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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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SUMO-1 influences the TDG-DNA interaction. (A) 15N-1H HSQC spectra of TDG at 20 μM without dsDNA substrate (black) or in presence of 50 μM of a 37-mer dsDNA substrate containing a G:T mismatch (red). (B) 15N-1H HSQC spectra of TDG in presence of 120 μM SUMO-1 without DNA (black) or in presence of 50 μM of DNA substrate containing a G:T mismatch (red). Resonances of TDG-RD residues are annotated in bold and resonances of C-terminal residues in italic.
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Figure 4: SUMO-1 influences the TDG-DNA interaction. (A) 15N-1H HSQC spectra of TDG at 20 μM without dsDNA substrate (black) or in presence of 50 μM of a 37-mer dsDNA substrate containing a G:T mismatch (red). (B) 15N-1H HSQC spectra of TDG in presence of 120 μM SUMO-1 without DNA (black) or in presence of 50 μM of DNA substrate containing a G:T mismatch (red). Resonances of TDG-RD residues are annotated in bold and resonances of C-terminal residues in italic.

Mentions: It has been shown that SUMO-1 intermolecular binding is strongly reduced by TDG's association with DNA [29]. Given our previous results concerning TDG-RD/DNA interactions [31], we have examined the effect of DNA heteroduplexes containing a G:U or a G:T mismatch on TDG conformation in the presence of SUMO-1. Some weak additional resonances matching with those of the isolated TDG N-terminus bound to DNA heteroduplexes are observed on the 15N-labeled TDG HSQC spectrum (Figure 4A and Additional file 6, Figure S6) suggesting that DNA substrates containing either a normal G:C pair or a G:T/U mismatch (in 2.5-fold excess) can displace similarly TDG-RD from its TDG-CAT interacting surface (see Additional file 6, Figure S6). Furthermore, no signal perturbation of TDG-RD or A328-A345 region was observed upon SUMO-1 addition (Figure 4B). These data indicate that a DNA heteroduplex containing either a G:U or a G:T mismatch induces a conformational modification of TDG-RD, this effect being independent of SUMO-1 being present or not, and prevents SUMO-1 binding to the C-terminal SBM which is in accordance with previous works [29]. DNA binding to TDG-CAT likely modifies the SBM2 conformation or accessibility so that it prevents any SUMO-1 interactions. We can not exclude that SUMO-1 could modify the binding affinity of TDG to DNA as it has been shown previously in an indirect manner [29]. However, given the dissociation constant of the TDG/DNA complex (in the nM range) and the relatively high protein concentrations that must be used for NMR studies (in the range of at least 10 μM), the SUMO-induced decrease of TDG/DNA affinity (leading to a shift or a decrease of RD resonances) is not strong enough to be detected since, with a 20 μM sample, TDG, and more particularly the RD, is still saturated with DNA whether SUMO is present or not.


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)

SUMO-1 influences the TDG-DNA interaction. (A) 15N-1H HSQC spectra of TDG at 20 μM without dsDNA substrate (black) or in presence of 50 μM of a 37-mer dsDNA substrate containing a G:T mismatch (red). (B) 15N-1H HSQC spectra of TDG in presence of 120 μM SUMO-1 without DNA (black) or in presence of 50 μM of DNA substrate containing a G:T mismatch (red). Resonances of TDG-RD residues are annotated in bold and resonances of C-terminal residues in italic.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: SUMO-1 influences the TDG-DNA interaction. (A) 15N-1H HSQC spectra of TDG at 20 μM without dsDNA substrate (black) or in presence of 50 μM of a 37-mer dsDNA substrate containing a G:T mismatch (red). (B) 15N-1H HSQC spectra of TDG in presence of 120 μM SUMO-1 without DNA (black) or in presence of 50 μM of DNA substrate containing a G:T mismatch (red). Resonances of TDG-RD residues are annotated in bold and resonances of C-terminal residues in italic.
Mentions: It has been shown that SUMO-1 intermolecular binding is strongly reduced by TDG's association with DNA [29]. Given our previous results concerning TDG-RD/DNA interactions [31], we have examined the effect of DNA heteroduplexes containing a G:U or a G:T mismatch on TDG conformation in the presence of SUMO-1. Some weak additional resonances matching with those of the isolated TDG N-terminus bound to DNA heteroduplexes are observed on the 15N-labeled TDG HSQC spectrum (Figure 4A and Additional file 6, Figure S6) suggesting that DNA substrates containing either a normal G:C pair or a G:T/U mismatch (in 2.5-fold excess) can displace similarly TDG-RD from its TDG-CAT interacting surface (see Additional file 6, Figure S6). Furthermore, no signal perturbation of TDG-RD or A328-A345 region was observed upon SUMO-1 addition (Figure 4B). These data indicate that a DNA heteroduplex containing either a G:U or a G:T mismatch induces a conformational modification of TDG-RD, this effect being independent of SUMO-1 being present or not, and prevents SUMO-1 binding to the C-terminal SBM which is in accordance with previous works [29]. DNA binding to TDG-CAT likely modifies the SBM2 conformation or accessibility so that it prevents any SUMO-1 interactions. We can not exclude that SUMO-1 could modify the binding affinity of TDG to DNA as it has been shown previously in an indirect manner [29]. However, given the dissociation constant of the TDG/DNA complex (in the nM range) and the relatively high protein concentrations that must be used for NMR studies (in the range of at least 10 μM), the SUMO-induced decrease of TDG/DNA affinity (leading to a shift or a decrease of RD resonances) is not strong enough to be detected since, with a 20 μM sample, TDG, and more particularly the RD, is still saturated with DNA whether SUMO is present or not.

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