (red). Resonances of the extreme N-terminus of TDG (residues 1 to 50) are annotated between brackets and indicated by arrows, resonances of TDG-RD and TDG C-terminus are in bold and in italic, respectively, and those of SUMO-1 in red.

Mentions: In our previous NMR study, we have shown that the TDG protein exhibits broad lines on the 15N-1H HSQC spectrum concerning the large majority of its residues and that only the N- and C-terminus resonances are detectable due to their high degree of flexibility in solution (corresponding to residues 1-50 and 328-410 respectively) [31]. We have also shown critical conformational dynamics for the regulatory domain of the N-terminus (TDG-RD, residues 51-111, see Figure 1). This region, coinciding with a functional domain implicated in specific G:T excision [1], adopts a residual structure in the context of the isolated N-terminus and undergoes a dramatic conformational and dynamic change in the context of the entire protein leading to the disappearance/broadening of corresponding resonances. The disappearance of resonances was shown to be due to intramolecular RD/CAT interactions [31]. As for the unconjugated TDG protein, the acquisition of a 15N-1H HSQC spectrum on SUMO-modified TDG leads to the detection of random coil regions. Only the 1-50 segment of the N-terminus and the extreme C-terminus display sufficiently sharp resonances (Figure 2). Furthermore, also for 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-CAT and TDG-RD domains (Figure 2). These data are consistent with the X-ray structure of TDG conjugated to SUMO1 where tight associations between SUMO-1 and TDG-CAT through the C-terminal SBM were highlighted [14]. The resonances of the TDG N-terminal region (residues 1-50) are not perturbed upon SUMO-1-conjugation when compared to non-modified TDG protein. In contrast, the resonances of residues 327 to 347, surrounding the K330 sumoylation site, are significantly broadened (Figure 2), indicating conformational modifications of the TDG C-terminus through covalent sumoylation and no remote perturbations of the N-terminal conformation. We cannot exclude, given the absence of detectable NMR signals that some conformational changes of the TDG regulatory and catalytic domains upon SUMO-1 conjugation occur. Note, however, that based on previous work a structural change of at least the TDG active site after SUMO conjugation is rather unlikely [20].


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)

15N-1H HSQC spectra of TDG (black) and TDG conjugated to SUMO-1 (red). Resonances of the extreme N-terminus of TDG (residues 1 to 50) are annotated between brackets and indicated by arrows, resonances of TDG-RD and TDG C-terminus are in bold and in italic, respectively, and those of SUMO-1 in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: 15N-1H HSQC spectra of TDG (black) and TDG conjugated to SUMO-1 (red). Resonances of the extreme N-terminus of TDG (residues 1 to 50) are annotated between brackets and indicated by arrows, resonances of TDG-RD and TDG C-terminus are in bold and in italic, respectively, and those of SUMO-1 in red.
Mentions: In our previous NMR study, we have shown that the TDG protein exhibits broad lines on the 15N-1H HSQC spectrum concerning the large majority of its residues and that only the N- and C-terminus resonances are detectable due to their high degree of flexibility in solution (corresponding to residues 1-50 and 328-410 respectively) [31]. We have also shown critical conformational dynamics for the regulatory domain of the N-terminus (TDG-RD, residues 51-111, see Figure 1). This region, coinciding with a functional domain implicated in specific G:T excision [1], adopts a residual structure in the context of the isolated N-terminus and undergoes a dramatic conformational and dynamic change in the context of the entire protein leading to the disappearance/broadening of corresponding resonances. The disappearance of resonances was shown to be due to intramolecular RD/CAT interactions [31]. As for the unconjugated TDG protein, the acquisition of a 15N-1H HSQC spectrum on SUMO-modified TDG leads to the detection of random coil regions. Only the 1-50 segment of the N-terminus and the extreme C-terminus display sufficiently sharp resonances (Figure 2). Furthermore, also for 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-CAT and TDG-RD domains (Figure 2). These data are consistent with the X-ray structure of TDG conjugated to SUMO1 where tight associations between SUMO-1 and TDG-CAT through the C-terminal SBM were highlighted [14]. The resonances of the TDG N-terminal region (residues 1-50) are not perturbed upon SUMO-1-conjugation when compared to non-modified TDG protein. In contrast, the resonances of residues 327 to 347, surrounding the K330 sumoylation site, are significantly broadened (Figure 2), indicating conformational modifications of the TDG C-terminus through covalent sumoylation and no remote perturbations of the N-terminal conformation. We cannot exclude, given the absence of detectable NMR signals that some conformational changes of the TDG regulatory and catalytic domains upon SUMO-1 conjugation occur. Note, however, that based on previous work a structural change of at least the TDG active site after SUMO conjugation is rather unlikely [20].

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