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Reactive oxygen species generated by thiopurine/UVA cause irreparable transcription-blocking DNA lesions.

Brem R, Li F, Karran P - Nucleic Acids Res. (2009)

Bottom Line: In vitro, 6-TG photoproducts, including the previously characterized guanine-6-sulfonate, in the transcribed DNA strand, are potent blocks to RNAPII transcription whereas 6-TG is only slightly inhibitory.In vivo, guanine-6-sulfonate is removed poorly from DNA and persists to a similar extent in the DNA of nucleotide excision repair-proficient and defective cells.Furthermore, transcription coupled repair-deficient Cockayne syndrome cells are not hypersensitive to UVA/6-TG, indicating that potentially lethal photoproducts are not selectively excised from transcribed DNA.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts, UK.

ABSTRACT
Long-term treatment with the anticancer and immunosuppressant thiopurines, azathioprine or 6-mercaptopurine, is associated with acute skin sensitivity to ultraviolet A (UVA) radiation and a high risk of skin cancer. 6-thioguanine (6-TG) that accumulates in the DNA of thiopurine-treated patients interacts with UVA to generate reactive oxygen species. These cause lethal and mutagenic DNA damage. Here we show that the UVA/DNA 6-TG interaction rapidly, and essentially irreversibly, inhibits transcription in cultured human cells and provokes polyubiquitylation of the major subunit of RNA polymerase II (RNAPII). In vitro, 6-TG photoproducts, including the previously characterized guanine-6-sulfonate, in the transcribed DNA strand, are potent blocks to RNAPII transcription whereas 6-TG is only slightly inhibitory. In vivo, guanine-6-sulfonate is removed poorly from DNA and persists to a similar extent in the DNA of nucleotide excision repair-proficient and defective cells. Furthermore, transcription coupled repair-deficient Cockayne syndrome cells are not hypersensitive to UVA/6-TG, indicating that potentially lethal photoproducts are not selectively excised from transcribed DNA. Since persistent transcription-blocking DNA lesions are associated with acute skin responses to sunlight and the development of skin cancer, our findings have implications for skin cancer in patients undergoing thiopurine therapy.

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Inhibition of RNAPII transcription in vitro by oxidised template 6-TG. (A) In vitro transcription set-up. The transcription system comprises a radiolabelled 9-mer RNA primer and two 124-mer DNA oligonucleotides. The 124-mer transcribed strands contain either a single G or 6-TG at position 87. (B) Oxidation products of 6-TG block in vitro transcription. Transcribed strand oligonucleotides were treated with MMPP or UVA prior to formation of the ternary complexes. These were supplemented with GTP, ATP and UTP and transcription was initiated by purified S. cerevisiae RNAPII in the presence or absence of CTP. Transcription products were analysed by gel electrophoresis.
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Figure 4: Inhibition of RNAPII transcription in vitro by oxidised template 6-TG. (A) In vitro transcription set-up. The transcription system comprises a radiolabelled 9-mer RNA primer and two 124-mer DNA oligonucleotides. The 124-mer transcribed strands contain either a single G or 6-TG at position 87. (B) Oxidation products of 6-TG block in vitro transcription. Transcribed strand oligonucleotides were treated with MMPP or UVA prior to formation of the ternary complexes. These were supplemented with GTP, ATP and UTP and transcription was initiated by purified S. cerevisiae RNAPII in the presence or absence of CTP. Transcription products were analysed by gel electrophoresis.

Mentions: UVA irradiation of 6-TG generates reactive oxygen species that can oxidize 6-TG to guanine sulfonate (GSO3). Since this photoproduct is a powerful block to DNA replication, we examined its effect on transcription. To do this, we used an established transcription system (19) with highly purified yeast RNAPII to address whether a single DNA GSO3 is sufficient to stall elongating transcription complexes. The active site of Rpb1 is 100% conserved from yeast to humans and both human and yeast RNAPIIs share a very similar subunit structure. Indeed, many human subunits can substitute for their yeast counterparts in a functional RNAPII (20). The in vitro transcription assay comprises RNAPII, a radiolabelled RNA primer, a nontranscribed 124-mer oligodeoxyribonucleotide and a transcribed 124-mer strand containing either a G or a 6-TG at position 87 (Figure 4A). To examine the effects of GSO3, the single 6-TG in the transcribed strand was selectively oxidized by treating the oligonucleotide with the mild oxidizing agent MMPP or with UVA. HPLC analysis of MMPP-treated oligonucleotides confirmed stoichiometric conversion of 6-TG to GSO3 (data not shown). Elongation-competent RNAPII/DNA/RNA ternary transcription complexes were assembled by annealing appropriate oligonucleotides and adding purified RNAPII. Elongation was started by the addition of nucleoside triphosphates. In the assembled complex, the 9-mer RNA primer places RNAPII 34 bases upstream of position 87. The sequence of the transcribed strand ensures that transcript elongation on undamaged DNA in the absence of CTP arrests at position 87 giving rise to an RNA product of 43 bases (Figure 4B, lane 2). In the presence of all four NTPs, the transcription run-off product is 80 nucleotides long (Figure 4B, lane 1). A single 6-TG at position 87 in the transcribed strand caused some reduction in the yield of run-off transcripts (Figure 4B, compare lanes 1 and 3). In contrast, GSO3 in the same position completely blocked transcript elongation. UVA irradiation of the 6-TG containing template induced a similar profound inhibition of transcription, even at the lowest dose of 20 kJ/m2. In each case, selective oxidation to GSO3 by MMPP or conversion to photoproducts by UVA, transcript termination occurred one base before the modified 6-TG. This is consistent with the inability of DNA GSO3 to form stable base pairs. As expected, MMPP treatment or UVA irradiation of the transcribed strand containing guanine had no detectable effect on transcription and levels of run-off products.Figure 4.


Reactive oxygen species generated by thiopurine/UVA cause irreparable transcription-blocking DNA lesions.

Brem R, Li F, Karran P - Nucleic Acids Res. (2009)

Inhibition of RNAPII transcription in vitro by oxidised template 6-TG. (A) In vitro transcription set-up. The transcription system comprises a radiolabelled 9-mer RNA primer and two 124-mer DNA oligonucleotides. The 124-mer transcribed strands contain either a single G or 6-TG at position 87. (B) Oxidation products of 6-TG block in vitro transcription. Transcribed strand oligonucleotides were treated with MMPP or UVA prior to formation of the ternary complexes. These were supplemented with GTP, ATP and UTP and transcription was initiated by purified S. cerevisiae RNAPII in the presence or absence of CTP. Transcription products were analysed by gel electrophoresis.
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Figure 4: Inhibition of RNAPII transcription in vitro by oxidised template 6-TG. (A) In vitro transcription set-up. The transcription system comprises a radiolabelled 9-mer RNA primer and two 124-mer DNA oligonucleotides. The 124-mer transcribed strands contain either a single G or 6-TG at position 87. (B) Oxidation products of 6-TG block in vitro transcription. Transcribed strand oligonucleotides were treated with MMPP or UVA prior to formation of the ternary complexes. These were supplemented with GTP, ATP and UTP and transcription was initiated by purified S. cerevisiae RNAPII in the presence or absence of CTP. Transcription products were analysed by gel electrophoresis.
Mentions: UVA irradiation of 6-TG generates reactive oxygen species that can oxidize 6-TG to guanine sulfonate (GSO3). Since this photoproduct is a powerful block to DNA replication, we examined its effect on transcription. To do this, we used an established transcription system (19) with highly purified yeast RNAPII to address whether a single DNA GSO3 is sufficient to stall elongating transcription complexes. The active site of Rpb1 is 100% conserved from yeast to humans and both human and yeast RNAPIIs share a very similar subunit structure. Indeed, many human subunits can substitute for their yeast counterparts in a functional RNAPII (20). The in vitro transcription assay comprises RNAPII, a radiolabelled RNA primer, a nontranscribed 124-mer oligodeoxyribonucleotide and a transcribed 124-mer strand containing either a G or a 6-TG at position 87 (Figure 4A). To examine the effects of GSO3, the single 6-TG in the transcribed strand was selectively oxidized by treating the oligonucleotide with the mild oxidizing agent MMPP or with UVA. HPLC analysis of MMPP-treated oligonucleotides confirmed stoichiometric conversion of 6-TG to GSO3 (data not shown). Elongation-competent RNAPII/DNA/RNA ternary transcription complexes were assembled by annealing appropriate oligonucleotides and adding purified RNAPII. Elongation was started by the addition of nucleoside triphosphates. In the assembled complex, the 9-mer RNA primer places RNAPII 34 bases upstream of position 87. The sequence of the transcribed strand ensures that transcript elongation on undamaged DNA in the absence of CTP arrests at position 87 giving rise to an RNA product of 43 bases (Figure 4B, lane 2). In the presence of all four NTPs, the transcription run-off product is 80 nucleotides long (Figure 4B, lane 1). A single 6-TG at position 87 in the transcribed strand caused some reduction in the yield of run-off transcripts (Figure 4B, compare lanes 1 and 3). In contrast, GSO3 in the same position completely blocked transcript elongation. UVA irradiation of the 6-TG containing template induced a similar profound inhibition of transcription, even at the lowest dose of 20 kJ/m2. In each case, selective oxidation to GSO3 by MMPP or conversion to photoproducts by UVA, transcript termination occurred one base before the modified 6-TG. This is consistent with the inability of DNA GSO3 to form stable base pairs. As expected, MMPP treatment or UVA irradiation of the transcribed strand containing guanine had no detectable effect on transcription and levels of run-off products.Figure 4.

Bottom Line: In vitro, 6-TG photoproducts, including the previously characterized guanine-6-sulfonate, in the transcribed DNA strand, are potent blocks to RNAPII transcription whereas 6-TG is only slightly inhibitory.In vivo, guanine-6-sulfonate is removed poorly from DNA and persists to a similar extent in the DNA of nucleotide excision repair-proficient and defective cells.Furthermore, transcription coupled repair-deficient Cockayne syndrome cells are not hypersensitive to UVA/6-TG, indicating that potentially lethal photoproducts are not selectively excised from transcribed DNA.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts, UK.

ABSTRACT
Long-term treatment with the anticancer and immunosuppressant thiopurines, azathioprine or 6-mercaptopurine, is associated with acute skin sensitivity to ultraviolet A (UVA) radiation and a high risk of skin cancer. 6-thioguanine (6-TG) that accumulates in the DNA of thiopurine-treated patients interacts with UVA to generate reactive oxygen species. These cause lethal and mutagenic DNA damage. Here we show that the UVA/DNA 6-TG interaction rapidly, and essentially irreversibly, inhibits transcription in cultured human cells and provokes polyubiquitylation of the major subunit of RNA polymerase II (RNAPII). In vitro, 6-TG photoproducts, including the previously characterized guanine-6-sulfonate, in the transcribed DNA strand, are potent blocks to RNAPII transcription whereas 6-TG is only slightly inhibitory. In vivo, guanine-6-sulfonate is removed poorly from DNA and persists to a similar extent in the DNA of nucleotide excision repair-proficient and defective cells. Furthermore, transcription coupled repair-deficient Cockayne syndrome cells are not hypersensitive to UVA/6-TG, indicating that potentially lethal photoproducts are not selectively excised from transcribed DNA. Since persistent transcription-blocking DNA lesions are associated with acute skin responses to sunlight and the development of skin cancer, our findings have implications for skin cancer in patients undergoing thiopurine therapy.

Show MeSH
Related in: MedlinePlus