Transcription Arrest at a Lesion in the Transcribed DNA Strand in Vitro Is Not Affected by a Nearby Lesion in the Opposite Strand*

Cis-syn cyclobutane pyrimidine dimers (CPDs) are the most frequently formed lesions in UV-irradiated DNA. CPDs are repaired by the nucleotide excision repair pathway. Additionally, they are subject to transcription-coupled DNA repair. In the general model for transcription-coupled DNA repair, an RNA polymerase arrested at a lesion on the transcribed DNA strand facilitates repair by recruiting the repair machinery to the site of the lesion. Consistent with this model, transcription experiments in vitro have shown that CPDs in the transcribed DNA strand interfere with the translocation of prokaryotic and eukaryotic RNA polymerases. Here, we study the behavior of RNA polymerase when transcribing a template that contains two closely spaced lesions, one on each DNA strand. Similar DNA templates containing no CPD, or a single CPD on either the transcribed or the nontranscribed strand were used as controls. Using an in vitro transcription system with purified T7 RNA polymerase (T7 RNAP) or rat liver RNAP II, we characterized transcript length and efficiency of transcription in vitro. We also tested the sensitivity of the arrested RNAP II-DNA-RNA ternary complex, at a CPD in the transcribed strand, to transcription factor TFIIS. The presence of a nearby CPD in the nontranscribed strand did not affect the behavior of either RNA polymerase nor did it affect the reverse translocation ability of the RNAP II-arrested complex. Our results additionally indicate that the sequence context of a CPD affects the efficiency of T7 RNAP arrest more significantly than that of RNAP II.

[1]  B. Hingerty,et al.  DNA adducts from a tumorigenic metabolite of benzo[a]pyrene block human RNA polymerase II elongation in a sequence- and stereochemistry-dependent manner. , 2002, Journal of molecular biology.

[2]  J. Svejstrup.  Transcription: Mechanisms of transcription-coupled DNA repair , 2002, Nature Reviews Molecular Cell Biology.

[3]  P. Hanawalt,et al.  Effect of Thymine Glycol on Transcription Elongation by T7 RNA Polymerase and Mammalian RNA Polymerase II* , 2001, The Journal of Biological Chemistry.

[4]  C. Martin,et al.  Fluorescence characterization of the transcription bubble in elongation complexes of T7 RNA polymerase. , 2001, Journal of molecular biology.

[5]  S. Amin,et al.  Bacteriophage T7 RNA polymerase transcription elongation is inhibited by site-specific, stereospecific benzo[c]phenanthrene diol epoxide DNA lesions. , 2001, Biochemistry.

[6]  P. Cramer,et al.  Structural Basis of Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution , 2001, Science.

[7]  D. Reines,et al.  Transcription elongation factor SII , 2000, BioEssays : news and reviews in molecular, cellular and developmental biology.

[8]  T. Steitz,et al.  Insights into transcription: structure and function of single-subunit DNA-dependent RNA polymerases. , 2000, Current opinion in structural biology.

[9]  P. Hanawalt,et al.  Structural Characterization of RNA Polymerase II Complexes Arrested by a Cyclobutane Pyrimidine Dimer in the Transcribed Strand of Template DNA* , 1999, The Journal of Biological Chemistry.

[10]  J. Essigmann,et al.  Inhibition of RNA polymerase II transcription in human cell extracts by cisplatin DNA damage. , 1999, Biochemistry.

[11]  P. Hanawalt,et al.  Effect of DNA lesions on transcription elongation. , 1999, Biochimie.

[12]  JohnB . Taylor,et al.  The Ability of a Variety of Polymerases to Synthesize Past Site-specific cis-syn, trans-syn-II, (6–4), and Dewar Photoproducts of Thymidylyl-(3′→5′)-thymidine* , 1998, The Journal of Biological Chemistry.

[13]  P. Hanawalt,et al.  Nucleotide Sequence Context Effect of a Cyclobutane Pyrimidine Dimer upon RNA Polymerase II Transcription* , 1997, The Journal of Biological Chemistry.

[14]  J. Taylor,et al.  Solid phase-supported thymine dimers for the construction of dimer-containing DNA by combined chemical and enzymatic synthesis: a potentially general method for the efficient incorporation of modified nucleotides into DNA. , 1997, Nucleic acids research.

[15]  Z. Wang,et al.  DNA damage-dependent transcriptional arrest and termination of RNA polymerase II elongation complexes in DNA template containing HIV-1 promoter. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[16]  D. Reinberg,et al.  RNA polymerase II stalled at a thymine dimer: footprint and effect on excision repair. , 1997, Nucleic acids research.

[17]  P. Hanawalt,et al.  Effects of Aminofluorene and Acetylaminofluorene DNA Adducts on Transcriptional Elongation by RNA Polymerase II (*) , 1996, The Journal of Biological Chemistry.

[18]  Wei Zhou,et al.  T7 RNA polymerase bypass of large gaps on the template strand reveals a critical role of the nontemplate strand in elongation , 1995, Cell.

[19]  P. Hanawalt.  Transcription-coupled repair and human disease. , 1994, Science.

[20]  P. Hanawalt,et al.  Transcript cleavage by RNA polymerase II arrested by a cyclobutane pyrimidine dimer in the DNA template. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[21]  G. P. Beardsley,et al.  Effects of arabinosylcytosine-substituted DNA on DNA/RNA hybrid stability and transcription by T7 RNA polymerase. , 1994, Biochemistry.

[22]  S. Wallace,et al.  Oxidative DNA Lesions as Blocks to in Vitro Transcription by Phage T7 RNA Polymerase , 1994, Annals of the New York Academy of Sciences.

[23]  N. Geacintov,et al.  Site-specific benzo[a]pyrene diol epoxide-DNA adducts inhibit transcription elongation by bacteriophage T7 RNA polymerase. , 1994, Biochemistry.

[24]  C. Job,et al.  Spectrum of DNA--platinum adduct recognition by prokaryotic and eukaryotic DNA-dependent RNA polymerases. , 1993, Biochemistry.

[25]  A. Sancar,et al.  Molecular mechanism of transcription-repair coupling. , 1993, Science.

[26]  D. Bogenhagen,et al.  Effects of DNA lesions on transcription elongation by T7 RNA polymerase. , 1993, The Journal of biological chemistry.

[27]  D. Lawrence,et al.  Strand-selective repair of DNA damage in the yeast GAL7 gene requires RNA polymerase II. , 1992, The Journal of biological chemistry.

[28]  P. Hanawalt,et al.  Preferential repair of cyclobutane pyrimidine dimers in the transcribed strand of a gene in yeast chromosomes and plasmids is dependent on transcription. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[29]  D. Luse,et al.  The RNA polymerase II ternary complex cleaves the nascent transcript in a 3'----5' direction in the presence of elongation factor SII. , 1992, Genes & development.

[30]  D. Reines.  RNA polymerase II elongation complex. Elongation complexes purified using an anti-RNA antibody do not contain initiation factor alpha. , 1991, The Journal of biological chemistry.

[31]  D. Kolakofsky,et al.  Pseudo-templated transcription in prokaryotic and eukaryotic organisms. , 1991, Genes & development.

[32]  C. Job,et al.  Transcription by eucaryotic and procaryotic RNA polymerases of DNA modified at a d(GG) or a d(AG) site by the antitumor drug cis-diamminedichloroplatinum(II). , 1991, Biochemistry.

[33]  P. Hanawalt,et al.  Induction of the Escherichia coli lactose operon selectively increases repair of its transcribed DNA strand , 1989, Nature.

[34]  J. Taylor,et al.  Synthesis of a trans-syn thymine dimer building block. Solid phase synthesis of CGTAT[t,s]TATGC. , 1988, Nucleic acids research.

[35]  J. Hearst,et al.  Interaction of T7 RNA polymerase with DNA in an elongation complex arrested at a specific psoralen adduct site. , 1988, The Journal of biological chemistry.

[36]  P. Hanawalt,et al.  Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene , 1987, Cell.

[37]  M. Khan,et al.  Genetic activity of trimethoprim in the Salmonella/microsomal screening system. , 1987, Mutation research.

[38]  P. Hanawalt,et al.  Preferential DNA repair of an active gene in human cells. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[39]  C. Y. Wang,et al.  Mutagenicity of the anticancer drug, caracemide, and related compounds for salmonella. , 1986, Mutation research.

[40]  R. Laskov,et al.  Antibodies to RNA from autoimmune NZB/NZW mice recognize a similar antigenic determinant and show a large idiotypic diversity. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[41]  P. Hanawalt,et al.  Sensitive determination of pyrimidine dimers in DNA of UV-irradiated mammalian cells. Introduction of T4 endonuclease V into frozen and thawed cells. , 1981, Mutation research.

[42]  M. O. Bradley,et al.  DNA double-strand breaks induced in normal human cells during the repair of ultraviolet light damage. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[43]  W. Haseltine,et al.  Comparison of the cleavage of pyrimidine dimers by the bacteriophage T4 and Micrococcus luteus UV-specific endonucleases. , 1980, The Journal of biological chemistry.

[44]  T. Bonura,et al.  QUANTITATIVE EVIDENCE FOR ENZYMATICALLY‐INDUCED DNA DOUBLE‐STRAND BREAKS AS LETHAL LESIONS IN UV IRRADIATED pol+ AND polAl STRAINS OF E. COLI K‐12 , 1975, Photochemistry and photobiology.

[45]  K. Minton,et al.  Letter: Evidence for clustering of pyrimidine dimers on opposite strands of U.V.-irradiated bacteriophage DNA. , 1974, International journal of radiation biology and related studies in physics, chemistry, and medicine.