Limits...
SUMO modification of the neuroprotective protein TDP1 facilitates chromosomal single-strand break repair.

Hudson JJ, Chiang SC, Wells OS, Rookyard C, El-Khamisy SF - Nat Commun (2012)

Bottom Line: Failure to reseal broken DNA strands results in protein-linked DNA breaks, causing neurodegeneration in humans.A TDP1 SUMOylation-deficient mutant displays a reduced rate of repair of chromosomal single-strand breaks arising from transcription-associated topoisomerase 1 activity or oxidative stress.These data identify a role for SUMO during single-strand break repair, and suggest a mechanism for protecting the nervous system from genotoxic stress.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, University of Sussex, Science Park Road, Falmer, Brighton BN1 9RQ, UK.

ABSTRACT
Breaking and sealing one strand of DNA is an inherent feature of chromosome metabolism to overcome torsional barriers. Failure to reseal broken DNA strands results in protein-linked DNA breaks, causing neurodegeneration in humans. This is typified by defects in tyrosyl DNA phosphodiesterase 1 (TDP1), which removes stalled topoisomerase 1 peptides from DNA termini. Here we show that TDP1 is a substrate for modification by the small ubiquitin-like modifier SUMO. We purify SUMOylated TDP1 from mammalian cells and identify the SUMOylation site as lysine 111. While SUMOylation exhibits no impact on TDP1 catalytic activity, it promotes its accumulation at sites of DNA damage. A TDP1 SUMOylation-deficient mutant displays a reduced rate of repair of chromosomal single-strand breaks arising from transcription-associated topoisomerase 1 activity or oxidative stress. These data identify a role for SUMO during single-strand break repair, and suggest a mechanism for protecting the nervous system from genotoxic stress.

Show MeSH

Related in: MedlinePlus

Human TDP1 is SUMOylated in vitro and in mammalian cells.(a) Yeast Y190 cells containing the indicated constructs were examined for the activation of His3 and β-Gal reporter genes. (b) Recombinant p53 (430 nM) was incubated with 50 nM SAE1/SAE2, 500 nM UBC9, 5 mM ATP and 30 μM SUMO1 'WT' or mutant SUMO1 that cannot be covalently conjugated 'MT'. Reactions were fractionated by SDS–PAGE and analysed by anti-p53 immunoblotting (Activemotif). (c) Human full-length TDP1 (500 nM) was subjected to in vitro SUMOylation reactions and analysed by anti-TDP1 (ab4166; Abcam) or anti-SUMO1 (Santa Cruz, SC-5308) immunoblotting. (d) SUMOylation reactions were conducted in the presence or absence of 5 mM ATP. (e) SUMOylation reactions conducted in the presence of SUMO1, SUMO2 or SUMO3 were divided into three fractions and analysed by immunoblotting. (f) Total cell extract from HEK293 (∼4×106 cells) transfected with Myc–TDP1 and/or GFP–SUMO1 was subjected to immunoprecipitation using anti-Myc monoclonal antibodies (9B11; Cell Signaling). Immunoprecipitates were analysed by immunoblotting using anti-Myc (9B11) or anti-SUMO1 (SC-5308) antibodies. Input is ∼5% of total cell extract. (g) HEK293 cells transfected with Myc–TDP1, HA-tagged His–SUMO1, His–SUMO2 or His–SUMO3 were analysed by anti-TDP1 (ab4166) or anti-His (H1029) immunoblotting. (h) Lysates from control HeLa cells 'C' or cells stably expressing His–SUMO1 'S1' were mixed with Ni2+ charged agarose beads and histidine-tagged SUMO conjugates 'bound' were analysed by anti-TDP1 immunoblotting. Input is ∼3% of total cell extract. (i) Total cell extract 'In' (∼20 μg) or Ni2+ beads enriched with histidine-tagged SUMO conjugates 'bound' were mixed with 32P-radiolabelled 18-mer duplex (50 nM) harbouring a 3′-phosphotyrosine terminus 'PY', inset. Repair products were analysed by denaturing PAGE and phosphorimaging. Positions of the 32P-radiolabelled substrate 'PY' and product 'P' are indicated. (j) Diagram depicting purification of SUMOylated TDP1 from mammalian cells. (k) Serial dilutions of purified Myc–TDP1 or Myc–TDP1–SUMO1–His–GFP were analysed by SDS–PAGE and anti-Myc immunoblotting. (l) Purified Myc-TDP1–SUMO1–His–GFP was mock-treated '−' or incubated with 250 ng of human sentrin/SUMO-specific protease-1 (SENP1) '+' and reaction products analysed by anti-Myc immunoblotting. Molecular weight size markers (kDa) are depicted. IB, immunoblot; IgG, immunoglobulin G; MT, mutant; WT, wild type.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3316882&req=5

f1: Human TDP1 is SUMOylated in vitro and in mammalian cells.(a) Yeast Y190 cells containing the indicated constructs were examined for the activation of His3 and β-Gal reporter genes. (b) Recombinant p53 (430 nM) was incubated with 50 nM SAE1/SAE2, 500 nM UBC9, 5 mM ATP and 30 μM SUMO1 'WT' or mutant SUMO1 that cannot be covalently conjugated 'MT'. Reactions were fractionated by SDS–PAGE and analysed by anti-p53 immunoblotting (Activemotif). (c) Human full-length TDP1 (500 nM) was subjected to in vitro SUMOylation reactions and analysed by anti-TDP1 (ab4166; Abcam) or anti-SUMO1 (Santa Cruz, SC-5308) immunoblotting. (d) SUMOylation reactions were conducted in the presence or absence of 5 mM ATP. (e) SUMOylation reactions conducted in the presence of SUMO1, SUMO2 or SUMO3 were divided into three fractions and analysed by immunoblotting. (f) Total cell extract from HEK293 (∼4×106 cells) transfected with Myc–TDP1 and/or GFP–SUMO1 was subjected to immunoprecipitation using anti-Myc monoclonal antibodies (9B11; Cell Signaling). Immunoprecipitates were analysed by immunoblotting using anti-Myc (9B11) or anti-SUMO1 (SC-5308) antibodies. Input is ∼5% of total cell extract. (g) HEK293 cells transfected with Myc–TDP1, HA-tagged His–SUMO1, His–SUMO2 or His–SUMO3 were analysed by anti-TDP1 (ab4166) or anti-His (H1029) immunoblotting. (h) Lysates from control HeLa cells 'C' or cells stably expressing His–SUMO1 'S1' were mixed with Ni2+ charged agarose beads and histidine-tagged SUMO conjugates 'bound' were analysed by anti-TDP1 immunoblotting. Input is ∼3% of total cell extract. (i) Total cell extract 'In' (∼20 μg) or Ni2+ beads enriched with histidine-tagged SUMO conjugates 'bound' were mixed with 32P-radiolabelled 18-mer duplex (50 nM) harbouring a 3′-phosphotyrosine terminus 'PY', inset. Repair products were analysed by denaturing PAGE and phosphorimaging. Positions of the 32P-radiolabelled substrate 'PY' and product 'P' are indicated. (j) Diagram depicting purification of SUMOylated TDP1 from mammalian cells. (k) Serial dilutions of purified Myc–TDP1 or Myc–TDP1–SUMO1–His–GFP were analysed by SDS–PAGE and anti-Myc immunoblotting. (l) Purified Myc-TDP1–SUMO1–His–GFP was mock-treated '−' or incubated with 250 ng of human sentrin/SUMO-specific protease-1 (SENP1) '+' and reaction products analysed by anti-Myc immunoblotting. Molecular weight size markers (kDa) are depicted. IB, immunoblot; IgG, immunoglobulin G; MT, mutant; WT, wild type.

Mentions: Yeast two-hybrid analyses conducted to identify novel TDP1 binding partners uncovered 14 independent clones encoding full-length UBE2I, the human homologue of the yeast SUMO-conjugating enzyme UBC9 (Fig. 1a). These observations suggested a previously unanticipated role for the SUMO modification pathway during TDP1-mediated repair. To test whether TDP1 is a substrate for covalent SUMO conjugation, we first reconstituted SUMOylation reactions in vitro using recombinant human p53, a known SUMO target20 (Fig. 1b). Parallel reactions conducted with human recombinant TDP1 revealed a slower migrating band, as detected by anti-TDP1 or anti-SUMO1 antibodies (Fig. 1c). These products were absent from control reactions conducted in the presence of a SUMO1 mutant that is incapable of forming the covalent conjugation reaction. Furthermore, their appearance was dependent on ATP (Fig. 1d), confirming that they are covalent TDP1–SUMO1 conjugates. Subsequent comparison of the different SUMO isoforms suggested that TDP1 is preferentially modified by SUMO1 (Fig. 1e). As most SUMO target proteins are modified at very low steady-state levels in vivo, we ectopically expressed TDP1 and SUMO1 in mammalian cells and examined the possibility of covalent TDP1–SUMO1 conjugations. For these experiments, we transfected HEK293 cells with Myc–TDP1 and/or green fluorescent protein (GFP)–SUMO1, followed by immunoprecipitation using anti-Myc antibodies. Probing the immunoprecipitate (IP) with anti-Myc antibodies revealed a discrete slower migrating band in Myc–IPs from extracts coexpressing Myc–TDP1 and GFP–SUMO1 (Fig. 1f). Importantly, this band was absent in Myc–IPs conducted on extracts expressing either Myc–TDP1 or GFP–SUMO1 alone. Furthermore, probing with anti-SUMO1 antibodies revealed a faint band in the input of extracts coexpressing Myc–TDP1 and GFP–SUMO1, but not in extracts expressing either Myc–TDP1 or GFP–SUMO1 alone. Enrichment of this band was observed following Myc–IPs, confirming that it is a TDP1–SUMO1 modification. Parallel experiments conducted using SUMO1, SUMO2 or SUMO3 isoforms were in agreement with the in vitro observations, suggesting that while TDP1 can be modified by all three SUMO isoforms its primary substrate appears to be SUMO1 (Fig. 1g).


SUMO modification of the neuroprotective protein TDP1 facilitates chromosomal single-strand break repair.

Hudson JJ, Chiang SC, Wells OS, Rookyard C, El-Khamisy SF - Nat Commun (2012)

Human TDP1 is SUMOylated in vitro and in mammalian cells.(a) Yeast Y190 cells containing the indicated constructs were examined for the activation of His3 and β-Gal reporter genes. (b) Recombinant p53 (430 nM) was incubated with 50 nM SAE1/SAE2, 500 nM UBC9, 5 mM ATP and 30 μM SUMO1 'WT' or mutant SUMO1 that cannot be covalently conjugated 'MT'. Reactions were fractionated by SDS–PAGE and analysed by anti-p53 immunoblotting (Activemotif). (c) Human full-length TDP1 (500 nM) was subjected to in vitro SUMOylation reactions and analysed by anti-TDP1 (ab4166; Abcam) or anti-SUMO1 (Santa Cruz, SC-5308) immunoblotting. (d) SUMOylation reactions were conducted in the presence or absence of 5 mM ATP. (e) SUMOylation reactions conducted in the presence of SUMO1, SUMO2 or SUMO3 were divided into three fractions and analysed by immunoblotting. (f) Total cell extract from HEK293 (∼4×106 cells) transfected with Myc–TDP1 and/or GFP–SUMO1 was subjected to immunoprecipitation using anti-Myc monoclonal antibodies (9B11; Cell Signaling). Immunoprecipitates were analysed by immunoblotting using anti-Myc (9B11) or anti-SUMO1 (SC-5308) antibodies. Input is ∼5% of total cell extract. (g) HEK293 cells transfected with Myc–TDP1, HA-tagged His–SUMO1, His–SUMO2 or His–SUMO3 were analysed by anti-TDP1 (ab4166) or anti-His (H1029) immunoblotting. (h) Lysates from control HeLa cells 'C' or cells stably expressing His–SUMO1 'S1' were mixed with Ni2+ charged agarose beads and histidine-tagged SUMO conjugates 'bound' were analysed by anti-TDP1 immunoblotting. Input is ∼3% of total cell extract. (i) Total cell extract 'In' (∼20 μg) or Ni2+ beads enriched with histidine-tagged SUMO conjugates 'bound' were mixed with 32P-radiolabelled 18-mer duplex (50 nM) harbouring a 3′-phosphotyrosine terminus 'PY', inset. Repair products were analysed by denaturing PAGE and phosphorimaging. Positions of the 32P-radiolabelled substrate 'PY' and product 'P' are indicated. (j) Diagram depicting purification of SUMOylated TDP1 from mammalian cells. (k) Serial dilutions of purified Myc–TDP1 or Myc–TDP1–SUMO1–His–GFP were analysed by SDS–PAGE and anti-Myc immunoblotting. (l) Purified Myc-TDP1–SUMO1–His–GFP was mock-treated '−' or incubated with 250 ng of human sentrin/SUMO-specific protease-1 (SENP1) '+' and reaction products analysed by anti-Myc immunoblotting. Molecular weight size markers (kDa) are depicted. IB, immunoblot; IgG, immunoglobulin G; MT, mutant; WT, wild type.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3316882&req=5

f1: Human TDP1 is SUMOylated in vitro and in mammalian cells.(a) Yeast Y190 cells containing the indicated constructs were examined for the activation of His3 and β-Gal reporter genes. (b) Recombinant p53 (430 nM) was incubated with 50 nM SAE1/SAE2, 500 nM UBC9, 5 mM ATP and 30 μM SUMO1 'WT' or mutant SUMO1 that cannot be covalently conjugated 'MT'. Reactions were fractionated by SDS–PAGE and analysed by anti-p53 immunoblotting (Activemotif). (c) Human full-length TDP1 (500 nM) was subjected to in vitro SUMOylation reactions and analysed by anti-TDP1 (ab4166; Abcam) or anti-SUMO1 (Santa Cruz, SC-5308) immunoblotting. (d) SUMOylation reactions were conducted in the presence or absence of 5 mM ATP. (e) SUMOylation reactions conducted in the presence of SUMO1, SUMO2 or SUMO3 were divided into three fractions and analysed by immunoblotting. (f) Total cell extract from HEK293 (∼4×106 cells) transfected with Myc–TDP1 and/or GFP–SUMO1 was subjected to immunoprecipitation using anti-Myc monoclonal antibodies (9B11; Cell Signaling). Immunoprecipitates were analysed by immunoblotting using anti-Myc (9B11) or anti-SUMO1 (SC-5308) antibodies. Input is ∼5% of total cell extract. (g) HEK293 cells transfected with Myc–TDP1, HA-tagged His–SUMO1, His–SUMO2 or His–SUMO3 were analysed by anti-TDP1 (ab4166) or anti-His (H1029) immunoblotting. (h) Lysates from control HeLa cells 'C' or cells stably expressing His–SUMO1 'S1' were mixed with Ni2+ charged agarose beads and histidine-tagged SUMO conjugates 'bound' were analysed by anti-TDP1 immunoblotting. Input is ∼3% of total cell extract. (i) Total cell extract 'In' (∼20 μg) or Ni2+ beads enriched with histidine-tagged SUMO conjugates 'bound' were mixed with 32P-radiolabelled 18-mer duplex (50 nM) harbouring a 3′-phosphotyrosine terminus 'PY', inset. Repair products were analysed by denaturing PAGE and phosphorimaging. Positions of the 32P-radiolabelled substrate 'PY' and product 'P' are indicated. (j) Diagram depicting purification of SUMOylated TDP1 from mammalian cells. (k) Serial dilutions of purified Myc–TDP1 or Myc–TDP1–SUMO1–His–GFP were analysed by SDS–PAGE and anti-Myc immunoblotting. (l) Purified Myc-TDP1–SUMO1–His–GFP was mock-treated '−' or incubated with 250 ng of human sentrin/SUMO-specific protease-1 (SENP1) '+' and reaction products analysed by anti-Myc immunoblotting. Molecular weight size markers (kDa) are depicted. IB, immunoblot; IgG, immunoglobulin G; MT, mutant; WT, wild type.
Mentions: Yeast two-hybrid analyses conducted to identify novel TDP1 binding partners uncovered 14 independent clones encoding full-length UBE2I, the human homologue of the yeast SUMO-conjugating enzyme UBC9 (Fig. 1a). These observations suggested a previously unanticipated role for the SUMO modification pathway during TDP1-mediated repair. To test whether TDP1 is a substrate for covalent SUMO conjugation, we first reconstituted SUMOylation reactions in vitro using recombinant human p53, a known SUMO target20 (Fig. 1b). Parallel reactions conducted with human recombinant TDP1 revealed a slower migrating band, as detected by anti-TDP1 or anti-SUMO1 antibodies (Fig. 1c). These products were absent from control reactions conducted in the presence of a SUMO1 mutant that is incapable of forming the covalent conjugation reaction. Furthermore, their appearance was dependent on ATP (Fig. 1d), confirming that they are covalent TDP1–SUMO1 conjugates. Subsequent comparison of the different SUMO isoforms suggested that TDP1 is preferentially modified by SUMO1 (Fig. 1e). As most SUMO target proteins are modified at very low steady-state levels in vivo, we ectopically expressed TDP1 and SUMO1 in mammalian cells and examined the possibility of covalent TDP1–SUMO1 conjugations. For these experiments, we transfected HEK293 cells with Myc–TDP1 and/or green fluorescent protein (GFP)–SUMO1, followed by immunoprecipitation using anti-Myc antibodies. Probing the immunoprecipitate (IP) with anti-Myc antibodies revealed a discrete slower migrating band in Myc–IPs from extracts coexpressing Myc–TDP1 and GFP–SUMO1 (Fig. 1f). Importantly, this band was absent in Myc–IPs conducted on extracts expressing either Myc–TDP1 or GFP–SUMO1 alone. Furthermore, probing with anti-SUMO1 antibodies revealed a faint band in the input of extracts coexpressing Myc–TDP1 and GFP–SUMO1, but not in extracts expressing either Myc–TDP1 or GFP–SUMO1 alone. Enrichment of this band was observed following Myc–IPs, confirming that it is a TDP1–SUMO1 modification. Parallel experiments conducted using SUMO1, SUMO2 or SUMO3 isoforms were in agreement with the in vitro observations, suggesting that while TDP1 can be modified by all three SUMO isoforms its primary substrate appears to be SUMO1 (Fig. 1g).

Bottom Line: Failure to reseal broken DNA strands results in protein-linked DNA breaks, causing neurodegeneration in humans.A TDP1 SUMOylation-deficient mutant displays a reduced rate of repair of chromosomal single-strand breaks arising from transcription-associated topoisomerase 1 activity or oxidative stress.These data identify a role for SUMO during single-strand break repair, and suggest a mechanism for protecting the nervous system from genotoxic stress.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, University of Sussex, Science Park Road, Falmer, Brighton BN1 9RQ, UK.

ABSTRACT
Breaking and sealing one strand of DNA is an inherent feature of chromosome metabolism to overcome torsional barriers. Failure to reseal broken DNA strands results in protein-linked DNA breaks, causing neurodegeneration in humans. This is typified by defects in tyrosyl DNA phosphodiesterase 1 (TDP1), which removes stalled topoisomerase 1 peptides from DNA termini. Here we show that TDP1 is a substrate for modification by the small ubiquitin-like modifier SUMO. We purify SUMOylated TDP1 from mammalian cells and identify the SUMOylation site as lysine 111. While SUMOylation exhibits no impact on TDP1 catalytic activity, it promotes its accumulation at sites of DNA damage. A TDP1 SUMOylation-deficient mutant displays a reduced rate of repair of chromosomal single-strand breaks arising from transcription-associated topoisomerase 1 activity or oxidative stress. These data identify a role for SUMO during single-strand break repair, and suggest a mechanism for protecting the nervous system from genotoxic stress.

Show MeSH
Related in: MedlinePlus