Characterizing requirements for small ubiquitin-like modifier (SUMO) modification and binding on base excision repair activity of thymine-DNA glycosylase in vivo

Dylan McLaughlin, Christopher T. Coey, Wei Chih Yang, Alexander C. Drohat, Michael J Matunis

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)9014-9024
Number of pages11
JournalJournal of Biological Chemistry
Volume291
Issue number17
DOIs
StatePublished - Apr 22 2016

Fingerprint

Thymine DNA Glycosylase
Ubiquitin
DNA Repair
Sumoylation
Repair
Assays
DNA
Biochemistry

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Characterizing requirements for small ubiquitin-like modifier (SUMO) modification and binding on base excision repair activity of thymine-DNA glycosylase in vivo. / McLaughlin, Dylan; Coey, Christopher T.; Yang, Wei Chih; Drohat, Alexander C.; Matunis, Michael J.

In: Journal of Biological Chemistry, Vol. 291, No. 17, 22.04.2016, p. 9014-9024.

Research output: Contribution to journalArticle

@article{e2c3795b9e444a4ab93b624b12901a48,
title = "Characterizing requirements for small ubiquitin-like modifier (SUMO) modification and binding on base excision repair activity of thymine-DNA glycosylase in vivo",
abstract = "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.",
author = "Dylan McLaughlin and Coey, {Christopher T.} and Yang, {Wei Chih} and Drohat, {Alexander C.} and Matunis, {Michael J}",
year = "2016",
month = "4",
day = "22",
doi = "10.1074/jbc.M115.706325",
language = "English (US)",
volume = "291",
pages = "9014--9024",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "17",

}

TY - JOUR

T1 - Characterizing requirements for small ubiquitin-like modifier (SUMO) modification and binding on base excision repair activity of thymine-DNA glycosylase in vivo

AU - McLaughlin, Dylan

AU - Coey, Christopher T.

AU - Yang, Wei Chih

AU - Drohat, Alexander C.

AU - Matunis, Michael J

PY - 2016/4/22

Y1 - 2016/4/22

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84965050663&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84965050663&partnerID=8YFLogxK

U2 - 10.1074/jbc.M115.706325

DO - 10.1074/jbc.M115.706325

M3 - Article

C2 - 26917720

AN - SCOPUS:84965050663

VL - 291

SP - 9014

EP - 9024

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 17

ER -