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

The K111R mutation does not alter TDP1 subcellular localization or interaction with Lig3α.(a) Yeast Y190 cells harbouring pGBKT7–TDP1 or pGBKT7–TDP1K111R and pACT or pACT–Lig3α constructs were plated onto selective media either containing 'control' or lacking 'His' histidine to test for the activation of the His3 reporter gene. Activation of the β-Gal reporter gene was determined using filter lifts from control plates. Expression levels of Myc–TDP1 or Myc–TDP1K111R (Gal4-binding domain fusion protein) and Lig3α (Gal4-activation domain fusion) were determined by immunoblotting with anti-Myc (9B11; Cell Signalling) or anti-Gal4 AD antibodies (06-283; Millipore). (b) HEK293 cells (∼4×106) were transfected with Myc–TDP1 and GFP–SUMO1, and total cell extract subjected to immunoprecipitation using anti-Myc monoclonal antibodies (9B11; Cell Signaling). Immunoprecipitates were fractionated by SDS–PAGE and analysed by immunoblotting using anti-Myc (top) or anti-Lig3α antibodies (bottom). Input is ∼5% of total cell extract used. (c) Human MRC5 cells were plated onto glass-bottom dishes and transfected with pMCEGFP–TDP1. Cells were incubated with DMSO 'DMSO' or 2 μM CPT 'CPT' for 1 h at 37 °C. DNA was counterstained with Hoechst 33285, and GFP-positive cells photographed with a ×40/1.2-W objective using a Zeiss Axiovert confocal microscope. Arrowheads point at the position of nucleoli. (d,e) A549 cells transiently transfected with pMCEGFPP–TDP1 'GFP–TDP1' or the SUMOylation-deficient mutant pMCEGFPP–TDP1K111R 'GFP–TDP1 K111R' were mock treated 'Mock' or incubated with 30 μM CPT 'CPT' for 30 min at 37 °C. Cells were then fixed, DNA counterstained with 4,6-diamidino-2-phenylindole, and analysed with a ×60/oil objective using a Deltavision microscope. A representative images depicting cells with GFP–TDP1 excluded from nucleoli 'excluded', concentrated in nucleoli 'enriched' or exhibited pan-nuclear distribution 'Pan-nuclear' are shown in d. The average number of cells with the indicated GFP–TDP1 localization pattern was measured from a total of ∼60 cells and presented as average±s.e.m. from n=3 biological replicates; Scale bar, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: The K111R mutation does not alter TDP1 subcellular localization or interaction with Lig3α.(a) Yeast Y190 cells harbouring pGBKT7–TDP1 or pGBKT7–TDP1K111R and pACT or pACT–Lig3α constructs were plated onto selective media either containing 'control' or lacking 'His' histidine to test for the activation of the His3 reporter gene. Activation of the β-Gal reporter gene was determined using filter lifts from control plates. Expression levels of Myc–TDP1 or Myc–TDP1K111R (Gal4-binding domain fusion protein) and Lig3α (Gal4-activation domain fusion) were determined by immunoblotting with anti-Myc (9B11; Cell Signalling) or anti-Gal4 AD antibodies (06-283; Millipore). (b) HEK293 cells (∼4×106) were transfected with Myc–TDP1 and GFP–SUMO1, and total cell extract subjected to immunoprecipitation using anti-Myc monoclonal antibodies (9B11; Cell Signaling). Immunoprecipitates were fractionated by SDS–PAGE and analysed by immunoblotting using anti-Myc (top) or anti-Lig3α antibodies (bottom). Input is ∼5% of total cell extract used. (c) Human MRC5 cells were plated onto glass-bottom dishes and transfected with pMCEGFP–TDP1. Cells were incubated with DMSO 'DMSO' or 2 μM CPT 'CPT' for 1 h at 37 °C. DNA was counterstained with Hoechst 33285, and GFP-positive cells photographed with a ×40/1.2-W objective using a Zeiss Axiovert confocal microscope. Arrowheads point at the position of nucleoli. (d,e) A549 cells transiently transfected with pMCEGFPP–TDP1 'GFP–TDP1' or the SUMOylation-deficient mutant pMCEGFPP–TDP1K111R 'GFP–TDP1 K111R' were mock treated 'Mock' or incubated with 30 μM CPT 'CPT' for 30 min at 37 °C. Cells were then fixed, DNA counterstained with 4,6-diamidino-2-phenylindole, and analysed with a ×60/oil objective using a Deltavision microscope. A representative images depicting cells with GFP–TDP1 excluded from nucleoli 'excluded', concentrated in nucleoli 'enriched' or exhibited pan-nuclear distribution 'Pan-nuclear' are shown in d. The average number of cells with the indicated GFP–TDP1 localization pattern was measured from a total of ∼60 cells and presented as average±s.e.m. from n=3 biological replicates; Scale bar, 10 μm.

Mentions: Next, we considered the possibility that SUMOylation may alter protein function by introducing structural changes that result in changes in enzymatic activity2930. To test this possibility, we subjected human recombinant TDP1 or TDP1K111R to SUMOylation reactions in the presence of wild-type SUMO1 or mutant SUMO1 (Fig. 4g) and incubated reaction products with the synthetic Top1 substrates (Fig. 4h–j). We also compared the effect of SUMOylation on TDP1 produced in mammalian cells (Fig. 5a,c,e,f). Furthermore, we subjected purified human SUMOylated TDP1 to SENP1 treatment and compared the activity of the resulting products to that of mock-treated fractions (Fig. 5b,d,g,h). Incubation of Myc–TDP1 with Top1-substrate mimics resulted in a dose-dependent conversion of 3′-phosphotyrosine to 3′-phosphate, indicating that TDP1 catalytic activity was not affected by Myc, His or GFP tags. Quantification of reaction products at different concentrations or at different time points revealed no marked impact of SUMOylation on TDP1 activity (Fig. 5e–h). We conclude from these experiments that SUMO1 conjugation to TDP1 does not affect its enzymatic activity. We also excluded the possibility that TDP1 SUMOylation might modulate interaction with DNA ligase IIIα (Lig3α), a known binding partner and a component of the SSB repair machinery (Fig. 6a,b). Using western blotting, we could not detect a measurable increase of TDP1 SUMOylation after exposure to exogenous DNA damage (Supplementary Fig. S1). This suggests that TDP1 SUMOylation is a housekeeping modification that occurs, as is also the case with other SUMO1-modified targets such as fission yeast Top1 and human Kap1 (refs 31 and 32), at low steady-state levels where cycles of conjugation and deconjugation are associated with endogenous levels of DNA breaks. Furthermore, SUMOylation did not alter TDP1 nucleolar exclusion after DNA damage, as determined by live and fixed cell fluorescence microscopy (Fig. 6c–e).


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)

The K111R mutation does not alter TDP1 subcellular localization or interaction with Lig3α.(a) Yeast Y190 cells harbouring pGBKT7–TDP1 or pGBKT7–TDP1K111R and pACT or pACT–Lig3α constructs were plated onto selective media either containing 'control' or lacking 'His' histidine to test for the activation of the His3 reporter gene. Activation of the β-Gal reporter gene was determined using filter lifts from control plates. Expression levels of Myc–TDP1 or Myc–TDP1K111R (Gal4-binding domain fusion protein) and Lig3α (Gal4-activation domain fusion) were determined by immunoblotting with anti-Myc (9B11; Cell Signalling) or anti-Gal4 AD antibodies (06-283; Millipore). (b) HEK293 cells (∼4×106) were transfected with Myc–TDP1 and GFP–SUMO1, and total cell extract subjected to immunoprecipitation using anti-Myc monoclonal antibodies (9B11; Cell Signaling). Immunoprecipitates were fractionated by SDS–PAGE and analysed by immunoblotting using anti-Myc (top) or anti-Lig3α antibodies (bottom). Input is ∼5% of total cell extract used. (c) Human MRC5 cells were plated onto glass-bottom dishes and transfected with pMCEGFP–TDP1. Cells were incubated with DMSO 'DMSO' or 2 μM CPT 'CPT' for 1 h at 37 °C. DNA was counterstained with Hoechst 33285, and GFP-positive cells photographed with a ×40/1.2-W objective using a Zeiss Axiovert confocal microscope. Arrowheads point at the position of nucleoli. (d,e) A549 cells transiently transfected with pMCEGFPP–TDP1 'GFP–TDP1' or the SUMOylation-deficient mutant pMCEGFPP–TDP1K111R 'GFP–TDP1 K111R' were mock treated 'Mock' or incubated with 30 μM CPT 'CPT' for 30 min at 37 °C. Cells were then fixed, DNA counterstained with 4,6-diamidino-2-phenylindole, and analysed with a ×60/oil objective using a Deltavision microscope. A representative images depicting cells with GFP–TDP1 excluded from nucleoli 'excluded', concentrated in nucleoli 'enriched' or exhibited pan-nuclear distribution 'Pan-nuclear' are shown in d. The average number of cells with the indicated GFP–TDP1 localization pattern was measured from a total of ∼60 cells and presented as average±s.e.m. from n=3 biological replicates; Scale bar, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: The K111R mutation does not alter TDP1 subcellular localization or interaction with Lig3α.(a) Yeast Y190 cells harbouring pGBKT7–TDP1 or pGBKT7–TDP1K111R and pACT or pACT–Lig3α constructs were plated onto selective media either containing 'control' or lacking 'His' histidine to test for the activation of the His3 reporter gene. Activation of the β-Gal reporter gene was determined using filter lifts from control plates. Expression levels of Myc–TDP1 or Myc–TDP1K111R (Gal4-binding domain fusion protein) and Lig3α (Gal4-activation domain fusion) were determined by immunoblotting with anti-Myc (9B11; Cell Signalling) or anti-Gal4 AD antibodies (06-283; Millipore). (b) HEK293 cells (∼4×106) were transfected with Myc–TDP1 and GFP–SUMO1, and total cell extract subjected to immunoprecipitation using anti-Myc monoclonal antibodies (9B11; Cell Signaling). Immunoprecipitates were fractionated by SDS–PAGE and analysed by immunoblotting using anti-Myc (top) or anti-Lig3α antibodies (bottom). Input is ∼5% of total cell extract used. (c) Human MRC5 cells were plated onto glass-bottom dishes and transfected with pMCEGFP–TDP1. Cells were incubated with DMSO 'DMSO' or 2 μM CPT 'CPT' for 1 h at 37 °C. DNA was counterstained with Hoechst 33285, and GFP-positive cells photographed with a ×40/1.2-W objective using a Zeiss Axiovert confocal microscope. Arrowheads point at the position of nucleoli. (d,e) A549 cells transiently transfected with pMCEGFPP–TDP1 'GFP–TDP1' or the SUMOylation-deficient mutant pMCEGFPP–TDP1K111R 'GFP–TDP1 K111R' were mock treated 'Mock' or incubated with 30 μM CPT 'CPT' for 30 min at 37 °C. Cells were then fixed, DNA counterstained with 4,6-diamidino-2-phenylindole, and analysed with a ×60/oil objective using a Deltavision microscope. A representative images depicting cells with GFP–TDP1 excluded from nucleoli 'excluded', concentrated in nucleoli 'enriched' or exhibited pan-nuclear distribution 'Pan-nuclear' are shown in d. The average number of cells with the indicated GFP–TDP1 localization pattern was measured from a total of ∼60 cells and presented as average±s.e.m. from n=3 biological replicates; Scale bar, 10 μm.
Mentions: Next, we considered the possibility that SUMOylation may alter protein function by introducing structural changes that result in changes in enzymatic activity2930. To test this possibility, we subjected human recombinant TDP1 or TDP1K111R to SUMOylation reactions in the presence of wild-type SUMO1 or mutant SUMO1 (Fig. 4g) and incubated reaction products with the synthetic Top1 substrates (Fig. 4h–j). We also compared the effect of SUMOylation on TDP1 produced in mammalian cells (Fig. 5a,c,e,f). Furthermore, we subjected purified human SUMOylated TDP1 to SENP1 treatment and compared the activity of the resulting products to that of mock-treated fractions (Fig. 5b,d,g,h). Incubation of Myc–TDP1 with Top1-substrate mimics resulted in a dose-dependent conversion of 3′-phosphotyrosine to 3′-phosphate, indicating that TDP1 catalytic activity was not affected by Myc, His or GFP tags. Quantification of reaction products at different concentrations or at different time points revealed no marked impact of SUMOylation on TDP1 activity (Fig. 5e–h). We conclude from these experiments that SUMO1 conjugation to TDP1 does not affect its enzymatic activity. We also excluded the possibility that TDP1 SUMOylation might modulate interaction with DNA ligase IIIα (Lig3α), a known binding partner and a component of the SSB repair machinery (Fig. 6a,b). Using western blotting, we could not detect a measurable increase of TDP1 SUMOylation after exposure to exogenous DNA damage (Supplementary Fig. S1). This suggests that TDP1 SUMOylation is a housekeeping modification that occurs, as is also the case with other SUMO1-modified targets such as fission yeast Top1 and human Kap1 (refs 31 and 32), at low steady-state levels where cycles of conjugation and deconjugation are associated with endogenous levels of DNA breaks. Furthermore, SUMOylation did not alter TDP1 nucleolar exclusion after DNA damage, as determined by live and fixed cell fluorescence microscopy (Fig. 6c–e).

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