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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.

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Accumulation of TDP1 at sites of DNA damage is in part transcription dependent.(a) MRC5 cells expressing GFP–TDP1 or GFP–TDP1 K111R were incubated with DMSO '-DRB' or with 50 μM 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside '+DRB' for 2 h at 37 °C. Cells expressing similar total GFP signal were irradiated with an ultraviolet A laser, and GFP–TDP1 accumulation at the site of damage was quantified. Data are the mean±s.e.m. of ∼40 cells from n=4 biological replicates. Note that the difference between the accumulation of GFP–TDP1 in the absence (yellow circles) and presence (yellow triangles) of DRB was statistically significant (P<0.05, Student's t-test). (b) Average total fluorescence±s.e.m. for cells analysed in a. (c) HEK293 cells were transfected with empty vector 'vector', Myc–TDP1 'TDP1' or Myc–TDP1K111R 'TDP1 K111R' and GFP–SUMO1. Cells were incubated with DMSO 'mock' or treated with 50 μM camptothecin 'CPT' with or without previous incubation with 50 μM DRB for 2 h. DNA strand breakage was quantified by alkaline comet assays and presented as mean tail moment. Data are the average±s.e.m. from n=3 biological replicates, where 50 cells per sample were blindly scored from each experiment. Statistical analyses (Student's t-test) were conducted to compare the difference between TDP1 and TDP1 K111R in the absence or presence of DRB, and the corresponding P-values are depicted. (d) Lysate from cells used for experiments in c were fractionated by SDS–PAGE and analysed by immunoblotting. (e) Collision of elongating RNA polymerases, such as RNA Pol II with Top1 intermediates, leads to stalling of the polymerase with subsequent proteasomal degradation of Top1, and possibly the stalled RNA Pol. We propose that these collision events recruit TDP1 to sites of polymerase stalling. TDP1 exists in equilibrium between unmodified (the majority) and a SUMOylated version (a tiny proportion), and the balance is maintained by the opposing activities of SUMO conjugation (SAE1/2, UBC9 and possibly a SUMO ligase) and deconjugation (SENPs). We show that TDP1 SUMOylation occurs primarily at K111 and propose that SUMOylated TDP1 is at least, in part, engaged in dealing with the transcription-blocking lesions.
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f8: Accumulation of TDP1 at sites of DNA damage is in part transcription dependent.(a) MRC5 cells expressing GFP–TDP1 or GFP–TDP1 K111R were incubated with DMSO '-DRB' or with 50 μM 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside '+DRB' for 2 h at 37 °C. Cells expressing similar total GFP signal were irradiated with an ultraviolet A laser, and GFP–TDP1 accumulation at the site of damage was quantified. Data are the mean±s.e.m. of ∼40 cells from n=4 biological replicates. Note that the difference between the accumulation of GFP–TDP1 in the absence (yellow circles) and presence (yellow triangles) of DRB was statistically significant (P<0.05, Student's t-test). (b) Average total fluorescence±s.e.m. for cells analysed in a. (c) HEK293 cells were transfected with empty vector 'vector', Myc–TDP1 'TDP1' or Myc–TDP1K111R 'TDP1 K111R' and GFP–SUMO1. Cells were incubated with DMSO 'mock' or treated with 50 μM camptothecin 'CPT' with or without previous incubation with 50 μM DRB for 2 h. DNA strand breakage was quantified by alkaline comet assays and presented as mean tail moment. Data are the average±s.e.m. from n=3 biological replicates, where 50 cells per sample were blindly scored from each experiment. Statistical analyses (Student's t-test) were conducted to compare the difference between TDP1 and TDP1 K111R in the absence or presence of DRB, and the corresponding P-values are depicted. (d) Lysate from cells used for experiments in c were fractionated by SDS–PAGE and analysed by immunoblotting. (e) Collision of elongating RNA polymerases, such as RNA Pol II with Top1 intermediates, leads to stalling of the polymerase with subsequent proteasomal degradation of Top1, and possibly the stalled RNA Pol. We propose that these collision events recruit TDP1 to sites of polymerase stalling. TDP1 exists in equilibrium between unmodified (the majority) and a SUMOylated version (a tiny proportion), and the balance is maintained by the opposing activities of SUMO conjugation (SAE1/2, UBC9 and possibly a SUMO ligase) and deconjugation (SENPs). We show that TDP1 SUMOylation occurs primarily at K111 and propose that SUMOylated TDP1 is at least, in part, engaged in dealing with the transcription-blocking lesions.

Mentions: A major source of Top1-breaks is the collision of Top1 intermediates with elongating RNA polymerases during transcription. Inhibiting RNA polymerase II by 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside (DRB) or α-amanitin has been shown to reduce the extent of CPT-induced DNA SSBs437. We reasoned that if TDP1 SUMOylation contributes to its recruitment to sites of transcription-associated SSBs, then inhibiting transcription should result in reduced accumulation of TDP1. Consistent with this prediction, pretreatment of GFP–TDP1-expressing cells with the transcription inhibitor DRB led to reduction in TDP1 accumulation at sites of laser damage (Fig. 8a,b). The reduction in recruitment was less than that observed for mock-treated GFP–TDPK111R, which could reflect a role for TDP1 SUMOylation during damage generated by DRB-resistant RNA polymerases. Alternatively, it could be due to roles for TDP1 SUMOylation unrelated to transcription. Importantly, DRB did not affect the initial recruitment of GFP–TDP1K111R, suggesting a role for TDP1 SUMOylation at K111 during the repair of transcription-blocking lesions. However, DRB ablated the time-dependent increase of GFP–TDP1K111R accumulation during the subsequent 90-s observation period. We reason that inhibiting transcription may reduce the frequency of collision of Top1 intermediates with elongating RNA polymerase II, thereby reducing the need for recruiting more GFP–TDP1K111R. We next quantified the extent of transcription-associated SSBs using alkaline comet assays. For these experiments, we used HEK293 cells to achieve ∼90% transfection efficiency. If the difference in repair capacity observed between TDP1 and TDP1K111R was, at least in part, due to a role for TDP1 SUMOylation in repairing transcription-associated SSBs, then inhibiting transcription should ablate or reduce this difference. While cells expressing TDP1K111R accumulated a higher level of SSBs compared with TDP1 expressing cells, they both decreased to a comparable level following incubation with DRB (Fig. 8c,d; P=0.78; t-test), suggesting that TDP1 SUMOylation contributes to the repair of transcription-associated SSBs. Taken together, these data suggest that TDP1 SUMOylation at K111 participates in the overall repair of transcription-dependent SSBs.


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)

Accumulation of TDP1 at sites of DNA damage is in part transcription dependent.(a) MRC5 cells expressing GFP–TDP1 or GFP–TDP1 K111R were incubated with DMSO '-DRB' or with 50 μM 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside '+DRB' for 2 h at 37 °C. Cells expressing similar total GFP signal were irradiated with an ultraviolet A laser, and GFP–TDP1 accumulation at the site of damage was quantified. Data are the mean±s.e.m. of ∼40 cells from n=4 biological replicates. Note that the difference between the accumulation of GFP–TDP1 in the absence (yellow circles) and presence (yellow triangles) of DRB was statistically significant (P<0.05, Student's t-test). (b) Average total fluorescence±s.e.m. for cells analysed in a. (c) HEK293 cells were transfected with empty vector 'vector', Myc–TDP1 'TDP1' or Myc–TDP1K111R 'TDP1 K111R' and GFP–SUMO1. Cells were incubated with DMSO 'mock' or treated with 50 μM camptothecin 'CPT' with or without previous incubation with 50 μM DRB for 2 h. DNA strand breakage was quantified by alkaline comet assays and presented as mean tail moment. Data are the average±s.e.m. from n=3 biological replicates, where 50 cells per sample were blindly scored from each experiment. Statistical analyses (Student's t-test) were conducted to compare the difference between TDP1 and TDP1 K111R in the absence or presence of DRB, and the corresponding P-values are depicted. (d) Lysate from cells used for experiments in c were fractionated by SDS–PAGE and analysed by immunoblotting. (e) Collision of elongating RNA polymerases, such as RNA Pol II with Top1 intermediates, leads to stalling of the polymerase with subsequent proteasomal degradation of Top1, and possibly the stalled RNA Pol. We propose that these collision events recruit TDP1 to sites of polymerase stalling. TDP1 exists in equilibrium between unmodified (the majority) and a SUMOylated version (a tiny proportion), and the balance is maintained by the opposing activities of SUMO conjugation (SAE1/2, UBC9 and possibly a SUMO ligase) and deconjugation (SENPs). We show that TDP1 SUMOylation occurs primarily at K111 and propose that SUMOylated TDP1 is at least, in part, engaged in dealing with the transcription-blocking lesions.
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Related In: Results  -  Collection

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f8: Accumulation of TDP1 at sites of DNA damage is in part transcription dependent.(a) MRC5 cells expressing GFP–TDP1 or GFP–TDP1 K111R were incubated with DMSO '-DRB' or with 50 μM 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside '+DRB' for 2 h at 37 °C. Cells expressing similar total GFP signal were irradiated with an ultraviolet A laser, and GFP–TDP1 accumulation at the site of damage was quantified. Data are the mean±s.e.m. of ∼40 cells from n=4 biological replicates. Note that the difference between the accumulation of GFP–TDP1 in the absence (yellow circles) and presence (yellow triangles) of DRB was statistically significant (P<0.05, Student's t-test). (b) Average total fluorescence±s.e.m. for cells analysed in a. (c) HEK293 cells were transfected with empty vector 'vector', Myc–TDP1 'TDP1' or Myc–TDP1K111R 'TDP1 K111R' and GFP–SUMO1. Cells were incubated with DMSO 'mock' or treated with 50 μM camptothecin 'CPT' with or without previous incubation with 50 μM DRB for 2 h. DNA strand breakage was quantified by alkaline comet assays and presented as mean tail moment. Data are the average±s.e.m. from n=3 biological replicates, where 50 cells per sample were blindly scored from each experiment. Statistical analyses (Student's t-test) were conducted to compare the difference between TDP1 and TDP1 K111R in the absence or presence of DRB, and the corresponding P-values are depicted. (d) Lysate from cells used for experiments in c were fractionated by SDS–PAGE and analysed by immunoblotting. (e) Collision of elongating RNA polymerases, such as RNA Pol II with Top1 intermediates, leads to stalling of the polymerase with subsequent proteasomal degradation of Top1, and possibly the stalled RNA Pol. We propose that these collision events recruit TDP1 to sites of polymerase stalling. TDP1 exists in equilibrium between unmodified (the majority) and a SUMOylated version (a tiny proportion), and the balance is maintained by the opposing activities of SUMO conjugation (SAE1/2, UBC9 and possibly a SUMO ligase) and deconjugation (SENPs). We show that TDP1 SUMOylation occurs primarily at K111 and propose that SUMOylated TDP1 is at least, in part, engaged in dealing with the transcription-blocking lesions.
Mentions: A major source of Top1-breaks is the collision of Top1 intermediates with elongating RNA polymerases during transcription. Inhibiting RNA polymerase II by 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside (DRB) or α-amanitin has been shown to reduce the extent of CPT-induced DNA SSBs437. We reasoned that if TDP1 SUMOylation contributes to its recruitment to sites of transcription-associated SSBs, then inhibiting transcription should result in reduced accumulation of TDP1. Consistent with this prediction, pretreatment of GFP–TDP1-expressing cells with the transcription inhibitor DRB led to reduction in TDP1 accumulation at sites of laser damage (Fig. 8a,b). The reduction in recruitment was less than that observed for mock-treated GFP–TDPK111R, which could reflect a role for TDP1 SUMOylation during damage generated by DRB-resistant RNA polymerases. Alternatively, it could be due to roles for TDP1 SUMOylation unrelated to transcription. Importantly, DRB did not affect the initial recruitment of GFP–TDP1K111R, suggesting a role for TDP1 SUMOylation at K111 during the repair of transcription-blocking lesions. However, DRB ablated the time-dependent increase of GFP–TDP1K111R accumulation during the subsequent 90-s observation period. We reason that inhibiting transcription may reduce the frequency of collision of Top1 intermediates with elongating RNA polymerase II, thereby reducing the need for recruiting more GFP–TDP1K111R. We next quantified the extent of transcription-associated SSBs using alkaline comet assays. For these experiments, we used HEK293 cells to achieve ∼90% transfection efficiency. If the difference in repair capacity observed between TDP1 and TDP1K111R was, at least in part, due to a role for TDP1 SUMOylation in repairing transcription-associated SSBs, then inhibiting transcription should ablate or reduce this difference. While cells expressing TDP1K111R accumulated a higher level of SSBs compared with TDP1 expressing cells, they both decreased to a comparable level following incubation with DRB (Fig. 8c,d; P=0.78; t-test), suggesting that TDP1 SUMOylation contributes to the repair of transcription-associated SSBs. Taken together, these data suggest that TDP1 SUMOylation at K111 participates in the overall repair of transcription-dependent SSBs.

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