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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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Schematic representation of TDG domains and the SUMO-conjugation site (upper panel). Three-dimensional structure of the SUMO-1-modified TDG-CAT protein (ribbon and surface representations) determined by X-ray diffraction (14) with, indicated between brackets, the junctions of TDG N- and C-terminus. TDG-CAT is represented in grey and the interface with SUMO-1 in blue, SUMO-1 is colored in yellow. The SUMO-binding motifs (SBM1 and SBM2) are indicated in red and the catalytic residues in blue. Molecular models were generated using the Protein Data Bank (PDB) structure (PDB code 1WYW) and processed with the MolMol software (42).
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Figure 1: Schematic representation of TDG domains and the SUMO-conjugation site (upper panel). Three-dimensional structure of the SUMO-1-modified TDG-CAT protein (ribbon and surface representations) determined by X-ray diffraction (14) with, indicated between brackets, the junctions of TDG N- and C-terminus. TDG-CAT is represented in grey and the interface with SUMO-1 in blue, SUMO-1 is colored in yellow. The SUMO-binding motifs (SBM1 and SBM2) are indicated in red and the catalytic residues in blue. Molecular models were generated using the Protein Data Bank (PDB) structure (PDB code 1WYW) and processed with the MolMol software (42).

Mentions: For both SUMO-1 conjugation and intermolecular SUMO-1 binding, the N-terminal domain of TDG was found to be targeted in the modification of TDG function in BER. We have previously reported that the regulatory domain, located in the N-terminus of TDG (see Figure 1), provides an additional non-sequence or mismatch specific DNA binding activity and furthermore established dynamic intramolecular interactions with the core catalytic domain [31]. This interface is altered in the presence of a DNA substrate. Moreover, the conformation of the regulatory domain modulates the TDG glycosylase activity and enzymatic turnover in a mismatch-dependent manner [31]. Here we describe the effects on the conformational dynamics of TDG, and in particular on the regulatory domain, of SUMO-1 conjugation on the one hand and non-covalent SUMO-1 binding on the other. The mechanism of stimulation of TDG glycosylase activity by SUMO-1 is described.


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)

Schematic representation of TDG domains and the SUMO-conjugation site (upper panel). Three-dimensional structure of the SUMO-1-modified TDG-CAT protein (ribbon and surface representations) determined by X-ray diffraction (14) with, indicated between brackets, the junctions of TDG N- and C-terminus. TDG-CAT is represented in grey and the interface with SUMO-1 in blue, SUMO-1 is colored in yellow. The SUMO-binding motifs (SBM1 and SBM2) are indicated in red and the catalytic residues in blue. Molecular models were generated using the Protein Data Bank (PDB) structure (PDB code 1WYW) and processed with the MolMol software (42).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Schematic representation of TDG domains and the SUMO-conjugation site (upper panel). Three-dimensional structure of the SUMO-1-modified TDG-CAT protein (ribbon and surface representations) determined by X-ray diffraction (14) with, indicated between brackets, the junctions of TDG N- and C-terminus. TDG-CAT is represented in grey and the interface with SUMO-1 in blue, SUMO-1 is colored in yellow. The SUMO-binding motifs (SBM1 and SBM2) are indicated in red and the catalytic residues in blue. Molecular models were generated using the Protein Data Bank (PDB) structure (PDB code 1WYW) and processed with the MolMol software (42).
Mentions: For both SUMO-1 conjugation and intermolecular SUMO-1 binding, the N-terminal domain of TDG was found to be targeted in the modification of TDG function in BER. We have previously reported that the regulatory domain, located in the N-terminus of TDG (see Figure 1), provides an additional non-sequence or mismatch specific DNA binding activity and furthermore established dynamic intramolecular interactions with the core catalytic domain [31]. This interface is altered in the presence of a DNA substrate. Moreover, the conformation of the regulatory domain modulates the TDG glycosylase activity and enzymatic turnover in a mismatch-dependent manner [31]. Here we describe the effects on the conformational dynamics of TDG, and in particular on the regulatory domain, of SUMO-1 conjugation on the one hand and non-covalent SUMO-1 binding on the other. The mechanism of stimulation of TDG glycosylase activity by SUMO-1 is described.

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