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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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The K111R mutation does not result in a measurable change of TDP1 structure or catalytic activity.(a) Recombinant TDP1 or TDP1K111R was mixed with SYPRO-Orange, and melting profiles were obtained between 20 and 70 °C using a ramping rate of 0.03 °C s−1. Data were normalized to a fraction of protein in its denatured state and presented as normalized relative fluorescence. Melting temperatures (Tm) were determined according to the Boltzmann model55. Error bars represent s.d. from n=3 independent replicates. (b,c) Recombinant TDP1 or TDP1K111R was placed in a 0.2-mm quartz cuvette and the circular dichroism spectrum determined using a JASCO J-715 spectropolarimeter. Scans were performed from 260 to 195 nm, buffer baselines were subtracted, and data recorded with a high-tension voltage 'HT [V]' <550 V. Data represent the average of n=4 independent replicates±s.e.m. (d) Subtraction of the TDP1 K111R spectra from that of TDP1 shows no gross structural difference. (e) Decreasing concentrations (30, 15, 7 and 3 nM) of purified recombinant TDP1 or TDP1K111R was incubated with a 32P-radiolabelled duplex-nicked substrate (50 nM) harbouring a 3′-phosphotyrosine 'PY' at the nick (inset). Repair products were analysed by denaturing PAGE and phosphorimaging. Positions of the 32P-radiolabelled substrate 'PY' and product 'P' are indicated by arrows. (f) Reaction products 'P' were quantified relative to total labelled substrate 'P+PY' and percentage conversion to 3′-P from experiments conducted in e was quantified. (g) Recombinant TDP1 or TDP1K111R was subjected to in vitro SUMOylation reactions in the presence of wild-type SUMO1 'WT' or mutant SUMO1 'MT' and analysed by immunoblotting. (h–j) Serial dilutions of SUMOylation reactions were subsequently mixed with Top1–DSBs (h,i) or Top1–SSBs (j) and analysed by denaturing PAGE and phosphorimaging. Reaction products were quantified relative to total labelled substrate and percentage conversion to 3′-P was quantified. Error bars, s.e.m. of n=3 independent experiments.
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f4: The K111R mutation does not result in a measurable change of TDP1 structure or catalytic activity.(a) Recombinant TDP1 or TDP1K111R was mixed with SYPRO-Orange, and melting profiles were obtained between 20 and 70 °C using a ramping rate of 0.03 °C s−1. Data were normalized to a fraction of protein in its denatured state and presented as normalized relative fluorescence. Melting temperatures (Tm) were determined according to the Boltzmann model55. Error bars represent s.d. from n=3 independent replicates. (b,c) Recombinant TDP1 or TDP1K111R was placed in a 0.2-mm quartz cuvette and the circular dichroism spectrum determined using a JASCO J-715 spectropolarimeter. Scans were performed from 260 to 195 nm, buffer baselines were subtracted, and data recorded with a high-tension voltage 'HT [V]' <550 V. Data represent the average of n=4 independent replicates±s.e.m. (d) Subtraction of the TDP1 K111R spectra from that of TDP1 shows no gross structural difference. (e) Decreasing concentrations (30, 15, 7 and 3 nM) of purified recombinant TDP1 or TDP1K111R was incubated with a 32P-radiolabelled duplex-nicked substrate (50 nM) harbouring a 3′-phosphotyrosine 'PY' at the nick (inset). Repair products were analysed by denaturing PAGE and phosphorimaging. Positions of the 32P-radiolabelled substrate 'PY' and product 'P' are indicated by arrows. (f) Reaction products 'P' were quantified relative to total labelled substrate 'P+PY' and percentage conversion to 3′-P from experiments conducted in e was quantified. (g) Recombinant TDP1 or TDP1K111R was subjected to in vitro SUMOylation reactions in the presence of wild-type SUMO1 'WT' or mutant SUMO1 'MT' and analysed by immunoblotting. (h–j) Serial dilutions of SUMOylation reactions were subsequently mixed with Top1–DSBs (h,i) or Top1–SSBs (j) and analysed by denaturing PAGE and phosphorimaging. Reaction products were quantified relative to total labelled substrate and percentage conversion to 3′-P was quantified. Error bars, s.e.m. of n=3 independent experiments.

Mentions: Why does a TDP1 SUMOylation-defective mutant display attenuated rates of SSB repair? Although unlikely for missense point mutations, we tested the possibility that the K111R mutation might lead to a gross distortion of TDP1 structure. We subjected recombinant TDP1 and TDP1K111R produced in Escherichia coli to thermal denaturation experiments, a widely used technique to examine structural changes of proteins24. The K111R mutation had no detectable impact on the thermal stability or unfolding profile of TDP1, as determined by comparing the denaturation curves and their corresponding melting temperatures (Fig. 4a). We also compared the circular dichroism absorption spectrum of TDP1 and TDP1K111R (Fig. 4b–d). The two proteins gave spectral shapes with negative bands at ∼210 and 220 nm, and positive bands at ∼195 nm. Analyses of secondary structure by the variable selection algorithm (CDSSTR), which provides superior fits for globular proteins25262728, revealed no significant difference in α-helical or β-sheet content (P>0.5; t-test), suggesting no apparent change in structure (Supplementary Table S1). This was also supported by using the CONTIN and K2D algorithms (Supplementary Tables S2 and S3). To further examine the impact on protein folding and catalytic activity, we incubated purified recombinant TDP1 or TDP1K111R with oligonucleotide duplexes harbouring 3′-phosphotyrosine that mimic Top1-linked breaks and quantified the 3′-phosphate products. The reactions showed a comparable concentration-dependent conversion of 3′-phosphotyrosine to 3′-phosphate, suggesting no impact of the K111R mutation on catalytic activity (Fig. 4e,f). Taken together, we conclude that mutation of the SUMOylation site of TDP1 to a non-SUMOylatable version results in delayed rate of SSB repair without a measurable impact on structure or catalytic activity.


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 result in a measurable change of TDP1 structure or catalytic activity.(a) Recombinant TDP1 or TDP1K111R was mixed with SYPRO-Orange, and melting profiles were obtained between 20 and 70 °C using a ramping rate of 0.03 °C s−1. Data were normalized to a fraction of protein in its denatured state and presented as normalized relative fluorescence. Melting temperatures (Tm) were determined according to the Boltzmann model55. Error bars represent s.d. from n=3 independent replicates. (b,c) Recombinant TDP1 or TDP1K111R was placed in a 0.2-mm quartz cuvette and the circular dichroism spectrum determined using a JASCO J-715 spectropolarimeter. Scans were performed from 260 to 195 nm, buffer baselines were subtracted, and data recorded with a high-tension voltage 'HT [V]' <550 V. Data represent the average of n=4 independent replicates±s.e.m. (d) Subtraction of the TDP1 K111R spectra from that of TDP1 shows no gross structural difference. (e) Decreasing concentrations (30, 15, 7 and 3 nM) of purified recombinant TDP1 or TDP1K111R was incubated with a 32P-radiolabelled duplex-nicked substrate (50 nM) harbouring a 3′-phosphotyrosine 'PY' at the nick (inset). Repair products were analysed by denaturing PAGE and phosphorimaging. Positions of the 32P-radiolabelled substrate 'PY' and product 'P' are indicated by arrows. (f) Reaction products 'P' were quantified relative to total labelled substrate 'P+PY' and percentage conversion to 3′-P from experiments conducted in e was quantified. (g) Recombinant TDP1 or TDP1K111R was subjected to in vitro SUMOylation reactions in the presence of wild-type SUMO1 'WT' or mutant SUMO1 'MT' and analysed by immunoblotting. (h–j) Serial dilutions of SUMOylation reactions were subsequently mixed with Top1–DSBs (h,i) or Top1–SSBs (j) and analysed by denaturing PAGE and phosphorimaging. Reaction products were quantified relative to total labelled substrate and percentage conversion to 3′-P was quantified. Error bars, s.e.m. of n=3 independent experiments.
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f4: The K111R mutation does not result in a measurable change of TDP1 structure or catalytic activity.(a) Recombinant TDP1 or TDP1K111R was mixed with SYPRO-Orange, and melting profiles were obtained between 20 and 70 °C using a ramping rate of 0.03 °C s−1. Data were normalized to a fraction of protein in its denatured state and presented as normalized relative fluorescence. Melting temperatures (Tm) were determined according to the Boltzmann model55. Error bars represent s.d. from n=3 independent replicates. (b,c) Recombinant TDP1 or TDP1K111R was placed in a 0.2-mm quartz cuvette and the circular dichroism spectrum determined using a JASCO J-715 spectropolarimeter. Scans were performed from 260 to 195 nm, buffer baselines were subtracted, and data recorded with a high-tension voltage 'HT [V]' <550 V. Data represent the average of n=4 independent replicates±s.e.m. (d) Subtraction of the TDP1 K111R spectra from that of TDP1 shows no gross structural difference. (e) Decreasing concentrations (30, 15, 7 and 3 nM) of purified recombinant TDP1 or TDP1K111R was incubated with a 32P-radiolabelled duplex-nicked substrate (50 nM) harbouring a 3′-phosphotyrosine 'PY' at the nick (inset). Repair products were analysed by denaturing PAGE and phosphorimaging. Positions of the 32P-radiolabelled substrate 'PY' and product 'P' are indicated by arrows. (f) Reaction products 'P' were quantified relative to total labelled substrate 'P+PY' and percentage conversion to 3′-P from experiments conducted in e was quantified. (g) Recombinant TDP1 or TDP1K111R was subjected to in vitro SUMOylation reactions in the presence of wild-type SUMO1 'WT' or mutant SUMO1 'MT' and analysed by immunoblotting. (h–j) Serial dilutions of SUMOylation reactions were subsequently mixed with Top1–DSBs (h,i) or Top1–SSBs (j) and analysed by denaturing PAGE and phosphorimaging. Reaction products were quantified relative to total labelled substrate and percentage conversion to 3′-P was quantified. Error bars, s.e.m. of n=3 independent experiments.
Mentions: Why does a TDP1 SUMOylation-defective mutant display attenuated rates of SSB repair? Although unlikely for missense point mutations, we tested the possibility that the K111R mutation might lead to a gross distortion of TDP1 structure. We subjected recombinant TDP1 and TDP1K111R produced in Escherichia coli to thermal denaturation experiments, a widely used technique to examine structural changes of proteins24. The K111R mutation had no detectable impact on the thermal stability or unfolding profile of TDP1, as determined by comparing the denaturation curves and their corresponding melting temperatures (Fig. 4a). We also compared the circular dichroism absorption spectrum of TDP1 and TDP1K111R (Fig. 4b–d). The two proteins gave spectral shapes with negative bands at ∼210 and 220 nm, and positive bands at ∼195 nm. Analyses of secondary structure by the variable selection algorithm (CDSSTR), which provides superior fits for globular proteins25262728, revealed no significant difference in α-helical or β-sheet content (P>0.5; t-test), suggesting no apparent change in structure (Supplementary Table S1). This was also supported by using the CONTIN and K2D algorithms (Supplementary Tables S2 and S3). To further examine the impact on protein folding and catalytic activity, we incubated purified recombinant TDP1 or TDP1K111R with oligonucleotide duplexes harbouring 3′-phosphotyrosine that mimic Top1-linked breaks and quantified the 3′-phosphate products. The reactions showed a comparable concentration-dependent conversion of 3′-phosphotyrosine to 3′-phosphate, suggesting no impact of the K111R mutation on catalytic activity (Fig. 4e,f). Taken together, we conclude that mutation of the SUMOylation site of TDP1 to a non-SUMOylatable version results in delayed rate of SSB repair without a measurable impact on structure or catalytic activity.

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