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[13] Assays for Transcription Elongation by RNA Polymerase II Using Oligo(dC)‐Tailed Template with Single DNA Damage By ISAO KURAOKA and KIYOJI TANAKA Abstract
A DNA molecule is vulnerable to many types of DNA‐damaging agents of endogenous and environmental origins. Although damage to DNA can interfere not only with replication but also transcription, the majority of DNA repair and mutagenesis studies are based on the actions of DNA polymerases in DNA replication. To investigate the actions of RNA polymerase II (RNAPII) encountering a single DNA lesion on transcription elongation, we employ a transcription elongation assay using purified RNAPII and oligo (dC)‐tailed templates containing a DNA lesion at a specific site. This chapter describes an analysis of whether elongating RNAPII stalls at a DNA lesion or whether RNAPII generates mutations in RNA transcripts. Introduction
When elongating RNA polymerase II (RNAPII) encounters DNA lesions on the transcribed strand in living cells, it basically has to choose between two actions (Doetsch, 2002; Tornaletti, 2005). One action is to stall at the DNA lesion. It is thought that this stalling recruits other DNA repair factors to remove the lesion. If DNA repair (maybe transcription‐ coupled DNA repair; TCR) does not occur to remove the lesion, which blocks transcription elongation, the stalling of RNAPII continues. Consequently, the cell cannot produce the mRNA transcripts and, if the transcripts are essential, the cell will die. The second action is to bypass the lesion and continue to synthesize the mRNA transcript. Even if RNAPII can bypass a lesion to produce the transcript, the process results in ribonucleotide misincorpration and will generate mutant transcripts. When there is one mutation in the termination codon of an mRNA transcript, the transcript will be subjected to nonsense‐mediated mRNA decay (Conti and Izaurralde, 2005; Lejeune and Maquat, 2005). However, when there is no mutation in the termination codon, the mutant transcript generated by damaged transcribed strands may be an important source of mutant proteins, particularly in nondividing cells. Therefore, DNA lesions on transcribed strand are a major challenge to RNAPII transcription. METHODS IN ENZYMOLOGY, VOL. 408 Copyright 2006, Elsevier Inc. All rights reserved.
0076-6879/06 $35.00 DOI: 10.1016/S0076-6879(06)08013-X
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To analyze the direct actions of RNAPII on a DNA lesion on the transcribed strand, we employed an in vitro transcription elongation assay using purified RNAPII and an oligo(dC)‐tailed template containing a single DNA lesion on the transcribed strand at a specific site (Kuraoka et al., 2003; Mei Kwei et al., 2004). This assay can be performed in the absence of any other transcription factors (e.g., in vitro transcription assays require TBP, TFIIB, THIIE, THIIF, and TFIIH) and does not need a specific promoter (Yamaguchi et al., 1999). Thus, there is little protein contamination and also few additional effects of other transcription factors on RNAPII in this assay. Only the enzymatic actions of RNAPII on damaged templates are observed. This chapter describes the generation of an oligo(dC)‐tailed template containing a DNA lesion on the transcribed strand at a specific site, an assay for RNAPII transcription elongation, and a technique for the analysis of RNA transcripts generated from damaged templates. Construction of Closed‐Circular Duplex DNA Substrates Containing a DNA Lesion at a Specific Site
To generate an oligo(dC)‐tailed template, closed‐circular duplex DNA (cccDNA) substrates containing a DNA lesion have to be prepared (Moggs et al., 1996; Shivji et al., 1999). An oligonucleotide containing a DNA lesion of interest is annealed to single‐stranded pBluescript DNA modified to obtain a sequence complementary to the oligonucleotide. The cccDNA substrates are synthesized by T4 DNA polymerases and T4 DNA ligases and purified by EtBr‐CsCl density gradient centrifugation. The DNA lesion is located on the transcribed strand at a specific site. Solutions and Materials Gel‐purified 24‐mer oligonucleotides containing a DNA lesion (several DNA lesions phophoramidite are available at GlenResearch) Single‐stranded pBluescript DNAs are prepared as described (Sambrook and Russell, 2001) 1. For the annealing reaction, a reaction mixture is set up in 200‐l aliquots containing 50 pmol of single‐stranded pBluescript DNA, 50 pmol of 50 ‐phosphorylated oligonucleotide containing a DNA lesion, and 20 l of 10 T4 DNA polymerase buffer (TaKaRa). Incubate the mixture at 70 for 5 min, 37 for 30 min, 25 for 20 min, and 4 for 20 min. 2. Add 30 l of 10 T4 DNA polymerase buffer, 50 l of 0.1% bovine serum albumin (BSA) (TaKaRa), 5 l of 100 mM ATP (Amersham
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Biosciences), 100 l of 2.5 mM dNTP (TaKaRa), 25 l of T4 DNA polymerase (4 U/l, TaKaRa), 10 l of T4 DNA ligase (350 U/l, TaKaRa), and 80 l of H2O. Incubate at 37 for 4 h. 3. Purify the synthesized closed‐circular duplex DNA substrates by EtBr‐CsCl density gradients with a final CsCl concentration of 1.55 g/ml. Dissolve 15 g of CsCl in 9.5 ml of H2O at 37 for 1 h. Mix 500 l of DNA sample, 1.352 ml of CsCl solution, and 148 l of a 10‐mg/mL EtBr solution (Invitrogen) in a 2‐ml Quick‐Seal tube. Perform EtBr/CsCl density gradient centrifugation at 90,000 rpm in a TLA 120.2 (Beckman) ultracentrifuge rotor at 18 for 16 h. 4. Use a handheld UV lamp (312 nm) to visualize the DNA. Collect the lower band of closed‐circular duplex DNA with a 21‐gauge hypodermic needle and 1‐ml syringe in a 1.5‐ml tube. Add an equal volume of isoamyl alcohol, vortex for 5 s, and centrifuge at 15,000 rpm for 1 min at room temperature. Remove and discard the upper phase (pink color). Repeat the extractions until no trace of pink remains. Dilute the DNA solution to 4.0 ml with 10 mM Tris–HCl (pH 8.5). 5. Add the sample to an Amicon Ultra‐4 Centrifugal 30K filter device unit, and centrifuge at 4000g for 20 min at 4 to concentrate the DNA to 50 l. Add more 10 mM Tris–HCl (pH 8.5) to 4 ml and repeat this step four more times. Quantify the final DNA solution using a spectrophotometer and confirm DNA purity. When there are restriction sites within a DNA lesion site, the presence of the lesion can be confirmed by digestion with a restriction endonuclease. Store the purified DNA in aliquots at 80 . Preparation of an Oligo(dc)‐Tailed Template from Closed‐Circular Duplex DNA Substrates
For transcription elongation by RNAPII, an oligo(dC)‐tailed template containing a lesion at a specific site on the transcribed strand is generated using EtBr/CsCl density gradient‐purified cccDNA substrates. To generate an oligo(dC) tail for RNAPII, we constructed a vector: pBluescript KS– GTG (Kuraoka et al., 2003). In our system, this vector is designed to generate 130 nucleotide RNA transcripts from a start point of transcription to the damaged region. Purified cccDNA substrates containing a lesion are digested with PstI, and the oligo(dC) tail is added to the 30 end by terminal deoxynucleotide transferase. After digestion with SmaI, the oligo (dC)‐tailed templates containing a lesion are purified. 1. A reaction mixture is set up in 50‐l aliquots. Add 5 l of 10 restriction endonuclease buffer (TOYOBO) and 5 l of PstI restriction endonuclease (TOYOBO) to 40 l of a DNA solution containing the cccDNA (5 pmol) with a lesion at a specific site.
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2. Incubate at 37 for 60 min. Add 50 l of phenol–chloroform–isoamyl alcohol, vortex for 1 min, and centrifuge at 15,000 rpm for 5 min at room temperature. Transfer 45 l of the upper phase to a new tube. Add 2 l of pellet paint Co‐Precipitant (Novagen) and 5 l of 3 M sodium acetate and 125 l of 100% ethanol. Incubate for 2 min at room temperature and centrifuge at 15,000 rpm for 5 min at 4 . Remove the ethanol solution carefully to retain the DNA pellets and rinse 100 l of 70% ethanol. Dry the DNA pellets. 3. Resuspend the DNA solution with 35 l of H2O and add 5 l of 10 buffer (Amersham Bioscience), 1 mM dCTP (Amersham Bioscience), and 5 l of terminal deoxynucleotide transferase (Amersham Bioscience). Repeat step 2. 4. Resuspend the DNA solution with 40 l of H2O and add 5 l of 10 restriction endonuclease buffer (TOYOBO) and 5 l of SmaI restriction endonuclease (TOYOBO). Incubate at 37 for 60 min, add 10 l of 6 gel loading buffer (TOYOBO), and then purify 3‐kbp products using 1% agarose gel electrophoresis. Dilute the DNA to a concentration of 10 ng/l and store the aliquots at 4 in the dark. RNAPII Elongation Reaction Using Oligo(dC)‐Tailed Template
Figure 1 shows an outline of the assay for RNAPII transcription elongation using an oligo(dC)‐tailed template containing a DNA lesion at a specific site. Hot Labeling Reaction (Fig. 1A)
To investigate whether RNAPII stalls at a lesion of interest, we employ a hot labeling reaction using 32P‐UTP and a denatured 6% polyacrylamide gel. When RNAPII stalls, 130 nucleotide RNA transcripts are observed, generated from the transcription start site to the damaged site in our system (Fig. 1, top). When RNAPII bypasses the lesion, elongated transcripts are observed giving products of around 3 kbp on the denatured gel. Solutions and Materials The RNAPII fraction is prepared from HeLa cells as described elsewhere (Hasegawa et al., 2003; Usuda et al., 1991) or purified RNAPII can be purchased from ProteinOne. NE()buffer: 20 mM Tris–HCl (pH 8.0), 20% glycerol, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol NE(þ) buffer: NE() buffer containing 12.5 mM MgCl2 X50 ACGUTP mixture: 2.5 mM ATP, 2.5 mM CTP, 2.5 mM GTP, 0.5 mM UTP (Amersham Bioscience)
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FIG. 1. Flow diagram of the analysis of RNAPII actions in transcription elongation using an oligo(dC)‐tailed template containing a DNA lesion at a specific site.
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Stop buffer: 7 M urea, 0.35 M NaCl, 10 mM Tris–HCl (pH 7.4), 10 mM EDTA, 1% SDS 1. Reaction mixtures are set up in 25‐l aliquots. Prepare DNA solutions, Pol II solutions, and NTP solutions as follows. DNA solutions (10 l): 1 l of 10 ng/l of oligo(dC)‐tailed template containing a lesion, 0.12 l of 1 M MgCl2, 2 l of 40% PEG, and 6.88 l of H2O. Pol II solutions (4 L): 1 l of RNAPII fraction (100 ng/l), 0.1 l of 50 mg/ml BSA (Nacalai), 0.2 l of ribonuclease inhibitor (RNaseIn: Ambion: 20 U/l), and 2.7 l of NE() buffer. NTP solutions (5 l): 0.5 l of 32P‐UTP (Amersham Biosciences), 0.5 l of 50 ACGUTP mixture, 4 l of NE(þ) buffer, and 5 l of H2O. 2. Add 20 l of DNA solution, 8 l of Pol II solution, and 12 l of NE() buffer to a fresh RNase‐free tube. 3. Preincubate the reaction mixtures at 30 for 30 min. Add 5 l of NTP solution to the reactions. (The elongation reaction will be started by this step.) Incubate for a further 2–60 min. 4. Add 100 l of stop buffer to the reaction tube, and mix with 100 l of phenol–chloroform–isoamyl alcohol. Vortex for 5 min and centrifuge for 5 min at maximum speed. 5. Carefully collect 90 l of the aqueous phase and transfer it to a fresh tube containing 2 l of yeast tRNA (50 g/ml), and 10 l of 3 M sodium acetate. Mix in 250 l of ice‐cold 100% ethanol and then place on dry ice for 20 min. 6. Centrifuge at maximum speed for 15 min at 4 and carefully remove the alcohol. Add 100 l of 70% ethanol and centrifuge at maximum speed for 5 min at 4 . 7. Carefully remove the alcohol and dry the RNA pellet in a centrifuge under vacuum for 5 min. Resuspend the RNA pellet in 5 l of formamide loading dye (USB). 8. Heat the samples at 95 for 2 min and load onto a denatured 6% polyacrylamide gel. 9. Transfer the gel to 3MM paper and dry the gel. Visualize by exposing the gel to X‐ray film or with a FUJIFILM BAS 2500 bioimage analyzer. An example of the type of image is shown in Fig. 1A. Cold Labeling Reaction
Cold labeling reactions are employed to analyze RNA transcripts produced from oligo(dC)‐tailed templates containing a DNA lesion by RNAPII. When RNAPII bypasses a lesion, it may misincorporate nucleotides opposite a lesion. For this, we sequence DNA fragments produced by RT‐PCR using bypassed transcription products (Fig. 1B). However, when
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RNAPII stalls at a DNA lesion, RNAPII‐stalled RNA transcripts are generated. To investigate whether the stalled RNAPII has the ability to incorporate nucleotides, we use 30 ligation‐mediated RT‐PCR to isolate and sequence the stalled transcripts (Fig. 1C). Preparation of RNA Transcripts from RNAPII Transcription Elongation Assay Using Oligo(dC)‐Tailed Template
Solutions and Materials Basically, we use the same reagents as in the hot labeling reaction. 1. Reaction mixtures are set up in 50‐l aliquots. Prepare DNA solutions, Pol II solutions, and NTP solutions as follows. DNA solutions (20 l): 2 l of 10 ng/l of oligo(dC) template containing a lesion, 0.24 l of 1 M MgCl2, 4 l of 40% PEG, and 13.76 l of H2O. Pol II solutions (8 l): 2 l of RNAPII fraction (100 ng/l), 0.2 l of 50 mg/ml BSA, 0.4 l of RNaseIn, and 5.4 l of NE() buffer. NTP solutions (10 l): 1 l of 50 ACGUTP mixture, 0.5 l of 2 mM UTP (Amersham Bioscience), 4 l of NE(þ) buffer, and 5 l of H2O. 2. Add 20 l of DNA solution, 8 l of Pol II solution, and 12 l of NE() buffer to a fresh RNase‐free tube. 3. Preincubate the reaction mixtures at 30 for 30 min. Add 10 l of NTP solution to the reaction. Incubate for a further 20–60 min to accumulate RNA transcripts. 4. Add 100 l of stop buffer to the reaction tubes. 5. Purify transcripts of RNA using an RNeasy minikit (Qiagen). At present, there is no way to purify RNA completely free of DNA, even if the DNA is not visible on an agarose gel. Therefore, we treat the purified RNA transcripts with RNase‐free DNase I. 6. Reaction mixtures are set up in 20‐l aliquots. Add 2 l of 10 DNase buffer (TaKaRa), 0.25 l of RNaseIn (40 units/l), 0.1 l of RNase‐ free DNase I (5 Kunitz units/l, TaKaRa), and 17.65 l of the purified RNA sample (must be less than 1 g). 7. Incubate at 37 for 30 min. Add 2 l of 50 mM EDTA. Incubate at 65 for 5 min. 8. Store the DNase I‐treated RNAs in aliquots at 20 or 80 . Analysis of Lesion‐Bypassed RNAPII Elongation Transcripts (Fig. 1B)
To examine the nucleotide preference for incorporation opposite a DNA lesion, DNA fragments produced by RT‐PCR from RNA transcripts are sequenced. It is important to have good experimental conditions
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because there is a possibility that the PCR itself will generate a mutation in the RNA and that the PCR may also produce excess DNA fragments from contaminated template DNAs. Reverse‐Transcription Reaction for First‐Strand CDNA Synthesis and PCR
1. Reaction mixtures are set up in 12‐l aliquots. Add 1 l of proper primer (10 pmol/l), 2 l of the DNase I‐treated RNA solution, and 9 l of RNase‐free H2O. 2. Incubate at 65 for 5 min and place on ice. 3. Add 4 l of 5 RT buffer (TOYOBO), 2 l of 10 mM dNTP solution, 1 l of ribonuclease inhibitor (10 units/l, TOYOBO), and 1 l of reverse transcriptase (4 units/l, TOYOBO). 4. Incubate at 42 for 60 min and at 85 for 5 min. 5. Store the first‐stranded cDNA in aliquots at 20 or 80 . 6. PCR mixtures are set up in 100‐l aliquots. Add 4 l of the RT solution or the mock solution, 10 l of 10 PCR buffer (TaKaRa), 8 l of 2.5 mM dNTP mixture, 6 l of 25 mM MgCl2, 0.5 l of primer A (100 pmol/ l), 0.5 l of primer B (100 pmol/l), and 0.5 l of Taq polymerase (5 U/l, TaKaRa). Also perform a mock reaction using DNase I‐treated RNA solutions without a reverse transcription reaction. 7. Perform 25, 30, and 35 cycles: 94 for 30 s, 55 for 30 s, and 72 for 30 s. Check PCR products on the 2% agarose gel. Here, it is important to decide on good PCR conditions (maybe PCR cycles). The PCR products should be detected in the RT solutions, but not the mock solution, because any PCR products of the mock solution must be from contaminated DNA templates. Usually, we use less than 30 cycles (see Fig. 1B, 2% agarose gel). 8. Purify the PCR products from the agarose gel. Sequence the products. Mutations should be detected at sites of DNA lesions. Analysis of 30 Ligation‐Mediated RT‐PCR (Fig. 1C)
To analyze the 30 end of RNAPII‐stalled transcripts on damaged templates, the transcripts must first be ligated with RNA oligonucleotides. The RNA oligonucleotide has 50 ‐phosphorylated residues for the ligation reaction and 30 NH3 residues for preventing self‐ligation and ligation with the 50 of transcripts. The ligated transcripts are amplified by RT‐PCR, subcloned into the plasmid vector, and then introduced into competent Escherichia coli cells. Sequence data of the plasmid DNA indicate that RNAPII incorporated nucleotides opposite the lesion (Mei Kwei et al., 2004).
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Materials RNA linker: the 50 end of RNA oligonucleotides is phosphorylated and the 30 end is NH2 modified. 1. Reaction mixtures are set up in 50‐l aliquots. Add 5 l of the DNase I‐treated RNA solution, 1 l of RNA linker (20 pmol/l), 3 l of 0.1% BSA (TaKaRa), 25 l of 50% PEG 8000, 5 l of 10 T4 RNA ligase buffer (TaKaRa), 1 l of T4 RNA ligase (40 U/l, TaKaRa), and 10 l of H2O. 2. Incubate at 16 for 14–16 h. 3. After ligation, incubate at 95 for 3 min. 4. Use 1.4 l of the ligated transcript solution and a Qiagen OneStep RT‐PCR kit (Qiagen). Follow the manufacturer’s instructions using primers specific for the 30 ‐ligated RNA transcripts. Perform a RT reaction: 50 for 30 min, 94 for 30 s, and PCR for 25 cycles: 94 for 30 s, 55 for 30 s, and 72 for 30 s. 5. Add 10 l of 6 gel‐loading buffer to reaction mixtures. Run the 2% agarose gel in 1 TAE buffer and purify the PCR products using the QIAquick gel extraction kit (Qiagen). 6. Ligate the purified PCR products into a TOPO TA cloning vector (Invitrogen) and transform competent E. coli cells. 7. Sequence the isolated plasmids. Sequencing data before the anti‐ RNA linker indicates the position of the 30 end of RNAPII‐stalled transcripts. Acknowledgments We thank the past and present members of our laboratory, including Joan Seah Mei Kwei, Katsuyoshi Horibata, Mika Hayashida, and Kyoko Suzuki, for their contributions to the methods described here.
References Conti, E., and Izaurralde, E. (2005). Nonsense‐mediated mRNA decay: Molecular insights and mechanistic variations across species. Curr. Opin. Cell Biol. 17, 316–325. Doetsch, P. W. (2002). Translesion synthesis by RNA polymerases: Occurrence and biological implications for transcriptional mutagenesis. Mutat. Res. 510, 131–140. Hasegawa, J., Endou, M., Narita, T., Yamada, T., Yamaguchi, Y., Wada, T., and Handa, H. (2003). A rapid purification method for human RNA polymerase II by two‐step affinity chromatography. J. Biochem. (Tokyo) 133, 133–138. Kuraoka, I., Endou, M., Yamaguchi, Y., Wada, T., Handa, H., and Tanaka, K. (2003). Effects of endogenous DNA base lesions on transcription elongation by mammalian RNA polymerase II. Implications for transcription‐coupled DNA repair and transcriptional mutagenesis. J. Biol. Chem. 278, 7294–7299.
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Lejeune, F., and Maquat, L. E. (2005). Mechanistic links between nonsense‐mediated mRNA decay and pre‐mRNA splicing in mammalian cells. Curr. Opin. Cell Biol. 17, 309–315. Mei Kwei, J. S., Kuraoka, I., Horibata, K., Ubukata, M., Kobatake, E., Iwai, S., Handa, H., and Tanaka, K. (2004). Blockage of RNA polymerase II at a cyclobutane pyrimidine dimer and 6–4 photoproduct. Biochem. Biophys. Res. Commun. 320, 1133–1138. Moggs, J. G., Yarema, K. J., Essigmann, J. M., and Wood, R. D. (1996). Analysis of incision sites produced by human cell extracts and purified proteins during nucleotide excision repair of a 1,3‐intrastrand d(GpTpG)‐cisplatin adduct. J. Biol. Chem. 271, 7177–7186. Sambrook, J., and Russell, D. W. (2001). ‘‘Molecular Cloning: A Laboratory Manual,’’ 3rd Ed., pp. 3.30–3.48. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Shivji, M. K., Moggs, J. G., Kuraoka, I., and Wood, R. D. (1999). Dual‐incision assays for nucleotide excision repair using DNA with a lesion at a specific site. Methods Mol. Biol. 113, 373–392. Tornaletti, S. (2005). Transcription arrest at DNA damage sites. Mutat. Res. Usuda, Y., Kubota, A., Berk, A. J., and Handa, H. (1991). Affinity purification of transcription factor IIA from HeLa cell nuclear extracts. EMBO J. 10, 2305–2310. Yamaguchi, Y., Takagi, T., Wada, T., Yano, K., Furuya, A., Sugimoto, S., Hasegawa, J., and Handa, H. (1999). NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation. Cell 97, 41–51.
[14] In Vivo Assays for Transcription‐Coupled Repair By GRACIELA SPIVAK, GERD P. PFEIFER , and PHILIP HANAWALT Abstract
This chapter describes the technologies used in our respective laboratories to study the incidence and repair of lesions induced in specific DNA sequences by ultraviolet light, chemical carcinogens, and products of cellular metabolism. The Southern blot method is suitable for analysis of damage and repair in the individual DNA strands of specific restriction fragments up to 25,000 nucleotides in length, whereas the ligation‐ mediated polymerase chain reaction approach permits analysis of shorter sequences at the nucleotide level. Both methods have unique advantages and limitations for particular applications. Introduction
In addition to the general excision repair pathways, there are dedicated mechanisms for the removal of some types of lesions from actively transcribed DNA. Transcription‐coupled repair (TCR), a specialized pathway for repair in the transcribed strands of active genes, has been demonstrated unequivocally for bulky DNA lesions such as cyclobutane pyrimidine METHODS IN ENZYMOLOGY, VOL. 408 Copyright 2006, Elsevier Inc. All rights reserved.
0076-6879/06 $35.00 DOI: 10.1016/S0076-6879(06)08014-1
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DNA repair, part A
[13] Assays for Transcription Elongation by RNA Polymerase II Using Oligo(dC)‐Tailed Template with Single DNA Damage By ISAO KURAOKA and KIYOJI TANAKA Abstract
A DNA molecule is vulnerable to many types of DNA‐damaging agents of endogenous and environmental origins. Although damage to DNA can interfere not only with replication but also transcription, the majority of DNA repair and mutagenesis studies are based on the actions of DNA polymerases in DNA replication. To investigate the actions of RNA polymerase II (RNAPII) encountering a single DNA lesion on transcription elongation, we employ a transcription elongation assay using purified RNAPII and oligo (dC)‐tailed templates containing a DNA lesion at a specific site. This chapter describes an analysis of whether elongating RNAPII stalls at a DNA lesion or whether RNAPII generates mutations in RNA transcripts. Introduction
When elongating RNA polymerase II (RNAPII) encounters DNA lesions on the transcribed strand in living cells, it basically has to choose between two actions (Doetsch, 2002; Tornaletti, 2005). One action is to stall at the DNA lesion. It is thought that this stalling recruits other DNA repair factors to remove the lesion. If DNA repair (maybe transcription‐ coupled DNA repair; TCR) does not occur to remove the lesion, which blocks transcription elongation, the stalling of RNAPII continues. Consequently, the cell cannot produce the mRNA transcripts and, if the transcripts are essential, the cell will die. The second action is to bypass the lesion and continue to synthesize the mRNA transcript. Even if RNAPII can bypass a lesion to produce the transcript, the process results in ribonucleotide misincorpration and will generate mutant transcripts. When there is one mutation in the termination codon of an mRNA transcript, the transcript will be subjected to nonsense‐mediated mRNA decay (Conti and Izaurralde, 2005; Lejeune and Maquat, 2005). However, when there is no mutation in the termination codon, the mutant transcript generated by damaged transcribed strands may be an important source of mutant proteins, particularly in nondividing cells. Therefore, DNA lesions on transcribed strand are a major challenge to RNAPII transcription. METHODS IN ENZYMOLOGY, VOL. 408 Copyright 2006, Elsevier Inc. All rights reserved.
0076-6879/06 $35.00 DOI: 10.1016/S0076-6879(06)08013-X
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To analyze the direct actions of RNAPII on a DNA lesion on the transcribed strand, we employed an in vitro transcription elongation assay using purified RNAPII and an oligo(dC)‐tailed template containing a single DNA lesion on the transcribed strand at a specific site (Kuraoka et al., 2003; Mei Kwei et al., 2004). This assay can be performed in the absence of any other transcription factors (e.g., in vitro transcription assays require TBP, TFIIB, THIIE, THIIF, and TFIIH) and does not need a specific promoter (Yamaguchi et al., 1999). Thus, there is little protein contamination and also few additional effects of other transcription factors on RNAPII in this assay. Only the enzymatic actions of RNAPII on damaged templates are observed. This chapter describes the generation of an oligo(dC)‐tailed template containing a DNA lesion on the transcribed strand at a specific site, an assay for RNAPII transcription elongation, and a technique for the analysis of RNA transcripts generated from damaged templates. Construction of Closed‐Circular Duplex DNA Substrates Containing a DNA Lesion at a Specific Site
To generate an oligo(dC)‐tailed template, closed‐circular duplex DNA (cccDNA) substrates containing a DNA lesion have to be prepared (Moggs et al., 1996; Shivji et al., 1999). An oligonucleotide containing a DNA lesion of interest is annealed to single‐stranded pBluescript DNA modified to obtain a sequence complementary to the oligonucleotide. The cccDNA substrates are synthesized by T4 DNA polymerases and T4 DNA ligases and purified by EtBr‐CsCl density gradient centrifugation. The DNA lesion is located on the transcribed strand at a specific site. Solutions and Materials Gel‐purified 24‐mer oligonucleotides containing a DNA lesion (several DNA lesions phophoramidite are available at GlenResearch) Single‐stranded pBluescript DNAs are prepared as described (Sambrook and Russell, 2001) 1. For the annealing reaction, a reaction mixture is set up in 200‐l aliquots containing 50 pmol of single‐stranded pBluescript DNA, 50 pmol of 50 ‐phosphorylated oligonucleotide containing a DNA lesion, and 20 l of 10 T4 DNA polymerase buffer (TaKaRa). Incubate the mixture at 70 for 5 min, 37 for 30 min, 25 for 20 min, and 4 for 20 min. 2. Add 30 l of 10 T4 DNA polymerase buffer, 50 l of 0.1% bovine serum albumin (BSA) (TaKaRa), 5 l of 100 mM ATP (Amersham
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Biosciences), 100 l of 2.5 mM dNTP (TaKaRa), 25 l of T4 DNA polymerase (4 U/l, TaKaRa), 10 l of T4 DNA ligase (350 U/l, TaKaRa), and 80 l of H2O. Incubate at 37 for 4 h. 3. Purify the synthesized closed‐circular duplex DNA substrates by EtBr‐CsCl density gradients with a final CsCl concentration of 1.55 g/ml. Dissolve 15 g of CsCl in 9.5 ml of H2O at 37 for 1 h. Mix 500 l of DNA sample, 1.352 ml of CsCl solution, and 148 l of a 10‐mg/mL EtBr solution (Invitrogen) in a 2‐ml Quick‐Seal tube. Perform EtBr/CsCl density gradient centrifugation at 90,000 rpm in a TLA 120.2 (Beckman) ultracentrifuge rotor at 18 for 16 h. 4. Use a handheld UV lamp (312 nm) to visualize the DNA. Collect the lower band of closed‐circular duplex DNA with a 21‐gauge hypodermic needle and 1‐ml syringe in a 1.5‐ml tube. Add an equal volume of isoamyl alcohol, vortex for 5 s, and centrifuge at 15,000 rpm for 1 min at room temperature. Remove and discard the upper phase (pink color). Repeat the extractions until no trace of pink remains. Dilute the DNA solution to 4.0 ml with 10 mM Tris–HCl (pH 8.5). 5. Add the sample to an Amicon Ultra‐4 Centrifugal 30K filter device unit, and centrifuge at 4000g for 20 min at 4 to concentrate the DNA to 50 l. Add more 10 mM Tris–HCl (pH 8.5) to 4 ml and repeat this step four more times. Quantify the final DNA solution using a spectrophotometer and confirm DNA purity. When there are restriction sites within a DNA lesion site, the presence of the lesion can be confirmed by digestion with a restriction endonuclease. Store the purified DNA in aliquots at 80 . Preparation of an Oligo(dc)‐Tailed Template from Closed‐Circular Duplex DNA Substrates
For transcription elongation by RNAPII, an oligo(dC)‐tailed template containing a lesion at a specific site on the transcribed strand is generated using EtBr/CsCl density gradient‐purified cccDNA substrates. To generate an oligo(dC) tail for RNAPII, we constructed a vector: pBluescript KS– GTG (Kuraoka et al., 2003). In our system, this vector is designed to generate 130 nucleotide RNA transcripts from a start point of transcription to the damaged region. Purified cccDNA substrates containing a lesion are digested with PstI, and the oligo(dC) tail is added to the 30 end by terminal deoxynucleotide transferase. After digestion with SmaI, the oligo (dC)‐tailed templates containing a lesion are purified. 1. A reaction mixture is set up in 50‐l aliquots. Add 5 l of 10 restriction endonuclease buffer (TOYOBO) and 5 l of PstI restriction endonuclease (TOYOBO) to 40 l of a DNA solution containing the cccDNA (5 pmol) with a lesion at a specific site.
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2. Incubate at 37 for 60 min. Add 50 l of phenol–chloroform–isoamyl alcohol, vortex for 1 min, and centrifuge at 15,000 rpm for 5 min at room temperature. Transfer 45 l of the upper phase to a new tube. Add 2 l of pellet paint Co‐Precipitant (Novagen) and 5 l of 3 M sodium acetate and 125 l of 100% ethanol. Incubate for 2 min at room temperature and centrifuge at 15,000 rpm for 5 min at 4 . Remove the ethanol solution carefully to retain the DNA pellets and rinse 100 l of 70% ethanol. Dry the DNA pellets. 3. Resuspend the DNA solution with 35 l of H2O and add 5 l of 10 buffer (Amersham Bioscience), 1 mM dCTP (Amersham Bioscience), and 5 l of terminal deoxynucleotide transferase (Amersham Bioscience). Repeat step 2. 4. Resuspend the DNA solution with 40 l of H2O and add 5 l of 10 restriction endonuclease buffer (TOYOBO) and 5 l of SmaI restriction endonuclease (TOYOBO). Incubate at 37 for 60 min, add 10 l of 6 gel loading buffer (TOYOBO), and then purify 3‐kbp products using 1% agarose gel electrophoresis. Dilute the DNA to a concentration of 10 ng/l and store the aliquots at 4 in the dark. RNAPII Elongation Reaction Using Oligo(dC)‐Tailed Template
Figure 1 shows an outline of the assay for RNAPII transcription elongation using an oligo(dC)‐tailed template containing a DNA lesion at a specific site. Hot Labeling Reaction (Fig. 1A)
To investigate whether RNAPII stalls at a lesion of interest, we employ a hot labeling reaction using 32P‐UTP and a denatured 6% polyacrylamide gel. When RNAPII stalls, 130 nucleotide RNA transcripts are observed, generated from the transcription start site to the damaged site in our system (Fig. 1, top). When RNAPII bypasses the lesion, elongated transcripts are observed giving products of around 3 kbp on the denatured gel. Solutions and Materials The RNAPII fraction is prepared from HeLa cells as described elsewhere (Hasegawa et al., 2003; Usuda et al., 1991) or purified RNAPII can be purchased from ProteinOne. NE()buffer: 20 mM Tris–HCl (pH 8.0), 20% glycerol, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol NE(þ) buffer: NE() buffer containing 12.5 mM MgCl2 X50 ACGUTP mixture: 2.5 mM ATP, 2.5 mM CTP, 2.5 mM GTP, 0.5 mM UTP (Amersham Bioscience)
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FIG. 1. Flow diagram of the analysis of RNAPII actions in transcription elongation using an oligo(dC)‐tailed template containing a DNA lesion at a specific site.
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Stop buffer: 7 M urea, 0.35 M NaCl, 10 mM Tris–HCl (pH 7.4), 10 mM EDTA, 1% SDS 1. Reaction mixtures are set up in 25‐l aliquots. Prepare DNA solutions, Pol II solutions, and NTP solutions as follows. DNA solutions (10 l): 1 l of 10 ng/l of oligo(dC)‐tailed template containing a lesion, 0.12 l of 1 M MgCl2, 2 l of 40% PEG, and 6.88 l of H2O. Pol II solutions (4 L): 1 l of RNAPII fraction (100 ng/l), 0.1 l of 50 mg/ml BSA (Nacalai), 0.2 l of ribonuclease inhibitor (RNaseIn: Ambion: 20 U/l), and 2.7 l of NE() buffer. NTP solutions (5 l): 0.5 l of 32P‐UTP (Amersham Biosciences), 0.5 l of 50 ACGUTP mixture, 4 l of NE(þ) buffer, and 5 l of H2O. 2. Add 20 l of DNA solution, 8 l of Pol II solution, and 12 l of NE() buffer to a fresh RNase‐free tube. 3. Preincubate the reaction mixtures at 30 for 30 min. Add 5 l of NTP solution to the reactions. (The elongation reaction will be started by this step.) Incubate for a further 2–60 min. 4. Add 100 l of stop buffer to the reaction tube, and mix with 100 l of phenol–chloroform–isoamyl alcohol. Vortex for 5 min and centrifuge for 5 min at maximum speed. 5. Carefully collect 90 l of the aqueous phase and transfer it to a fresh tube containing 2 l of yeast tRNA (50 g/ml), and 10 l of 3 M sodium acetate. Mix in 250 l of ice‐cold 100% ethanol and then place on dry ice for 20 min. 6. Centrifuge at maximum speed for 15 min at 4 and carefully remove the alcohol. Add 100 l of 70% ethanol and centrifuge at maximum speed for 5 min at 4 . 7. Carefully remove the alcohol and dry the RNA pellet in a centrifuge under vacuum for 5 min. Resuspend the RNA pellet in 5 l of formamide loading dye (USB). 8. Heat the samples at 95 for 2 min and load onto a denatured 6% polyacrylamide gel. 9. Transfer the gel to 3MM paper and dry the gel. Visualize by exposing the gel to X‐ray film or with a FUJIFILM BAS 2500 bioimage analyzer. An example of the type of image is shown in Fig. 1A. Cold Labeling Reaction
Cold labeling reactions are employed to analyze RNA transcripts produced from oligo(dC)‐tailed templates containing a DNA lesion by RNAPII. When RNAPII bypasses a lesion, it may misincorporate nucleotides opposite a lesion. For this, we sequence DNA fragments produced by RT‐PCR using bypassed transcription products (Fig. 1B). However, when
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RNAPII stalls at a DNA lesion, RNAPII‐stalled RNA transcripts are generated. To investigate whether the stalled RNAPII has the ability to incorporate nucleotides, we use 30 ligation‐mediated RT‐PCR to isolate and sequence the stalled transcripts (Fig. 1C). Preparation of RNA Transcripts from RNAPII Transcription Elongation Assay Using Oligo(dC)‐Tailed Template
Solutions and Materials Basically, we use the same reagents as in the hot labeling reaction. 1. Reaction mixtures are set up in 50‐l aliquots. Prepare DNA solutions, Pol II solutions, and NTP solutions as follows. DNA solutions (20 l): 2 l of 10 ng/l of oligo(dC) template containing a lesion, 0.24 l of 1 M MgCl2, 4 l of 40% PEG, and 13.76 l of H2O. Pol II solutions (8 l): 2 l of RNAPII fraction (100 ng/l), 0.2 l of 50 mg/ml BSA, 0.4 l of RNaseIn, and 5.4 l of NE() buffer. NTP solutions (10 l): 1 l of 50 ACGUTP mixture, 0.5 l of 2 mM UTP (Amersham Bioscience), 4 l of NE(þ) buffer, and 5 l of H2O. 2. Add 20 l of DNA solution, 8 l of Pol II solution, and 12 l of NE() buffer to a fresh RNase‐free tube. 3. Preincubate the reaction mixtures at 30 for 30 min. Add 10 l of NTP solution to the reaction. Incubate for a further 20–60 min to accumulate RNA transcripts. 4. Add 100 l of stop buffer to the reaction tubes. 5. Purify transcripts of RNA using an RNeasy minikit (Qiagen). At present, there is no way to purify RNA completely free of DNA, even if the DNA is not visible on an agarose gel. Therefore, we treat the purified RNA transcripts with RNase‐free DNase I. 6. Reaction mixtures are set up in 20‐l aliquots. Add 2 l of 10 DNase buffer (TaKaRa), 0.25 l of RNaseIn (40 units/l), 0.1 l of RNase‐ free DNase I (5 Kunitz units/l, TaKaRa), and 17.65 l of the purified RNA sample (must be less than 1 g). 7. Incubate at 37 for 30 min. Add 2 l of 50 mM EDTA. Incubate at 65 for 5 min. 8. Store the DNase I‐treated RNAs in aliquots at 20 or 80 . Analysis of Lesion‐Bypassed RNAPII Elongation Transcripts (Fig. 1B)
To examine the nucleotide preference for incorporation opposite a DNA lesion, DNA fragments produced by RT‐PCR from RNA transcripts are sequenced. It is important to have good experimental conditions
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because there is a possibility that the PCR itself will generate a mutation in the RNA and that the PCR may also produce excess DNA fragments from contaminated template DNAs. Reverse‐Transcription Reaction for First‐Strand CDNA Synthesis and PCR
1. Reaction mixtures are set up in 12‐l aliquots. Add 1 l of proper primer (10 pmol/l), 2 l of the DNase I‐treated RNA solution, and 9 l of RNase‐free H2O. 2. Incubate at 65 for 5 min and place on ice. 3. Add 4 l of 5 RT buffer (TOYOBO), 2 l of 10 mM dNTP solution, 1 l of ribonuclease inhibitor (10 units/l, TOYOBO), and 1 l of reverse transcriptase (4 units/l, TOYOBO). 4. Incubate at 42 for 60 min and at 85 for 5 min. 5. Store the first‐stranded cDNA in aliquots at 20 or 80 . 6. PCR mixtures are set up in 100‐l aliquots. Add 4 l of the RT solution or the mock solution, 10 l of 10 PCR buffer (TaKaRa), 8 l of 2.5 mM dNTP mixture, 6 l of 25 mM MgCl2, 0.5 l of primer A (100 pmol/ l), 0.5 l of primer B (100 pmol/l), and 0.5 l of Taq polymerase (5 U/l, TaKaRa). Also perform a mock reaction using DNase I‐treated RNA solutions without a reverse transcription reaction. 7. Perform 25, 30, and 35 cycles: 94 for 30 s, 55 for 30 s, and 72 for 30 s. Check PCR products on the 2% agarose gel. Here, it is important to decide on good PCR conditions (maybe PCR cycles). The PCR products should be detected in the RT solutions, but not the mock solution, because any PCR products of the mock solution must be from contaminated DNA templates. Usually, we use less than 30 cycles (see Fig. 1B, 2% agarose gel). 8. Purify the PCR products from the agarose gel. Sequence the products. Mutations should be detected at sites of DNA lesions. Analysis of 30 Ligation‐Mediated RT‐PCR (Fig. 1C)
To analyze the 30 end of RNAPII‐stalled transcripts on damaged templates, the transcripts must first be ligated with RNA oligonucleotides. The RNA oligonucleotide has 50 ‐phosphorylated residues for the ligation reaction and 30 NH3 residues for preventing self‐ligation and ligation with the 50 of transcripts. The ligated transcripts are amplified by RT‐PCR, subcloned into the plasmid vector, and then introduced into competent Escherichia coli cells. Sequence data of the plasmid DNA indicate that RNAPII incorporated nucleotides opposite the lesion (Mei Kwei et al., 2004).
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Materials RNA linker: the 50 end of RNA oligonucleotides is phosphorylated and the 30 end is NH2 modified. 1. Reaction mixtures are set up in 50‐l aliquots. Add 5 l of the DNase I‐treated RNA solution, 1 l of RNA linker (20 pmol/l), 3 l of 0.1% BSA (TaKaRa), 25 l of 50% PEG 8000, 5 l of 10 T4 RNA ligase buffer (TaKaRa), 1 l of T4 RNA ligase (40 U/l, TaKaRa), and 10 l of H2O. 2. Incubate at 16 for 14–16 h. 3. After ligation, incubate at 95 for 3 min. 4. Use 1.4 l of the ligated transcript solution and a Qiagen OneStep RT‐PCR kit (Qiagen). Follow the manufacturer’s instructions using primers specific for the 30 ‐ligated RNA transcripts. Perform a RT reaction: 50 for 30 min, 94 for 30 s, and PCR for 25 cycles: 94 for 30 s, 55 for 30 s, and 72 for 30 s. 5. Add 10 l of 6 gel‐loading buffer to reaction mixtures. Run the 2% agarose gel in 1 TAE buffer and purify the PCR products using the QIAquick gel extraction kit (Qiagen). 6. Ligate the purified PCR products into a TOPO TA cloning vector (Invitrogen) and transform competent E. coli cells. 7. Sequence the isolated plasmids. Sequencing data before the anti‐ RNA linker indicates the position of the 30 end of RNAPII‐stalled transcripts. Acknowledgments We thank the past and present members of our laboratory, including Joan Seah Mei Kwei, Katsuyoshi Horibata, Mika Hayashida, and Kyoko Suzuki, for their contributions to the methods described here.
References Conti, E., and Izaurralde, E. (2005). Nonsense‐mediated mRNA decay: Molecular insights and mechanistic variations across species. Curr. Opin. Cell Biol. 17, 316–325. Doetsch, P. W. (2002). Translesion synthesis by RNA polymerases: Occurrence and biological implications for transcriptional mutagenesis. Mutat. Res. 510, 131–140. Hasegawa, J., Endou, M., Narita, T., Yamada, T., Yamaguchi, Y., Wada, T., and Handa, H. (2003). A rapid purification method for human RNA polymerase II by two‐step affinity chromatography. J. Biochem. (Tokyo) 133, 133–138. Kuraoka, I., Endou, M., Yamaguchi, Y., Wada, T., Handa, H., and Tanaka, K. (2003). Effects of endogenous DNA base lesions on transcription elongation by mammalian RNA polymerase II. Implications for transcription‐coupled DNA repair and transcriptional mutagenesis. J. Biol. Chem. 278, 7294–7299.
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Lejeune, F., and Maquat, L. E. (2005). Mechanistic links between nonsense‐mediated mRNA decay and pre‐mRNA splicing in mammalian cells. Curr. Opin. Cell Biol. 17, 309–315. Mei Kwei, J. S., Kuraoka, I., Horibata, K., Ubukata, M., Kobatake, E., Iwai, S., Handa, H., and Tanaka, K. (2004). Blockage of RNA polymerase II at a cyclobutane pyrimidine dimer and 6–4 photoproduct. Biochem. Biophys. Res. Commun. 320, 1133–1138. Moggs, J. G., Yarema, K. J., Essigmann, J. M., and Wood, R. D. (1996). Analysis of incision sites produced by human cell extracts and purified proteins during nucleotide excision repair of a 1,3‐intrastrand d(GpTpG)‐cisplatin adduct. J. Biol. Chem. 271, 7177–7186. Sambrook, J., and Russell, D. W. (2001). ‘‘Molecular Cloning: A Laboratory Manual,’’ 3rd Ed., pp. 3.30–3.48. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Shivji, M. K., Moggs, J. G., Kuraoka, I., and Wood, R. D. (1999). Dual‐incision assays for nucleotide excision repair using DNA with a lesion at a specific site. Methods Mol. Biol. 113, 373–392. Tornaletti, S. (2005). Transcription arrest at DNA damage sites. Mutat. Res. Usuda, Y., Kubota, A., Berk, A. J., and Handa, H. (1991). Affinity purification of transcription factor IIA from HeLa cell nuclear extracts. EMBO J. 10, 2305–2310. Yamaguchi, Y., Takagi, T., Wada, T., Yano, K., Furuya, A., Sugimoto, S., Hasegawa, J., and Handa, H. (1999). NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation. Cell 97, 41–51.
[14] In Vivo Assays for Transcription‐Coupled Repair By GRACIELA SPIVAK, GERD P. PFEIFER , and PHILIP HANAWALT Abstract
This chapter describes the technologies used in our respective laboratories to study the incidence and repair of lesions induced in specific DNA sequences by ultraviolet light, chemical carcinogens, and products of cellular metabolism. The Southern blot method is suitable for analysis of damage and repair in the individual DNA strands of specific restriction fragments up to 25,000 nucleotides in length, whereas the ligation‐ mediated polymerase chain reaction approach permits analysis of shorter sequences at the nucleotide level. Both methods have unique advantages and limitations for particular applications. Introduction
In addition to the general excision repair pathways, there are dedicated mechanisms for the removal of some types of lesions from actively transcribed DNA. Transcription‐coupled repair (TCR), a specialized pathway for repair in the transcribed strands of active genes, has been demonstrated unequivocally for bulky DNA lesions such as cyclobutane pyrimidine METHODS IN ENZYMOLOGY, VOL. 408 Copyright 2006, Elsevier Inc. All rights reserved.
0076-6879/06 $35.00 DOI: 10.1016/S0076-6879(06)08014-1