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Alexander C Drohat - One of the best experts on this subject based on the ideXlab platform.
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defining the impact of Sumoylation on substrate binding and catalysis by thymine dna glycosylase
Nucleic Acids Research, 2018Co-Authors: Christopher T Coey, Alexander C DrohatAbstract:: Thymine DNA glycosylase (TDG) excises thymine from mutagenic G·T mispairs generated by deamination of 5-methylcytosine (mC) and it removes two mC derivatives, 5-formylcytosine (fC) and 5-carboxylcytosine (caC), in a multistep pathway for DNA demethylation. TDG is modified by small ubiquitin-like modifier (SUMO) proteins, but the impact of Sumoylation on TDG activity is poorly defined and the functions of TDG Sumoylation remain unclear. We determined the effect of TDG Sumoylation, by SUMO-1 or SUMO-2, on substrate binding and catalytic parameters. Single turnover experiments reveal that Sumoylation dramatically impairs TDG base-excision activity, such that G·T activity is reduced by ≥45-fold and fC and caC are excised slowly, with a reaction half-life of ≥9 min (37°C). Fluorescence anisotropy studies reveal that unmodified TDG binds tightly to G·fC and G·caC substrates, with dissociation constants in the low nanomolar range. While Sumoylation of TDG weakens substrate binding, the residual affinity is substantial and is comparable to that of biochemically-characterized readers of fC and caC. Our findings raise the possibility that Sumoylation enables TDG to function, at least transiently, as reader of fC and caC. Notably, Sumoylation could potentially facilitate TDG recruitment of other proteins, including transcription factors or epigenetic regulators, to these sites in DNA.
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characterizing requirements for small ubiquitin like modifier sumo modification and binding on base excision repair activity of thymine dna glycosylase in vivo
Journal of Biological Chemistry, 2016Co-Authors: Dylan Mclaughlin, Alexander C Drohat, Christopher T Coey, Weichih Yang, Michael J. MatunisAbstract:: Thymine-DNA glycosylase (TDG) plays critical roles in DNA base excision repair and DNA demethylation. It has been proposed, based on structural studies and in vitro biochemistry, that Sumoylation is required for efficient TDG enzymatic turnover following base excision. However, whether Sumoylation is required for TDG activity in vivo has not previously been tested. We have developed an in vivo assay for TDG activity that takes advantage of its recently discovered role in DNA demethylation and selective recognition and repair of 5-carboxylcytosine. Using this assay, we investigated the role of Sumoylation in regulating TDG activity through the use of TDG mutants defective for Sumoylation and Small Ubiquitin-like Modifier (SUMO) binding and by altering TDG Sumoylation through SUMO and SUMO protease overexpression experiments. Our findings indicate that Sumoylation and SUMO binding are not essential for TDG-mediated excision and repair of 5-carboxylcytosine bases. Moreover, in vitro assays revealed that apurinic/apyrimidinic nuclease 1 provides nearly maximum stimulation of TDG processing of G·caC substrates. Thus, under our assay conditions, apurinic/apyrimidinic nuclease 1-mediated stimulation or other mechanisms sufficiently alleviate TDG product inhibition and promote its enzymatic turnover in vivo.
Christopher T Coey - One of the best experts on this subject based on the ideXlab platform.
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defining the impact of Sumoylation on substrate binding and catalysis by thymine dna glycosylase
Nucleic Acids Research, 2018Co-Authors: Christopher T Coey, Alexander C DrohatAbstract:: Thymine DNA glycosylase (TDG) excises thymine from mutagenic G·T mispairs generated by deamination of 5-methylcytosine (mC) and it removes two mC derivatives, 5-formylcytosine (fC) and 5-carboxylcytosine (caC), in a multistep pathway for DNA demethylation. TDG is modified by small ubiquitin-like modifier (SUMO) proteins, but the impact of Sumoylation on TDG activity is poorly defined and the functions of TDG Sumoylation remain unclear. We determined the effect of TDG Sumoylation, by SUMO-1 or SUMO-2, on substrate binding and catalytic parameters. Single turnover experiments reveal that Sumoylation dramatically impairs TDG base-excision activity, such that G·T activity is reduced by ≥45-fold and fC and caC are excised slowly, with a reaction half-life of ≥9 min (37°C). Fluorescence anisotropy studies reveal that unmodified TDG binds tightly to G·fC and G·caC substrates, with dissociation constants in the low nanomolar range. While Sumoylation of TDG weakens substrate binding, the residual affinity is substantial and is comparable to that of biochemically-characterized readers of fC and caC. Our findings raise the possibility that Sumoylation enables TDG to function, at least transiently, as reader of fC and caC. Notably, Sumoylation could potentially facilitate TDG recruitment of other proteins, including transcription factors or epigenetic regulators, to these sites in DNA.
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characterizing requirements for small ubiquitin like modifier sumo modification and binding on base excision repair activity of thymine dna glycosylase in vivo
Journal of Biological Chemistry, 2016Co-Authors: Dylan Mclaughlin, Alexander C Drohat, Christopher T Coey, Weichih Yang, Michael J. MatunisAbstract:: Thymine-DNA glycosylase (TDG) plays critical roles in DNA base excision repair and DNA demethylation. It has been proposed, based on structural studies and in vitro biochemistry, that Sumoylation is required for efficient TDG enzymatic turnover following base excision. However, whether Sumoylation is required for TDG activity in vivo has not previously been tested. We have developed an in vivo assay for TDG activity that takes advantage of its recently discovered role in DNA demethylation and selective recognition and repair of 5-carboxylcytosine. Using this assay, we investigated the role of Sumoylation in regulating TDG activity through the use of TDG mutants defective for Sumoylation and Small Ubiquitin-like Modifier (SUMO) binding and by altering TDG Sumoylation through SUMO and SUMO protease overexpression experiments. Our findings indicate that Sumoylation and SUMO binding are not essential for TDG-mediated excision and repair of 5-carboxylcytosine bases. Moreover, in vitro assays revealed that apurinic/apyrimidinic nuclease 1 provides nearly maximum stimulation of TDG processing of G·caC substrates. Thus, under our assay conditions, apurinic/apyrimidinic nuclease 1-mediated stimulation or other mechanisms sufficiently alleviate TDG product inhibition and promote its enzymatic turnover in vivo.
Chun Yang Zhang - One of the best experts on this subject based on the ideXlab platform.
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simultaneous measurement of Sumoylation using snap clip tag mediated translation at the single molecule level
Angewandte Chemie, 2013Co-Authors: Yong Yang, Chun Yang ZhangAbstract:Two opposing SUMO wrestlers: simultaneous measurement of multiple Sumoylations was achieved at the single-molecule level by integrating SNAP/CLIP-tag-mediated translation with single-molecule detection (SUMO=small ubiquitin-like modifier). This method gives exceptional sensitivity, and is capable of measuring the Sumoylation of different proteins under various physiological conditions.
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Simultaneous Measurement of Sumoylation using SNAP/CLIP‐Tag‐Mediated Translation at the Single‐Molecule Level
Angewandte Chemie, 2012Co-Authors: Yong Yang, Chun Yang ZhangAbstract:Two opposing SUMO wrestlers: simultaneous measurement of multiple Sumoylations was achieved at the single-molecule level by integrating SNAP/CLIP-tag-mediated translation with single-molecule detection (SUMO=small ubiquitin-like modifier). This method gives exceptional sensitivity, and is capable of measuring the Sumoylation of different proteins under various physiological conditions.
Michael J. Matunis - One of the best experts on this subject based on the ideXlab platform.
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Detection of Sumoylation in Plasmodium falciparum.
Methods of Molecular Biology, 2016Co-Authors: Katherine H. Reiter, Michael J. MatunisAbstract:: Reversible protein modification by small ubiquitin-related modifiers (SUMOs) regulates many cellular processes, including transcription, protein quality control, cell division, and oxidative stress. Sumoylation is therefore essential for normal cell function and represents a potentially valuable target for the development of inhibitors of pathogenic eukaryotic organisms, including the malaria parasite, Plasmodium falciparum (Pf). The specific and essential functions of Sumoylation in Pf, however, remain largely uncharacterized. The further development of antimalarial drugs targeting Sumoylation would benefit significantly from a more detailed understanding of its functions and regulation during the parasite life cycle. The recent development of antibodies specific for Pf SUMO provides a valuable tool to study the functions and regulation of Sumoylation. In preliminary studies, we have used immunoblot analysis to demonstrate that Sumoylation levels vary significantly in parasites during different stages of the red blood cell cycle and also in response to oxidative stress. Owing to the dynamic nature of Sumoylation and to the robust activity of SUMO isopeptidases, analysis of Sumoylation in cultured Pf parasites requires a number of precautions during parasite purification and lysis. Here, we outline methods for preserving SUMO conjugates during isolation of Pf parasites from human red blood cell cultures, and for their detection by immunoblot analysis using PfSUMO-specific antibodies.
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characterizing requirements for small ubiquitin like modifier sumo modification and binding on base excision repair activity of thymine dna glycosylase in vivo
Journal of Biological Chemistry, 2016Co-Authors: Dylan Mclaughlin, Alexander C Drohat, Christopher T Coey, Weichih Yang, Michael J. MatunisAbstract:: Thymine-DNA glycosylase (TDG) plays critical roles in DNA base excision repair and DNA demethylation. It has been proposed, based on structural studies and in vitro biochemistry, that Sumoylation is required for efficient TDG enzymatic turnover following base excision. However, whether Sumoylation is required for TDG activity in vivo has not previously been tested. We have developed an in vivo assay for TDG activity that takes advantage of its recently discovered role in DNA demethylation and selective recognition and repair of 5-carboxylcytosine. Using this assay, we investigated the role of Sumoylation in regulating TDG activity through the use of TDG mutants defective for Sumoylation and Small Ubiquitin-like Modifier (SUMO) binding and by altering TDG Sumoylation through SUMO and SUMO protease overexpression experiments. Our findings indicate that Sumoylation and SUMO binding are not essential for TDG-mediated excision and repair of 5-carboxylcytosine bases. Moreover, in vitro assays revealed that apurinic/apyrimidinic nuclease 1 provides nearly maximum stimulation of TDG processing of G·caC substrates. Thus, under our assay conditions, apurinic/apyrimidinic nuclease 1-mediated stimulation or other mechanisms sufficiently alleviate TDG product inhibition and promote its enzymatic turnover in vivo.
Yong Yang - One of the best experts on this subject based on the ideXlab platform.
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simultaneous measurement of Sumoylation using snap clip tag mediated translation at the single molecule level
Angewandte Chemie, 2013Co-Authors: Yong Yang, Chun Yang ZhangAbstract:Two opposing SUMO wrestlers: simultaneous measurement of multiple Sumoylations was achieved at the single-molecule level by integrating SNAP/CLIP-tag-mediated translation with single-molecule detection (SUMO=small ubiquitin-like modifier). This method gives exceptional sensitivity, and is capable of measuring the Sumoylation of different proteins under various physiological conditions.
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Simultaneous Measurement of Sumoylation using SNAP/CLIP‐Tag‐Mediated Translation at the Single‐Molecule Level
Angewandte Chemie, 2012Co-Authors: Yong Yang, Chun Yang ZhangAbstract:Two opposing SUMO wrestlers: simultaneous measurement of multiple Sumoylations was achieved at the single-molecule level by integrating SNAP/CLIP-tag-mediated translation with single-molecule detection (SUMO=small ubiquitin-like modifier). This method gives exceptional sensitivity, and is capable of measuring the Sumoylation of different proteins under various physiological conditions.