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Cited by 124DNA Repair MechanismsUbiquitin and proteasome pathwaysNuclear Structure and Function

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Regulation of nucleotide excision repair activity by transcriptional and post-transcriptional control of the XPA protein

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Kang, T.-H., Reardon, J. T., Sancar, A.·2011·Nucleic Acids Research
DOI 10.1093/nar/gkq1318PMC PMC3082913
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Abstract

The XPA (Xeroderma pigmentosum A) protein is one of the six core factors of the human nucleotide excision repair system. In this study we show that XPA is a rate-limiting factor in all human cell lines tested, including a normal human fibroblast cell line. The level of XPA is controlled at the transcriptional level by the molecular circadian clock and at the post-translational level by a HECT domain family E3 ubiquitin ligase called HERC2. Stabilization of XPA by downregulation of HERC2 moderately enhances excision repair activity. Conversely, downregulation of XPA by siRNA reduces excision repair activity in proportion to the level of XPA. Ubiquitination and proteolysis of XPA are inhibited by DNA damage that promotes tight association of the protein with chromatin and its dissociation from the HERC2 E3 ligase. Finally, in agreement with a recent report, we find that XPA is post-translationally modified by acetylation. However, contrary to the previous claim, we find that in mouse liver only a small fraction of XPA is acetylated and that downregulation of SIRT1 deacetylase in two human cell lines does not affect the overall repair rate. Collectively, the data reveal that XPA is a limiting factor in excision repair and that its level is coordinately regulated by the circadian clock, the ubiquitin-proteasome system and DNA damage.

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Figure 1. Protein level of XPA is a determinant of the DNA excision repair activity. (A) Protein levels in human cells were analyzed by immunoblotting with the indicated antibodies from cells transfected with 100 nM siRNA targeting XPA or cyclophylin B (Control, CTRL). (B and C) Residual CPD (B) or (6–4)PP (C) damage in genomic DNA was detected by immunoslot blotting using damage specific monoclonal antibodies. Genomic DNA was prepared from cells transfected with indicated siRNAs for 24 h and repair was allowed for the indicated times. (D and E) Averages and error bars (standard deviations) are shown for data from two independent biological experiments and two technical repeats from each biological experiment.
Figure 1. Protein level of XPA is a determinant of the DNA excision repair activity. (A) Protein levels in human cells were analyzed by immunoblotting with the indicated antibodies from cells transfected with 100 nM siRNA targeting XPA or cyclophylin B (Control, CTRL). (B and C) Residual CPD (B) or (6–4)PP (C) damage in genomic DNA was detected by immunoslot blotting using damage specific monoclonal antibodies. Genomic DNA was prepared from cells transfected with indicated siRNAs for 24 h and repair was allowed for the indicated times. (D and E) Averages and error bars (standard deviations) are shown for data from two independent biological experiments and two technical repeats from each biological experiment.CC BY-NC · PubMed Central
Figure 2. Correlation between the XPA level and DNA excision repair activity in human cells. (A) Protein levels in NHF-1 cells transfected with different amounts of siRNA targeting XPA for 24 h were analyzed by immunoblotting with the indicated antibodies. CPB, cyclophilin B. (B and C) Residual (6–4)PP (B) or CPD (C) damage in genomic DNA was detected by immunoslot blotting using damage specific monoclonal antibodies. (D and E) Average repair values and standard deviations are shown for data from three independent experiments for the (6–4)PP (D) and CPD (E). (F and G) Initial repair rate for (6–4)PP (F) and CPD (G) were measured as a function of XPA protein level which was manipulated by knockdown of XPA using siRNA. The initial rates were calculated from the linear range of the kinetic data in D and E.
Figure 2. Correlation between the XPA level and DNA excision repair activity in human cells. (A) Protein levels in NHF-1 cells transfected with different amounts of siRNA targeting XPA for 24 h were analyzed by immunoblotting with the indicated antibodies. CPB, cyclophilin B. (B and C) Residual (6–4)PP (B) or CPD (C) damage in genomic DNA was detected by immunoslot blotting using damage specific monoclonal antibodies. (D and E) Average repair values and standard deviations are shown for data from three independent experiments for the (6–4)PP (D) and CPD (E). (F and G) Initial repair rate for (6–4)PP (F) and CPD (G) were measured as a function of XPA protein level which was manipulated by knockdown of XPA using siRNA. The initial rates were calculated from the linear range of the kinetic data in D and E.CC BY-NC · PubMed Central
Figure 3. Regulation of DNA excision repair by HERC2 via XPA stability control. (A) Protein levels were analyzed by immunoblotting with the indicated antibodies from A549 cells transfected with the indicated siRNAs for 96 h. Asterisk in the RNF8 blot indicates non-specific (NS) signal. CPB, cyclophilin B. (B) cDNA was prepared from cells transfected with the indicated siRNAs and was used for the amplification of the indicated genes using PCR. (C) HERC2 was immunoprecipitated from cells transfected with the indicated siRNAs and was used for the ubiquitination assay. Human Flag-XPA purified from 293T cells was used as a substrate. Total and ubiquitinated XPA were detected by immunoblotting with the anti-Flag antibody. The amount of HERC2 immunoprecipitated from each siRNA treated cell was analyzed by immunoblotting with the anti-HERC2 antibody. Asterisk in the Flag blot indicates NS signal. (D) Wild-type (WT) and mutant (Mut) HECT domain of HERC2 were transiently expressed in 293T cells, immuno-purified using Flag-agarose beads and used for the ubiquitination assay. Flag-XPA was used as a substrate. Total and ubiquitinated XPA were detected by immunoblotting with the anti-XPA antibody. The amount of HECT immunoprecipitated was analyzed by immunoblotting with anti-Flag antibody. (E) Residual (6–4)PP or CPD damage in the genomic DNA was detected by immunoslot blotting using damage specific monoclonal antibodies. (F) Average values and standard deviations from three independent experiments are shown. Protein levels were analyzed from cells transfected with siRNAs with the indicated antibodies (inset).
Figure 3. Regulation of DNA excision repair by HERC2 via XPA stability control. (A) Protein levels were analyzed by immunoblotting with the indicated antibodies from A549 cells transfected with the indicated siRNAs for 96 h. Asterisk in the RNF8 blot indicates non-specific (NS) signal. CPB, cyclophilin B. (B) cDNA was prepared from cells transfected with the indicated siRNAs and was used for the amplification of the indicated genes using PCR. (C) HERC2 was immunoprecipitated from cells transfected with the indicated siRNAs and was used for the ubiquitination assay. Human Flag-XPA purified from 293T cells was used as a substrate. Total and ubiquitinated XPA were detected by immunoblotting with the anti-Flag antibody. The amount of HERC2 immunoprecipitated from each siRNA treated cell was analyzed by immunoblotting with the anti-HERC2 antibody. Asterisk in the Flag blot indicates NS signal. (D) Wild-type (WT) and mutant (Mut) HECT domain of HERC2 were transiently expressed in 293T cells, immuno-purified using Flag-agarose beads and used for the ubiquitination assay. Flag-XPA was used as a substrate. Total and ubiquitinated XPA were detected by immunoblotting with the anti-XPA antibody. The amount of HECT immunoprecipitated was analyzed by immunoblotting with anti-Flag antibody. (E) Residual (6–4)PP or CPD damage in the genomic DNA was detected by immunoslot blotting using damage specific monoclonal antibodies. (F) Average values and standard deviations from three independent experiments are shown. Protein levels were analyzed from cells transfected with siRNAs with the indicated antibodies (inset).CC BY-NC · PubMed Central
Figure 4. Effect of DNA damage on the interaction of XPA and HERC2. (A) Protein levels were analyzed by immunoblotting with the indicated antibodies. A549 cells were treated with UV or mock irradiated and then times were allowed as indicated for repair in the presence of cycloheximide (CHX). (B) Quantitative analysis of the XPA expression from the blot shown in panel (A). Averages and standard deviations were plotted from three independent experiments. (C) cDNA was prepared from cells allowed to repair UV damage for the indicated times and was used for PCR amplification of the genes indicated. (D) UV treated A549 cells were allowed to repair for the indicated times and cells were fractionated into soluble and chromatin fractions. Equal amounts of soluble and chromatin fractions were analyzed by immunoblotting with the indicated antibodies. H2AX was used as a chromatin loading control. (E) HERC2 interaction with XPA was analyzed by immunoprecipitation of XPA from UV treated A549 cells followed by immunoblotting of HERC2. Average values and standard deviations from three independent experiments are shown. HERC2 levels are shown as arbitrary units (AU) with 100% defined as HERC2 in XPA complexes from mock-irradiated cells.
Figure 4. Effect of DNA damage on the interaction of XPA and HERC2. (A) Protein levels were analyzed by immunoblotting with the indicated antibodies. A549 cells were treated with UV or mock irradiated and then times were allowed as indicated for repair in the presence of cycloheximide (CHX). (B) Quantitative analysis of the XPA expression from the blot shown in panel (A). Averages and standard deviations were plotted from three independent experiments. (C) cDNA was prepared from cells allowed to repair UV damage for the indicated times and was used for PCR amplification of the genes indicated. (D) UV treated A549 cells were allowed to repair for the indicated times and cells were fractionated into soluble and chromatin fractions. Equal amounts of soluble and chromatin fractions were analyzed by immunoblotting with the indicated antibodies. H2AX was used as a chromatin loading control. (E) HERC2 interaction with XPA was analyzed by immunoprecipitation of XPA from UV treated A549 cells followed by immunoblotting of HERC2. Average values and standard deviations from three independent experiments are shown. HERC2 levels are shown as arbitrary units (AU) with 100% defined as HERC2 in XPA complexes from mock-irradiated cells.CC BY-NC · PubMed Central
Figure 5. Circadian oscillation of the acetylation of XPA. (A) Protein levels in liver cytosol and nuclear extracts were analyzed by immunoblotting with the indicated antibodies including histone H3 (H3). (B) Acetylated proteins from mouse liver cytosol and nuclear extracts were immunoprecipitated using acetyl-lysine antibody and the level of XPA was analyzed by immunoblotting for XPA. (C) Quantitative analysis of the acetylated XPA in the liver nuclear extract. Average values and standard deviations are shown from two independent experiments with two technical measurements on each together with XPA protein expression and DNA excision repair activity data from a previous report (14). (D) Acetylated proteins from mouse liver nuclear extracts were immunoprecipitated with anti-acetyl-lysine antibodies (1° IP). The immunodepleted-lysate from 1° IP was used for second round of immunoprecipitation (2° IP). The level of acetylated XPA from each IP was analyzed and compared with the input signal (10% of nuclear extracts used for IP) by immunoblotting for XPA.
Figure 5. Circadian oscillation of the acetylation of XPA. (A) Protein levels in liver cytosol and nuclear extracts were analyzed by immunoblotting with the indicated antibodies including histone H3 (H3). (B) Acetylated proteins from mouse liver cytosol and nuclear extracts were immunoprecipitated using acetyl-lysine antibody and the level of XPA was analyzed by immunoblotting for XPA. (C) Quantitative analysis of the acetylated XPA in the liver nuclear extract. Average values and standard deviations are shown from two independent experiments with two technical measurements on each together with XPA protein expression and DNA excision repair activity data from a previous report (14). (D) Acetylated proteins from mouse liver nuclear extracts were immunoprecipitated with anti-acetyl-lysine antibodies (1° IP). The immunodepleted-lysate from 1° IP was used for second round of immunoprecipitation (2° IP). The level of acetylated XPA from each IP was analyzed and compared with the input signal (10% of nuclear extracts used for IP) by immunoblotting for XPA.CC BY-NC · PubMed Central
Figure 6. Effect of SIRT1 on DNA excision repair. (A) Protein levels in the indicated cells transfected with the indicated siRNAs were analyzed by immunoblotting with the indicated antibodies. (B) Residual CPD damage in genomic DNA from cells transfected with siRNA for 36 h was detected by immunoslot blotting using damage specific monoclonal antibodies. (C) Quantitative analysis of data shown in B. Average values and standard deviations from two independent experiments with two technical repeats for each are plotted.
Figure 6. Effect of SIRT1 on DNA excision repair. (A) Protein levels in the indicated cells transfected with the indicated siRNAs were analyzed by immunoblotting with the indicated antibodies. (B) Residual CPD damage in genomic DNA from cells transfected with siRNA for 36 h was detected by immunoslot blotting using damage specific monoclonal antibodies. (C) Quantitative analysis of data shown in B. Average values and standard deviations from two independent experiments with two technical repeats for each are plotted.CC BY-NC · PubMed Central

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  1. Boyer, 1991, Cancer Research, 51, 2960
  2. Cleaver, 1995, Cancer Research, 55, 6152

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Metadata

Authors
Kang, T.-H., Reardon, J. T., & Sancar, A.
Affiliations
University of North Carolina at Chapel Hill (US)
Corresponding author
Sancar, A.
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2010-12-30
Volume
39
Issue
8
Pages
3176-3187
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gkq1318
PMC
PMC3082913
OpenAlex
W2116594363
Cited by
124
References
45
Citations by year
2026: 4 · 2025: 4 · 2024: 5 · 2023: 2 · 2022: 4 · 2021: 15 · 2020: 6 · 2019: 5 · 2018: 9 · 2017: 13 · 2016: 11 · 2015: 11 · 2014: 12 · 2013: 7 · 2012: 11
FWCI
2.99
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by-nc)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • Carolina Digital Repository (University of North Carolina at Chapel Hill) (submittedVersion)
  • UNC Libraries
Funders
  • National Institutes of Health
  • National Institute of General Medical Sciences (R01GM032833, R01GM031082, GM310.82, R37GM031082, GM32833)
  • National Institute of Environmental Health Sciences (P30ES010126)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Nuclear Structure and Function
Keywords
XPA, nucleotide excision repair, xeroderma pigmentosum, HERC2, E3 ubiquitin ligase, ubiquitination, protein stability, circadian clock, SIRT1, protein acetylation, DNA damage response, chromatin association, transcriptional regulation, post-transcriptional regulation, mouse liver
MeSH terms
Acetylation, Animals, Cell Line, DNA Damage, DNA Repair, Fibroblasts, Humans, Transcription, Genetic, Ubiquitin-Protein Ligases, Mice, Xeroderma Pigmentosum Group A Protein, Ubiquitination, Circadian Clocks

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