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Behavior of T7 RNA Polymerase and Mammalian RNA Polymerase II at Site-specific Cisplatin Adducts in the Template DNA

2003; Elsevier BV; Volume: 278; Issue: 37 Linguagem: Inglês

10.1074/jbc.m305394200

ISSN

1083-351X

Autores

Silvia Tornaletti, Steve M. Patrick, John J. Turchi, Philip C. Hanawalt,

Tópico(s)

Bacterial Genetics and Biotechnology

Resumo

Transcription-coupled DNA repair is dedicated to the removal of DNA lesions from transcribed strands of expressed genes. RNA polymerase arrest at a lesion has been proposed as a sensitive signal for recruitment of repair enzymes to the lesion site. To understand how initiation of transcription-coupled repair may occur, we have characterized the properties of the transcription complex when it encounters a lesion in its path. Here we have compared the effect of cisplatin-induced intrastrand cross-links on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. We found that a single cisplatin 1,2-d(GG) intrastrand cross-link or a single cisplatin 1,3-d(GTG) intrastrand cross-link is a strong block to both polymerases. Furthermore, the efficiency of the block at a cisplatin 1,2-d(GG) intrastrand cross-link was similar in several different nucleotide sequence contexts. Interestingly, some blockage was also observed when the single cisplatin 1,3-d(GTG) intrastrand cross-link was located in the non-transcribed strand. Transcription complexes arrested at the cisplatin adducts were substrates for the transcript cleavage reaction mediated by the elongation factor TFIIS, indicating that the RNA polymerase II complexes arrested at these lesions are not released from template DNA. Addition of TFIIS yielded a population of transcripts up to 30 nucleotides shorter than those arrested at the lesion. In the presence of nucleoside triphosphates, these shortened transcripts could be re-elongated up to the site of the lesion, indicating that the arrested complexes are stable and competent to resume elongation. These results show that cisplatin-induced lesions in the transcribed DNA strand constitute a strong physical barrier to RNA polymerase progression, and they support current models of transcription arrest and initiation of transcription-coupled repair. Transcription-coupled DNA repair is dedicated to the removal of DNA lesions from transcribed strands of expressed genes. RNA polymerase arrest at a lesion has been proposed as a sensitive signal for recruitment of repair enzymes to the lesion site. To understand how initiation of transcription-coupled repair may occur, we have characterized the properties of the transcription complex when it encounters a lesion in its path. Here we have compared the effect of cisplatin-induced intrastrand cross-links on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. We found that a single cisplatin 1,2-d(GG) intrastrand cross-link or a single cisplatin 1,3-d(GTG) intrastrand cross-link is a strong block to both polymerases. Furthermore, the efficiency of the block at a cisplatin 1,2-d(GG) intrastrand cross-link was similar in several different nucleotide sequence contexts. Interestingly, some blockage was also observed when the single cisplatin 1,3-d(GTG) intrastrand cross-link was located in the non-transcribed strand. Transcription complexes arrested at the cisplatin adducts were substrates for the transcript cleavage reaction mediated by the elongation factor TFIIS, indicating that the RNA polymerase II complexes arrested at these lesions are not released from template DNA. Addition of TFIIS yielded a population of transcripts up to 30 nucleotides shorter than those arrested at the lesion. In the presence of nucleoside triphosphates, these shortened transcripts could be re-elongated up to the site of the lesion, indicating that the arrested complexes are stable and competent to resume elongation. These results show that cisplatin-induced lesions in the transcribed DNA strand constitute a strong physical barrier to RNA polymerase progression, and they support current models of transcription arrest and initiation of transcription-coupled repair. Transcription-coupled repair (TCR) 1The abbreviations used are: TCR, transcription-coupled repair; CPD, cyclobutane pyrimidine dimer; cisplatin, cis-diamminedichloroplatinum(II); RNAP, RNA polymerase; T7RNAP, T7RNA polymerase; RNAPII, RNA polymerase II; AdMLP, adenovirus major late promoter; BPDE, benzo[a]pyrene diol epoxide; cis 1,2-d(GG), cis-[Pt-(NH3)2{d(GpG)-N7(1),N7(2)}]; cis 1,3-d(GTG), cis-[Pt-(NH3)2{d(GpTpG)-N7(1),N7(3)}]; nt, nucleotide.1The abbreviations used are: TCR, transcription-coupled repair; CPD, cyclobutane pyrimidine dimer; cisplatin, cis-diamminedichloroplatinum(II); RNAP, RNA polymerase; T7RNAP, T7RNA polymerase; RNAPII, RNA polymerase II; AdMLP, adenovirus major late promoter; BPDE, benzo[a]pyrene diol epoxide; cis 1,2-d(GG), cis-[Pt-(NH3)2{d(GpG)-N7(1),N7(2)}]; cis 1,3-d(GTG), cis-[Pt-(NH3)2{d(GpTpG)-N7(1),N7(3)}]; nt, nucleotide. operates on DNA lesions located in the transcribed strands of expressed genes. Several lines of evidence indicate that an RNA polymerase in the elongating mode is required to initiate TCR. Induction of the lac operon of Escherichia coli is necessary to observe preferential repair of cyclobutane pyrimidine dimers (CPD) in the transcribed strand (1Mellon I. Hanawalt P.C. Nature. 1989; 342: 95-98Crossref PubMed Scopus (463) Google Scholar). Treatment of mammalian cells with α-amanitin to specifically inhibit RNA polymerase (RNAP) II elongation abolishes the preferential repair of CPDs in expressed genes (2Carreau M. Hunting D. Mutat. Res. 1992; 274: 57-64Crossref PubMed Scopus (37) Google Scholar, 3Christians F.C. Hanawalt P.C. Mutat. Res. 1992; 274: 93-101Crossref PubMed Scopus (91) Google Scholar). In yeast with temperature-sensitive mutations in the gene encoding a subunit of RNAPII, a loss of TCR is observed at the non-permissive temperature (4Sweder K.S. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10696-10700Crossref PubMed Scopus (160) Google Scholar). Mammalian ribosomal genes, transcribed by RNA polymerase I, are not preferentially repaired (5Vos J.M.H. Wauthier E.L. Mol. Cell. Biol. 1991; 11: 2245-2252Crossref PubMed Scopus (52) Google Scholar, 6Christians F.C. Hanawalt P.C. Biochemistry. 1993; 32: 10512-10518Crossref PubMed Scopus (74) Google Scholar, 7Fritz L.K. Smerdon M.J. Biochemistry. 1995; 34: 13117-13124Crossref PubMed Scopus (38) Google Scholar), although more recent studies suggest that in yeast there is TCR of ribosomal genes (8Conconi A. Bespalov V.A. Smerdon M.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 649-654Crossref PubMed Scopus (70) Google Scholar). Genes transcribed by RNA polymerase III are also not subject to TCR (9Dammann R. Pfeifer G.P. Mol. Cell. Biol. 1997; 17: 219-229Crossref PubMed Scopus (41) Google Scholar).A current model for TCR proposes that RNA polymerase arrested at a lesion in DNA constitutes a signal for the repair proteins to initiate repair. This model assumes that the polymerase must be removed from the damaged site to provide access for the repair complex to the lesion (10Mellon I. Bohr V.A. Smith C.A. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8878-8882Crossref PubMed Scopus (379) Google Scholar). In E. coli, the mfd gene product participates in this process (11Selby C.P. Sancar A. Science. 1993; 260: 53-58Crossref PubMed Scopus (560) Google Scholar). The Mfd protein can promote the release of the RNA polymerase and the incomplete transcript from the DNA template and then can target components of nucleotide excision repair to the site of transcription blockage (11Selby C.P. Sancar A. Science. 1993; 260: 53-58Crossref PubMed Scopus (560) Google Scholar, 12Park J.-S. Marr M.T. Roberts J. Cell. 2002; 109: 757-767Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). In human cells, the CSB gene product is implicated in this process. However, it remains unclear whether the polymerase is released or translocated away from the site of damage without dissociating from the template DNA (13Hanawalt P.C. Bohr V.A. Wasserman K. Kraemer K.H. Proceedings Alfred Benton Symposium 35 on DNA Repair Mechanisms. Munksgaard, Copenhagen1993: 231-242Google Scholar, 14Hanawalt P.C. Science. 1994; 266: 1957-1958Crossref PubMed Scopus (452) Google Scholar, 15Svejstrup J.Q. Nat. Rev. Mol. Cell Biol. 2002; 3: 21-29Crossref PubMed Scopus (305) Google Scholar).As a first step in elucidating how initiation of TCR occurs, we have characterized the properties of the transcription complex when it encounters a lesion. The analysis of different types of arrested complexes should help us understand how an RNA polymerase arrested at a lesion signals the repair proteins to initiate a repair event. Previously, we have shown that a CPD, located in the transcribed strand of template DNA in different sequence contexts, is an absolute block to transcription elongation by mammalian RNAPII (16Donahue B.A. Yin S. Taylor J.-S. Reines D. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8502-8506Crossref PubMed Scopus (308) Google Scholar, 17Tornaletti S. Donahue B.A. Reines D. Hanawalt P.C. J. Biol. Chem. 1997; 272: 31719-31724Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 18Tornaletti S. Reines D. Hanawalt P.C. J. Biol. Chem. 1999; 274: 24124-24130Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 19Kalogeraki V.S. Tornaletti S. Hanawalt P.C. J. Biol. Chem. 2003; 278: 19558-19564Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The arrested complexes are stable (16Donahue B.A. Yin S. Taylor J.-S. Reines D. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8502-8506Crossref PubMed Scopus (308) Google Scholar, 20Selby C.P. Drapkin R. Reinberg D. Sancar A. Nucleic Acids Res. 1997; 25: 787-793Crossref PubMed Scopus (154) Google Scholar) and competent to resume elongation after reversal of the lesion by the repair enzyme photolyase (18Tornaletti S. Reines D. Hanawalt P.C. J. Biol. Chem. 1999; 274: 24124-24130Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar).Here we describe the effect of cisplatin-induced intrastrand cross-links on transcription elongation by T7 RNA polymerase (T7 RNAP) and mammalian RNAPII from rat liver. cis-Diamminedichloroplatinum (II) (cisplatin) preferentially reacts with purine bases in the DNA in vitro and in vivo to form the cis-[Pt-(NH3)2{d(GpG)-N7(1),N7(2)}] (cis 1,2-d(GG)), with a frequency of 65%, the cis-[Pt-(NH3)2{d(ApG)-N7(1),N7(2)}], with a frequency of 25%, the cis-[Pt-(NH3)2{d(GpTpG)-N7-(1),N7(3)}] (cis 1,3-d(GTG)) with a frequency of 5–10%, and a small percentage of interstrand cross-links and monofunctional adducts (21Kartalou M. Essigmann J.M. Mutat. Res. 2001; 478: 1-21Crossref PubMed Scopus (329) Google Scholar). Cisplatin-induced adducts are repaired by global genomic nucleotide excision repair and by TCR (22Jones J.C. Zhen W. Reed E. Parker R.J. Sancar A. Bohr V. J. Biol. Chem. 1991; 266: 7101-7107Abstract Full Text PDF PubMed Google Scholar, 23May A. Nairn R.S. Okumoto D.S. Wassermann K. Stevnsner T. Jones J.C. Bohr V. J. Biol. Chem. 1993; 268: 1650-1657Abstract Full Text PDF PubMed Google Scholar, 24Zhen W. Evans M.K. Haggerty C.M. Bohr V.A. Carcinogenesis. 1993; 14: 919-924Crossref PubMed Scopus (44) Google Scholar). These adducts may impose a more serious problem for an elongating RNA polymerase compared with a CPD, because they cause substantial unwinding and bending of the DNA helix (reviewed in Ref. 21Kartalou M. Essigmann J.M. Mutat. Res. 2001; 478: 1-21Crossref PubMed Scopus (329) Google Scholar). These lesions have been shown to block transcription by T3, E. coli, and wheat germ RNAP (25Corda Y. Job C. Anin M.-F. Leng M. Job D. Biochemistry. 1991; 30: 222-230Crossref PubMed Scopus (79) Google Scholar, 26Corda Y. Job C. Anin M.-F. Leng M. Job D. Biochemistry. 1993; 32: 8582-8588Crossref PubMed Scopus (71) Google Scholar, 27Cullinane C. Mazur S.J. Essigmann J.M. Phillips D.R. Bohr V.A. Biochemistry. 1999; 38: 6204-6212Crossref PubMed Scopus (75) Google Scholar). The 1,3-d(GTG) intrastrand cross-link also blocks RNAPII transcription in extracts of human cells. In addition, the presence of cisplatin-induced lesions in plasmids transfected into human or hamster cells almost completely inhibits RNAPII transcription of a reporter gene (28Mello J.A. Lippard S.J. Essigmann J.M. Biochemistry. 1995; 34: 14783-14791Crossref PubMed Scopus (95) Google Scholar).To study the effect of a single cis 1,2-d(GG) or a single cis 1,3-d(GTG) on transcription, we have developed an in vitro transcription system consisting of DNA substrates containing a single cis 1,2-d(GG) in two different sequence contexts and a single cis 1,3-d(GTG) located in the transcribed or the non-transcribed strand downstream of the T7 promoter or the adenovirus major late promoter (AdMLP), with purified T7 RNAP or rat liver RNAPII and initiation factors, respectively. We show that a single cis 1,2-d(GG) or a single cis 1,3-d(GTG) located in the transcribed strand is a strong block to both T7 RNAP and RNAPII. Furthermore, the efficiency of the block at a cis 1,2-d(GG) is not affected by the sequence context around the lesion. Interestingly, we also observed partial blockage when a single cis 1,3-d(GTG) was located in the non-transcribed strand. The arrested RNAPII complex was stable, as indicated by the ability of elongation factor TFIIS to induce transcript cleavage, producing a population of transcripts up to 30 nts shorter than those arrested at the lesion, which could then be re-elongated up to the lesion when the nucleoside triphosphate precursors were added.EXPERIMENTAL PROCEDURESProteins and Reagents—T7 RNAP was purchased from Promega. RNAPII, transcription initiation factors, and elongation factor TFIIS, purified from rat liver or recombinant sources as described previously (29Gu W. Reines D. J. Biol. Chem. 1995; 270: 11238-11244Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), were obtained from Dr. Daniel Reines (Emory University, Atlanta, GA). T4 polynucleotide kinase and T4 DNA ligase were obtained from Invitrogen. E. coli strain MV1184 was a gift of Dr. Joachim Messing (Rutgers University, Piscataway, NJ). D44 IgG anti-RNA antibodies (30Eilat D. Hochberg M. Fischel R. Laskov R. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 3818-3822Crossref PubMed Scopus (42) Google Scholar) were purified from rodent ascites fluid as described previously (31Reines D. J. Biol. Chem. 1991; 266: 10510-10517Abstract Full Text PDF PubMed Google Scholar). Highly purified NTPs and radiolabeled nucleotides were purchased from Amersham Biosciences. Formalin-fixed Staphylococcus aureus was obtained from Calbiochem. Custom-made DNA oligonucleotides were obtained from Qiagen (Chatsworth, CA) or Integrated DNA Technologies (Coralville, IA).Preparation of DNA Oligonucleotides Containing a Single Platinated Adduct—DNA oligonucleotides of sequence 5′-TCTTCTTCTGTGCACTCTTCTTCT-3′(GTG), 5′-CTTCTCTTCTGGCCTTCTCT-3′ (GG1), and 5′-TCTTCTTCTAGGCCTTCTTCTTCT3′ (GG2) were incubated with a 3:1 ratio of cisplatin to GG or GTG sites in 1 mm NaHPO4 (pH 7.5) and 3 mm NaCl for 16 h at 37 °C in the dark as described (32Turchi J. Patrick S. Henkels K. Biochemistry. 1997; 36: 7586-7593Crossref PubMed Scopus (41) Google Scholar). After annealing to a complementary DNA, the presence of the lesion was confirmed by resistance of the modified oligonucleotides to digestion with restriction enzymes ApaLI or HaeIII.DNA Templates for Transcription—GG-adducted DNA templates used for transcription reactions with T7 RNAP consisted of 159-bp DNA fragments containing a single GG adduct in the transcribed (GG1TST7) (Fig. 1A) or in the non-transcribed (GGNTST7) strand downstream of the T7 promoter. These substrates were constructed from 8 oligonucleotides, 5 in the damage-containing strand and 3 in the opposite strand (Fig. 1A) (33Shi Y.-b. Gamper H. Hearst J. J. Biol. Chem. 1988; 263: 527-534Abstract Full Text PDF PubMed Google Scholar). 50 pmol of each oligonucleotide were phosphorylated with T4 polynucleotide kinase. The mixture was heated for 3 min at 95 °C and slowly cooled to room temperature. ATP to a final concentration of 1 mm and 10 units of T4 DNA ligase were added to the mixture containing 50 mm Tris-HCl, pH 7.6, 10 mm MgCl2, 1 mm dithiothreitol, and 5 mm polyethyleneglycol-8000. The DNA was ligated overnight at 16 °C. The ligation products were treated with proteinase K and further purified by ethanol precipitation. The DNA samples were resuspended in formamide dye. The single-stranded 159-nt DNA fragments were purified by electrophoresis on an 8% denaturing polyacrylamide gel. To ensure that all samples were double-stranded after reannealing of the complementary strands, the DNA was digested with appropriate restriction enzymes and, if necessary, further purified by electrophoresis on an 8% non-denaturing gel. GTG-adducted DNA templates used for transcription reactions with T7 RNAP consisted of HindIII linearized plasmid DNA or a 1160-bp ApaLI-HindIII DNA fragment containing a single cis 1,3-d(GTG) downstream of the T7 promoter and the AdMLP (Fig. 1C). The presence of a single cis 1,3-d(GTG) or a single cis 1,2-d(GG) in the T7 and RNAPII DNA templates was confirmed by resistance to cleavage by the restriction enzymes ApaLI, HaeIII, or StuI (data not shown).DNA templates used for transcription reactions with RNAPII consisted of HindIII linearized plasmid DNA containing a single cis 1,2-d(GG) or a single cis 1,3-d(GTG) downstream of the AdMLP (Fig. 1, B and C). To separate molecules containing a single cis 1,3-d(GTG) from undamaged molecules, HindIII-digested substrates were further treated with ApaLI followed by purification of an 1160-bp fragment containing the lesion from an agarose gel.To construct plasmids to receive cisplatin-adducted oligonucleotides, oligomers with the sequence 5′-TCGAGTCTTCTTCTGTGCACTCTTCTTCTG-3′, 5′-TCGAGCTTCTCTTCTGGCCTTCTCTG-3′, and 5′-TCGAGCTTCTCTTCAGGCCTTCTCTG-3′ were annealed to the complementary strand and ligated to a BamHI fragment from pUCTgTS (34Tornaletti S. Maeda L.S. Lloyd D.R. Reines D. Hanawalt P.C. J. Biol. Chem. 2001; 276: 45367-45371Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) to yield pUCGTG-TS, pUCGG1-TS, or pUCGG2-TS, or they were ligated to a BamHI fragment of pUCTgNTS (34Tornaletti S. Maeda L.S. Lloyd D.R. Reines D. Hanawalt P.C. J. Biol. Chem. 2001; 276: 45367-45371Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) to yield pUCGTG-NTS, pUCGG1-NTS, or pUCGG2-NTS. These plasmids were transformed into the F′ E. coli strain MV1184 to produce single-stranded DNA for primer extension, as described (18Tornaletti S. Reines D. Hanawalt P.C. J. Biol. Chem. 1999; 274: 24124-24130Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar).Covalently closed circular DNA containing a single GTG or a single GG on either the transcribed or the non-transcribed strand was generated by priming 10 μg of plus strand of pUCGTG-TS, pUCGG1-TS, and pUCGG2-TS or pUCGTG-NTS, pUCGG1-NTS, and pUCGG2-NTS with a 5-fold molar excess of GTG- or GG-containing oligonucleotide phosphorylated at the 5′ end in a 300-μl reaction mixture containing 10 mm Tris-HCl (pH 7.9), 50 mm NaCl, 10 mm MgCl2, 1 mm dithiothreitol, 600 μm each of dATP, dCTP, dTTP, and dGTP, 1 mm ATP, 30 units of T4 DNA polymerase, and 5 units of T4 DNA ligase. Covalently closed circular molecules were purified after electrophoresis in an agarose gel containing 0.3 μg/ml ethidium bromide. Under our conditions, covalently closed circular DNA migrated as supercoiled DNA and could be resolved from single-stranded closed circular and nicked double-stranded plasmids. Closed circular DNA molecules containing GG2 were further separated from those lacking a site-specific lesion by digestion with StuI followed by purification from agarose gels.T7 RNAP Transcription Reactions—DNA templates were incubated for 5 min. at 37 °C in a 10-μl mixture containing 40 mm Tris-HCl (pH 7.9), 6 mm MgCl2, 2 mm spermidine, 10 mm dithiothreitol, 1 μm [α-32P]-GTP (800 Ci/mmol), 100 μm ATP, 212 units of RNasin, and 50 units of T7 RNAP. Elongation proceeded until T7 RNAP reached nucleotide 7, at which time the first UTP was required for incorporation. Heparin was added to prevent further initiation, and then 100 μm each of CTP, UTP, and GTP were added to allow elongation to continue, typically for 30 min. Reactions were stopped with SDS and proteinase K, and nucleic acids were precipitated with ethanol. Samples were resuspended in formamide loading dye, heat-denatured, and electrophoresed through an 8% polyacrylamide gel in TBE (89 mm Tris, 89 mm boric acid, 1 mm EDTA, pH 8.0) containing 8.3 m urea. Gels were dried and autoradiographed using intensifying screens. Transcripts were quantified by using a Bio-Rad GS-363 phosphorimaging device. All transcripts were labeled up to nucleotide 6, making quantitation independent of their subsequent length and G content.RNAPII Transcription Reactions—DNA templates were incubated for 30 min at 28 °C with rat liver protein fractions D (2 μg, containing TFIID and TFIIH) and rat liver RNAPII (0.5 μg) in a 20-μl mixture containing 20 mm Hepes-NaOH, pH 7.9, 20 mm Tris-HCl, pH 7.9, 2.2% polyvinyl alcohol, 212 units of RNasin, 0.5 mg/ml acetylated bovine serum albumin, 150 mm KCl, 2 mm dithiothreitol, and 3% glycerol. After incubation, 33 μl of a solution containing fraction B′ (1 μg, containing TFIIF and TFIIE) and recombinant rat TFIIB (3 ng) in the same buffer without KCl were added, and incubation continued for 20 min. at 28 °C to form preinitiation complexes. 7 mm MgCl2, 20 μm ATP, 20 μm UTP, and 0.8 μm of [α-32P]CTP (800 Ci/mmol) were added, and incubation continued for 20 min. Elongation proceeded until RNAPII reached nucleotide 15, at which time the first GTP was required for incorporation. Heparin was added to prevent further initiation, and then 800 μm each of ATP, CTP, UTP, and GTP was added to allow elongation to continue, typically for 15 min. Elongation complexes were immunoprecipitated with D44 anti-RNA antibodies and formalin-fixed S. aureus and then washed three times in reaction buffer containing 20 mm Tris-HCl, pH 7.9, 3 mm Hepes-NaOH, pH 7.9, 60 mm KCl, 0.5 mm EDTA, 2 mm dithiothreitol, 0.2 mg/ml acetylated bovine serum albumin, 2.2% (w/v) polyvinyl alcohol. Washed complexes were resuspended in 60 μl of reaction buffer for further treatment. For TFIIS-mediated transcript cleavage, arrested complexes were incubated with TFIIS for 1hat28 °Cin60 μl of reaction buffer containing 7 mm MgCl2. Reactions were stopped with SDS and proteinase K, and nucleic acids were precipitated with ethanol. Samples were resuspended in formamide loading dye, heat-denatured, and electrophoresed through a 6% polyacrylamide gel in TBE (89 mm Tris, 89 mm boric acid, 1 mm EDTA, pH 8.0) with 8.3 m urea. Gels were dried and autoradiographed using intensifying screens.RESULTSEffect of a Single Cisplatin Intrastrand Cross-link in the Transcribed or Non-transcribed Strand of Template DNA on Transcription Elongation by T7 RNAP and Mammalian RNA-PII—DNA substrates containing a single platinated adduct in the transcribed or non-transcribed strand downstream of the T7 promoter or the AdMLP were constructed as described previously (34Tornaletti S. Maeda L.S. Lloyd D.R. Reines D. Hanawalt P.C. J. Biol. Chem. 2001; 276: 45367-45371Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The presence of the lesion in either strand was confirmed by resistance to cleavage with restriction enzymes HaeIII, StuI, or ApaLI that cleave the DNA substrates at the site of the lesion (data not shown). T7 RNAP or RNAPII was stalled downstream of the T7 promoter or AdMLP, respectively, after synthesis of a short 32P-labeled RNA, followed by the addition of heparin to prevent further initiation. As a result, the transcription products represented a single promoter-dependent elongation event (35Viswanathan A. Doetsch P.W. J. Biol. Chem. 1998; 273: 21276-21281Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). All 4 NTPs were then added to allow elongation to continue. In this transcription system, repair of the lesion cannot occur because of a lack of repair proteins. The effect of either lesion on transcription was then monitored as formation of transcripts shorter than those observed with the undamaged template. We found that when a cis 1,3-d(GTG) was located in the transcribed strand, 70% of transcripts produced after T7 RNAP transcription were shorter than the full-length RNA present in the control (Fig. 2, lane 3). Comparison of the size of these transcripts with those obtained from an undamaged template digested at the site of the lesion with ApaLI indicated that these RNAs were extended up to the site of the cis 1,3-d(GTG) (Fig. 2, lane 7). To rule out the possibility that the full-length RNA originated from some undamaged template contaminating the DNA preparation, an ApaLI-HindIII fragment containing the lesion was further purified from the HindIII-digested plasmid previously utilized as a DNA substrate. This fragment originates from resistance to cleavage at the damage-containing site and therefore, contains 100% lesion. We also found that with this DNA template, some full-length transcripts were observed (Fig. 2, lane 5), confirming that the full-length RNA we observed previously was indeed due to lesion bypass. Interestingly, a cis 1,3-d(GTG) located in the non-transcribed strand produced 10% of transcripts shorter than the full-length RNA (Fig. 2, lanes 4 and 6); these were extended up to the site of the lesion (Fig. 2, lane 8). Similarly, a cis 1,2-d(GG) in the transcribed strand in two sequence contexts blocked transcription by T7 RNAP, as indicated by formation of 90% of transcripts shorter than the full-length product (Fig. 3, lanes 3 and 7). A small fraction of transcripts arrested around the lesion was also observed; it was likely the product of nucleotide addition or loss by T7 RNAP (36Jacques J.P. Kolakofsky D. Genes Dev. 1991; 5: 707-713Crossref PubMed Scopus (89) Google Scholar). This lesion in the non-transcribed strand was bypassed by T7 RNAP (Fig. 3, lanes 4 and 8).Fig. 2Effect of a single cis 1,3-d(GTG) on transcription by T7 RNAP. DNA templates were transcribed in vitro such that transcripts were labeled with 32P as described in the text. Elongation was allowed to proceed for 30 min after the addition of NTPs to the reaction mixture. RNA was isolated and electrophoresed through an 8% denaturing polyacrylamide gel. Lanes 1 and 2, unadducted templates (C); lanes 3–4, 5–6, templates containing a specific cis 1,3-d(GTG) in the transcribed or in the non-transcribed strand, respectively; lanes 7 and 8, unadducted templates digested with ApaLI. The position of the full-length runoff transcript is indicated by RO; transcripts arrested at a cis 1,3-d(GTG) are indicated by GTG. TS, transcribed strand; NTS, non-transcribed strand.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3The sequence context of the lesion does not affect the extent of T7 RNAP blockage at a single cis 1,2-d(GG) intrastrand cross-link. DNA templates were transcribed in vitro as described in Fig. 2. RNA was isolated and electrophoresed through an 8% denaturing polyacrylamide gel. Lanes 1–2 and 5–6, unadducted templates (C); lanes 3–4 and 7–8, templates containing a specific cis 1,2-d(GG) in the transcribed or in the non-transcribed strand, respectively. The position of the full-length runoff transcript is indicated by RO; transcripts arrested at a cis 1,2-d(GG) are indicated by GG. TS, transcribed strand; NTS, non-transcribed strand; M, 10-bp ladder.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To study the effect of a platinated lesion on transcription by RNAPII, we used an in vitro reconstituted system containing purified RNAPII and initiation factors. When a cis 1,3-d(GTG) was in the transcribed strand, 90% of the transcripts were shorter than the full-length RNA. Comparison of these RNAs with those obtained after transcription of an ApaLI-digested undamaged DNA indicated that these transcripts were arrested at the lesion (Fig. 4, lanes 3 and 5). Similarly, when a cis 1,2-d(GG) was located in the transcribed strand in the sequence contexts 5′-CTGGCC-3′ or 5′-TAGGCC-3′, most of the transcripts produced arrested at the lesion (Figs. 5A and 6), with a readthrough frequency of up to 10% (Fig. 5B) and 5%, respectively. When a cis 1,3-d(GTG) was in the non-transcribed strand, 15% of the transcripts were arrested at a lesion (Fig. 4, lane 4). However, a cis 1,2-d(GG) in the non-transcribed strand was completely bypassed (data not shown).Fig. 4Effect of a single cis 1,3-d(GTG) on transcription by RNAPII. Templates were transcribed in vitro such that transcripts were labeled with 32P as described in the text. Elongation was allowed for 15 min after the addition of NTPs to the reaction mixture. RNA was then isolated and electrophoresed through a 5% polyacrylamide gel. Lanes 1 and 2, unadducted templates (C). Lanes 3 and 4, templates containing a single cis 1,3-d(GTG) in the transcribed or in the non-transcribed strand, respectively; transcripts arrested at a cis 1,3-d(GTG) are indicated by GTG. Lanes 5 and 6, unadducted templates digested with ApaLI. RO, full-length runoff transcript; M, 10-bp ladder.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Time course of RNAPII transcription of templates containing a single cis 1,2-d(GG) in the sequence context 5′-CTGGCC-3′. A, templates containin

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