Proc. Natl. Acad. Sci. USA Vol. 91, pp. 8502-8506, August 1994 Biochemistry Transcript cleavage by RNA polymerase II arrested by a cyclobutane pyrimidine dimer in the DNA template (DNA damage/DNA repair/transcription) BRIAN A. DONAHUE*, SHANG YINt, JOHN-STEPHEN TAYLORt, DANIEL REINES*, AND PHILIP C. HANAWALT* *Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020; tDepartment of Chemistry, Washington University, St. Louis, MO 63130; and tDepartment of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322 Contributed by Philip C. Hanawalt, May 9, 1994

ABSTRACT A current model for transcription-coupled strands has been demonstrated in E. coli (6) and Saccharo- DNA repair is that RNA polymerase, arrested at a DNA lesion, myces cerevisiae (7-9), so it is likely to be a universal directs the repair machinery to the transcribed strand of an phenomenon. active gene. To help elucidate this role of RNA polymerase, we Several lines of evidence have suggested that an active constructed DNA templates containing the major late promoter RNA polymerase elongation complex is necessary for pref- of adenovirus and a cyclobutane pyrimidine dimer (CPD) at a erential repair of transcribed DNA strands. Induction of the specific site. CPDs, the predominant DNA lesions formed by lac operon of E. coli is necessary to observe preferential ultraviolet radiation, are good substrates for transcription- repair of the transcribed strand in the lacZ gene (6). Treat- coupled repair. A CPD located on the transcribed strand ofthe ment of mammalian cells with a-amanitin to specifically template was a strong block to polymerase movement, whereas inhibit RNA polymerase II (pol II) elongation abolishes the a CPD located on the nontranscribed strand had no effect on preferential repair of expressed genes (10-12). Temperature- transcription. Furthermore, the arrested polymerase shielded sensitive mutations in the gene encoding a subunit of RNA the CPD from recognition by photolyase, a bacterial DNA pol II ofS. cerevisiae have been used to demonstrate the loss repair protein. Transcription elongation factor SIU (also called of preferential repair at the nonpermissive temperature at TFIIS) facilitates read-through of a variety of transcriptional which transcription is inactive (8, 9). Ribosomal genes, pause sites by a process in which RNA polymerase II cleaves the transcribed by RNA polymerase I, are not preferentially nascent transcript before elongation resumes. We show that SU1 repaired (13, 14). A model was proposed for transcription- induces nascent transcript cleavage by RNA polymerase H coupled repair in which RNA polymerase, stalled at a DNA stalled at a CPD. However, this cleavage does not remove the lesion, directs repair to the transcribed strand of an active arrested polymerase from the site of the DNA lesion, nor does gene (5, 6). This model assumes that the polymerase must be it facilitate translesional bypass by the polymerase. The ar- removed from the site ofthe lesion so that the repair complex rested ternary complex is stable and competent to resume would have access to the DNA damage and so that the DNA elongation, demonstrating that neither the polymerase nor the strands could reanneal to form a proper substrate for repair. RNA product dissociates from the DNA template. Otherwise, the large polymerase complex would shield the DNA lesion and inhibit rather than promote efficient repair. Helix-distorting lesions are produced in cellular DNA by Indeed, it has been subsequently shown that the E. coli RNA various endogenous and exogenous agents. Among such polymerase, stalled at a CPD, inhibits the repair ofthat CPD lesions is the cyclobutane pyrimidine dimer (CPD), the most by purified repair enzymes in vitro (15). Thus, it appears prevalent lesion formed by short-wavelength UV radiation. essential to change the conformation and/or localization of CPDs can block DNA replication and transcription, leading the arrested polymerase in order to facilitate repair of the to cell death. If unrepaired, the DNA damage can lead to damaged template. Removal of the polymerase could occur mutagenesis, activation of protooncogenes, and ultimately by dissociation of the transcription complex from the DNA, carcinogenesis. One mechanism to remove these lesions is and this has been documented in E. coli extracts (16). nucleotide excision repair (NER), common to a wide range of Alternatively, the polymerase might translocate upstream species from Escherichia coli to humans. The basic mecha- from the lesion, without loss of the nascent transcript, to nism ofNER is well understood in E. coli (1). Recognition of provide access for the repair complex and then eventually the damage is followed by incision of the damaged DNA resume elongation past the site of the repaired DNA. strand on both sides of the lesion. The damage-containing There are many examples of elongation through transcrip- oligonucleotide is removed, the resultant gap is filled in by tional arrest sites that might provide clues to the mechanism DNA polymerase, and DNA ligase completes the process by of transcription-coupled DNA repair. One of these is pro- joining the repair patch to the contiguous DNA strand. vided by the transcription factor SH1 (also called TFIlS), Although the detailed mechanism of NER in eukaryotes is which facilitates elongation by RNA pol II through transcrip- not established as firmly, it appears to have the same essen- tional pause sites (17). This activity has been demonstrated in tial features. A striking property of NER is the intragenomic yeast (18), Drosophila (19, 20), rat (21), and human cells (22, heterogeneity of repair efficiency (2). Expressed genes are 23) and it is thought to be involved in transcriptional regu- repaired more rapidly than the overall genome in rodent (3) lation. SH1 binds to RNA pol II (24-26) and may also bind and human (4) cells in culture. Furthermore, this preferential nucleic acids by way of a cryptic "zinc ribbon" nucleotide- repair is largely due to efficient repair of the transcribed binding domain (27). The mechanism of 51-mediated read- strand of an active gene compared to the nontranscribed through is not understood, but several key features of this strand or unexpressed DNA sequences (5). In addition to activity have been elucidated (17). Predominant among these mammalian cells, preferential repair of transcribed DNA features are the requirements for nascent transcript cleavage

The publication costs ofthis article were defrayed in part by page charge Abbreviations: CPD, cyclobutane pyrimidine dimer; NER, nucleo- payment. This article must therefore be hereby marked "advertisement" tide excision repair; RNA pol II, RNA polymerase II; TRCF, in accordance with 18 U.S.C. §1734 solely to indicate this fact. transcription repair coupling factor; GHD, gapped heteroduplexes. 8502 Downloaded by guest on September 30, 2021 Biochemistry: Donahue et al. Proc. Natl. Acad. Sci. USA 91 (1994) 8503 and the removal of a short stretch of nucleotides from the 3' Hpal end prior to elongation beyond the transcriptional block. CCCCGGTTAACGCGGG Nascent transcript cleavage is sensitive to a-amanitin, sug- a. GGGGCCAATTGCGCCC gesting that this activity resides in the polymerase, but there a- pAdHpa is a strong requirement for 51. Polymerase translocation is not required for transcriptional readthrough of a sequence- specific pause site, but under some conditions upstream EcoRI translocation of RNA pol II has been observed after tran- / EcoRi digest script cleavage (28). It remains to be determined whether Smal digest \ upstream translocation of polymerase is necessary for elon- Heat gation past other transcriptional blocks. Reanneal We have constructed DNA templates in which thymine- thymine CPDs were situated at specific sites downstream of ccc GGG CCCCGGTTAACGCGGG the major late promoter of adenovirus to investigate the K GGGGCCAATTGCGCCC GGG CCC properties of a transcription complex arrested by a DNA lesion. We show that a CPD on the transcribed strand of the GHD-nts + GHD-s template is a strong block to RNA pol II. The arrested polymerase inhibits repair of the CPD by photolyase, sug- gesting that the polymerase shields the CPD. The transcript is not released from the CPD-arrested complex, since SIT 5' CGG4AACGC-3 3 ' -GCCAk4GG-5 ' induces nascent transcript cleavage by the arrested polymer- ase, and these shortened transcripts can be reelongated up to the point of blockage. However, S51 and transcript cleavage CCCCGQfAACGCGGG CCCCGGTTAACGCGGG . aI GGGGCCAATTGCGCCC do not facilitate detectable readthrough of this potent block. t r GGGGCCAATTGCGCCC pAdT=T-nts pAdT=T-ts MATERIAL AND METHODS Proteins and Reagents. RNA pol II, transcription initiation factors, and SUI were purified from rat liver as described (21), FIG. 1. Scheme for constructing DNA templates containing specifically located CPDs. GHD were prepared as shown. Oligonu- as was D44 IgG (29). Photolyase from Anacystis nidulans was A a gift from Gilbert Chu (Stanford University, Stanford, CA) cleotides with the sequence 5'-GCGTTAACCG-3' can be inserted and Anders Eker (Erasmus University, Rotterdam, The specifically into GHD-ts to form pAdTT-ts containing a CPD in the transcribed stand as shown. An oligonucleotide with the sequence Netherlands). A Plasmids. Plasmid pAdBam was constructed by replacing 5'-CGGTTAACGC-3' can be inserted specifically into GHD-nts to the 18-bp Xba I-Sph I fragment of pAdLac (30) with the form pAdT=T-nts in which the CPD is located in the nontranscribed 333-bp Nhe I-Sph I fragment of pBR322. pAdSma was strand. MLP indicates the position of the major late promoter of created by ligating BamHI-Sma I adaptors (New England adenovirus. Biolabs) into the unique BamHI site of pAdBam. Two 20-base oligonucleotides with the sequences 5'-GATC- then irradiated 10 cm from a Sylvania F15T8/B lamp (A.., CCCCGGTTAACGCGGG-3' and 5'-GATCCCCGCGT- 425 nM) for 20 min. The DNA was extracted with phenol/ TAACCGGGG-3' were annealed and ligated into the BamHI chloroform to remove photolyase, digested with Hpa I and site of pAdBam to yield pAdHpa. Plasmid pAdHpa is iden- electrophoresed in a 1.5% agarose gel in TAE (40 mM Tris tical to pAdSma except that the Sma I recognition sequence acetate, pH 7.5/1 mM EDTA) containing 0.5 jig of ethidium is interrupted by a 10-base fragment containing the recogni- bromide per ml. The DNA was transferred to a nylon tion sequence of Hpa I (Fig. 1). membrane (Hybond-N+, Amersham), hybridized with 32P_ Synthesis of Oligonudodes Containing a Single CPD. labeled nick-translated pAdHpa, and autoradiographed. A ~~~A Trnscipin Reactis and Nascent Transit Cleavage. Oligonucleotides 5'-GCGTTAACCG-3' and 5'-CGGTT- Transcription reactions were performed as described (21). AACGC-3' were prepared by automated DNA synthesis. A DNA templates, typically SOng, were linearized withHindI cis-syn thymine dimer building block (31) was used in these and incubated with rat liver protein fractions D (containing syntheses to place CPDs at positions 4 and 5 of both oligo- transcription factors TFUD, -E, and -H) and B' (coning nucleotides. Oligonucleotides were purified by HPLC. transcription factor TFI1F), recombinant rat transcription Insertion of Adducted Oligonucleotides into pAdHpa. The factor TFIIB (33), and RNA pol II to form preinitiation scheme for constructing DNA templates containing a single complexes. Nascent transcripts were radiolabeled at their 5' CPD utilized gapped heteroduplexes (GHD) as described (32) ends by the addition ofATP, UTP, and 40 GCi of [a-32P]CTP. (Fig. 1). Phosphorylated oligonucleotides were inserted into Elongation proceeds until position 15, where the first GTP is GHD by using T4 DNA ligase, and covalently closed circular required. Heparin was added to prevent further initiation, DNA ligation products were purified from an agarose gel. and 800 pM of each NTP was added to allow elongation to Plasmids in which adducted or unadducted oligonucleotides continue. Elongation complexes were precipitated with D44 were inserted into the transcribed strand (ts) were named anti-RNA antibodies and washed in reaction buffer (20 mM pAdT=T-ts and pAdUn-ts, respectively. Plasmids receiving Tris HCl, pH 7.9/3 mM Hepes-NaOH, pH 7.9/60 mM KCl/ adducted and unadducted oligonucleotides into the nontran- 0.5 mM EDTA/2 mM dithiothreitol/0.2 mg of acetylated scribed strand (nts) were labeled pAdT=T-nts and pAdUn- bovine serum albumin per ml/3% glycerol/2.2% polyvinyl nts, respectively. alcohol). The reaction buffer was adjusted to contain 5 mM Characterization ofTemplates. The presence ofCPDs at the dithiothreitol in experiments with photolyase. Purified elon- expected positions in the DNA templates was verified by gation complexes were then treated with SI1 and/or photo- using photolyase from A. nidulans. Covalently closed circu- lyase as described in each experiment. Reactions were lar templates were prepared, dissolved in 20 mM potassium stopped with SDS and proteinase K. RNA was precipitated, phosphate, pH 7.0/100 mM NaCl/5 mM 2-mercaptoethanol, denatured, and electrophoresed on 5% polyacrylamide (19:1 incubated with photolyase at 25TC for 20 min in the dark, and acrylamide/methylenebisacrylamide) gels in TBE (89 mM Downloaded by guest on September 30, 2021 8504 Biochemistry: Donahue et al. Proc. NatL Acad Sci. USA 91 (1994) Tris/89 mM boric acid/1 mM EDTA, pH 8) with 8.3 M urea. 1 2 3 4 5 6 7 Gels were dried and autoradiographed with an intensifying t _ An b screen.

RESULTS Characterization of DNA Templates Containing a CPD at a Specific Site. To study the interactions of an RNA pol II ..~ _ _ _1 4: 4 R elongation complex with a damaged DNA template, we IAd...0 constructed templates containing single CPDs at specific sites. Decanucleotides containing CPDs in the recognition site of the restriction endonuclease Hpa I were inserted into gapped heteroduplexes so that the CPD was located on either a the transcribed or nontranscribed strand downstream of the major late promoter of adenovirus. To confirm that a CPD was located at these sites, templates were analyzed by using A. nidulans photolyase, a repair enzyme that is specific for CPDs. In the presence of 437-nm light, photolyase reverses the cyclobutane ring and restores the original structure ofthe ** _ TzT pyrimidines. The presence of a CPD in the Hpa I sequence of pAdT=T was demonstrated by its resistance to cleavage by Hpa I (Fig. 2, lane 2), but treatment of the plasmid with activated photolyase rendered pAdT=T-ts sensitive to Hpa I (Fig. 2, lane 4). The unmodified plasmid was sensitive to cleavage by Hpa I in the absence of the repair enzyme (Fig. 2, lane 6), indicating that this plasmid did not contain a dimer at this site. The presence of a single CPD at this site was also FIG. 3. Transcription oftemplates containing specifically located demonstrated by using T4 endonuclease V, another repair CPDs. Transcription ofHindIII-linearized DNA templates. Lanes: 1, enzyme that is specific for CPDs (data not shown). Taken 4X174/Hae III fragments; 2, pAdHpa (Hpa I digest); 3, pAdT=T-ts; together, these results show that pAdT=T-ts contained a 4, pAdUn-ts; 5, pAdT=T-nts; 6, pAdUn-nts; 7, pAdHpa. RO, 369-nt single CPD located in the Hpa I restriction sequence on the run-off transcripts; T=T, 169-nt arrested transcripts. transcribed strand 169 nucleotides (nt) downstream from the start site ofthe major late promoter. The presence or absence low level of undamaged template in the preparation. A CPD of a CPD in templates in which modified or unmodified in the nontranscribed strand of the DNA template was not a oligonucleotides were inserted into the nontranscribed strand block to RNA pol II (Fig. 3, lane 5). Transcripts from were verified by using photolyase and T4 endonuclease V templates in which undamaged oligonucleotides were ligated (data not shown). into either strand were all full length, indicating that con- A CPD is a Strong Block to RNA pol II. DNA templates struction of the templates did not introduce blocks to RNA were used in an in vitro transcription assay to determine the pol IT. extent to which a CPD blocks RNA pol II (Fig. 3). Most SIT Induces Nascent Transcript Cleavage by RNA pol IT transcripts derived from templates containing a CPD on the Arrested by a CPD. The transcription elongation factor SII transcribed strand migrated through the gel as a single short facilitates read-through past certain types of transcriptional species, indicating that the CPD is a potent arrest for RNA pause sites by a process that requires cleavage of the 3' end pol II (Fig. 3, lane 3). Comparison of the size of these of the nascent transcript before elongation can resume. To transcripts with RNAs transcribed from Hpa I-cleaved see if an analogous process occurs when RNA pol II is pAdHpa (Fig. 3, lane 2) indicate that the arrested transcripts arrested at a CPD, DNA templates containing a single CPD (T=T) were -169 nt long; thus, the polymerase transcribed on the template strand were first transcribed in vitro, and very close to the site of the CPD. A small portion of arrested elongation complexes were purified and then incu- full-length run-off (RO) transcripts are also seen comprising bated with SII. Most SII-treated transcripts were shortened about 3.6% of the total transcripts from this reaction. Al- at the =10 nt though these transcripts may result from a small amount of 3' end by (Fig. 4, lanes 3 and 6). The extent of the could a transcript cleavage increased with time of incubation in the transcriptional bypass of CPD, they also indicate presence of SII. In other experiments we have observed pAdT=T pAdUn increased RNA cleavage with up to 4 hr of incubation (data not shown). Addition ofNTPs after the SHI treatment allowed PL - - + + elongation of the cleaved transcripts up to but not past the 437 nm - - - + CPD (Fig. 4, lanes 4 and 7), indicating that by itself, SII does HpaII + + + + + + not facilitate read-through past the site of DNA damage. 1 2 3 4 5 6 7 8 RNA pol IT Shields the CPD. To investigate whether the arrested RNA polymerase shields the CPD from recognition

N by DNA repair enzymes, arrested transcription complexes were purified and then probed with A. nidulans photolyase S - - and 437-nm light to try to reverse the lesion in the complex, and then NTPs were added to allow transcription to proceed on any repaired templates. No evidence for reversal was FIG. 2. Characterization of DNA templates. Plasmids receiving obtained (Fig. 5, lanes 1-3). These results were confirmed by the adducted oligonucleotide (lanes 1-4) or unmodified oligonucle- analyzing the DNA templates after transcription by digestion otide (lanes 5-8) were treated with photolyase (PL), 437-nm light, with Hpa I. The DNA template remained resistant to cleav- and Hpa I as indicated. N, L, and S indicate the migration of nicked age by Hpa I, indicating that the CPD had not been repaired circular, linear, and supercoiled plasmids, respectively. (data not shown). Downloaded by guest on September 30, 2021 Biochemistry: Donahue et al. Proc. Nati. Acad. Sci. USA 91 (1994) 8505 30' 60' pAdT=T A14 SIl - + +45+ + SI' _ +- PL NTPs _ + - - g+ -- +-+ - + NTPs 12 3 4 5 6 7 12 3 4 5 6 7 8 9 *wwm w,.*

:* am - RO

* -_ T=T

FIG. 4. SU1 induces nascent transcript cleavage by RNA pol II arrested at a CPD. Arrested complexes were purified and treated with SR1 or mock treated for 30 (lanes 2-4) or 60 (lanes 5-7) min. NTPs were added, and incubation was continued for 15 min (lanes 4 and 7). Lane 1 contains 4X174/Hae III fragments. FIG. 5. RNA pol II shields the CPD from recognition by photo- We also investigated whether the photolyase could reverse Iyase. Lanes: 1-3, arrested complexes purified and treated with CPDs after the nascent photolyase (PL) and NTPs as indicated; 4-7, arrested complexes induction of transcript cleavage by purified and incubated with 51 for 2 hr before photolyase and NTP treating arrested complexes with SII before the addition of treatment; 8 and9, complexes arrested at position 14 and treated with photolyase (Fig. 5, lanes 4-7). The lack of an increase in the photolyase and NTPs. RO, run-off transcript; T=T, arrested tran- amount of full-length transcripts in lane 8 suggests that script. elongation after SI and photolyase treatments only pro- ceeded up to the site of the DNA damage. Therefore, the CPD. Transcripts that were cleaved by the stalled RNA conformation or position of the arrested polymerase was not polymerase remained competent to elongate for at least 2 hr altered sufficiently to allow access to the CPD by the repair (Fig. 5, lane 8), demonstrating that the arrested complex is enzyme. To verify that the photolyase was active under the quite stable. It is likely, however, that some transcripts are conditions of the transcription assay, elongation complexes released following arrest. Transcripts isolated from UV- were arrested at position 14 on the template by omission of irradiated eukaryotic cells have been utilized to map tran- GTP, then treated with photolyase to repair the CPD, and scription units (35). The size ofthe transcripts were inversely finally incubated with NTPs to allow elongation to resume. proportional to the UV dose, suggesting that UV-induced Photolyase treatment of these templates enabled full-length lesions arrested the polymerase releasing the aborted tran- RNA synthesis (Fig. 5, compare lanes 8 and 9), thereby scripts. Under the disruptive conditions of RNA isolation, demonstrating that the photolyase was active. however, it could not be distinguished whether transcripts were released or remained as part of stalled elongation complexes. It is possible that in our reconstituted assay DISCUSSION system, the portion of transcripts that remained resistant to We have examined transcription by rat liver RNA pol II in SfI-mediated cleavage (Fig. 4) had actually been released vitro on DNA templates containing a single, specifically from the elongation complex, but it is clear that a large located CPD. A CPD on the transcribed strand was a strong portion ofthe transcripts remained as part of a stable ternary block to transcription; nearly all elongation complexes were complex. Furthermore, the CPDs in the templates remained arrested at the site ofthe damage. On the other hand, a CPD resistant to repair by photolyase, suggesting that the arrested on the nontranscribed strand had no effect on the synthesis polymerases did not dissociate from the complex. of full-length transcripts. Previous studies using UV- We have shown that when RNA pol'II is blocked at a CPD, irradiated DNA have demonstrated that UV-induced DNA it shields the CPD from repair by A. nidulans photolyase. To lesions are strong blocks to RNA pol 11 (34, 35). Photore- the extent to which photolyase damage recognition resembles versal ofthe randomly modified DNA to specifically remove NER damage recognition, these results have important im- CPDs demonstrated that among the DNA lesions formed by plications for the mechanism oftranscription-coupled repair. UV radiation, CPDs were blocks to the polymerase. Our In addition, E. coli RNA polymerase blocked at a CPD results show directly that a CPD is a strong block to a inhibited repair by the E. coli UvrABC NER complex in an mammalian RNA pol II. Similar results have also been in vitro assay (15). Clearly, the polymerase must be moved reported for an analogous assay with E. coli RNA polymerase away from the CPD to allow access to the DNA lesion by the (15). repair complex. A transcription repair coupling factor Our results suggest that at least a portion of the nascent (TRCF) has been identified in E. coli that appears to resolve transcripts are not released immediately following arrest at a this problem (16). It has been proposed that the TRCF Downloaded by guest on September 30, 2021 8506 Biochemistry: Donahue et al. Proc. NatL Acad. Sci. USA 91 (1994) dissociates RNA polymerase from the site ofdamage and that transcription after the DNA damage is repaired. The purifi- the TRCF remains bound at the lesion as a signal to direct the cation of arrested transcription elongation complexes will be UvrA2B complex to the transcribed strand of active genes. useful in testing this and other models of transcription- To resume transcription after the lesion has been removed, coupled repair. RNA polymerase must reinitiate at the promoter. No known homologs ofthe TRCF have yet been identified We thank C. A. Smith, A. K. Ganesan, and K. S. Sweder for is unknown if a similar mechanism of helpful discussions and critical reading of this manuscript. We are in eukaryotes, and it indebted to J. Hunt and J. Mote, Jr., for technical assistance. This transcription-coupled repair is utilized in eukaryotes. Tran- work was supported by a postdoctoral fellowship from the American scripts in most eukaryotes are much larger than those of Cancer Society (PF-3594) to B.A.D., by an Outstanding Investigator prokaryotes, and it might be more costly to the cell to Grant from the National Cancer Institute (CA44349) to P.C.H., and abandon these larger nascent transcripts blocked by DNA by a grant from the National Institutes of Health (GM 46331) to D.R. lesions. Ifthe polymerase translocates upstream ofthe site of damage without release of the nascent transcript, transcrip- 1. Van Houten, B. (1990) Microbiol. Rev. 54, 18-51. 3' 2. Hanawalt, P. & Mellon, I. (1993) Curr. Blol. 3, 67-69. tion could be resumed after the completion of repair. The 3. Bohr, V. A., Smith, C. A., Okumoto, D. S. & Hanawalt, P. C. incision of NER in eukaryotes is made on the sixth phos- (1985) Cell 40, 359-369. phodiester bond from the DNA lesion (36), suggesting that 4. Mellon, I., Bohr, V. A., Smith, C. A. & Hanawalt, P. C. (1986) the polymerase might have to translocate only a short dis- Proc. Natd. Acad. Sci. USA 83, 8878-8882. tance to allow repair proteins access to the lesion. 5. Mellon, I., Spivak, G. & Hanawalt, P. C. (1987) Cell 51, 241-249. 6. Mellon, I. & Hanawalt, P. C. (1989) Nature (London) 342, 95-98. SII induces nascent transcript cleavage by RNA pol II 7. Smerdon, M. J. & Thoma, F. (1990) Cell 61, 675-684. arrested by a CPD on the transcribed strand. Previously it has 8. Sweder, K. S. & Hanawalt, P. C. (1992) Proc. Nadl. Acad. Sci. been shown that SII induced transcript shortening by RNA USA 89, 10696-10700. polymerase arrested at intrinsic arrest sites (21-23) coinci- 9. Leadon, S. A. & Lawrence, D. A. (1992) J. Biol. Chem. 267, dent, with altered DNA structures (37) or for RNA pol II 23175-23182. 10. Leadon, S. A. & Lawrence, D. A. (1991) Mutat. Res. 255, 67-78. stopped by DNA binding proteins (30) or drugs (38). Unlike 11. Carreau, M. & Hunting, D. (1992) Mutat. Res. 274, 57-64. the case for polymerase stalled at a CPD, a significant portion 12. Christians, F. C. & Hanawalt, P. C. (1992) Mutat. Res. 274,93-101. ofthe polymerase molecules were able to bypass these pause 13. Vos, J. M. H. & Wauthier, E. L. (1991) Mol. Cell. Biol. 11, 2245- sites in the absence of ST. It is unlikely that SII removes the 2252. 14. Christians, F. C. & Hanawalt, P. C. (1993) Biochemistry 32, 10512- transcriptional block to facilitate read-through. More likely, 10518. SIT induces transcript shortening to allow the polymerase to 15. Selby, C. P. & Sancar, A. (1990) J. Biol. Chem. 265, 21330-21336. reelongate, thereby increasing the number of chances a 16. Selby, C. P. & Sancar, A. (1993) Science 260, 53-58. polymerase molecule has of bypassing the block (17). Since 17. Reines, D. (1994) in Transcription: Mechanisms and Regulation, CPDs appear to be complete blocks to RNA pol II, it is not eds. Conaway, R. C. & Conaway, J. W. (Raven, New York), pp. surprising that SII did not by itself facilitate transcriptional 263-278. 18. Christie, K. R., Awrey, D. E., Edwards, A. M. & Kane, C. M. bypass of the DNA lesion. In addition, the induction of (1994) J. Biol. Chem. 269, 936-943. nascent transcript cleavage did not alter the conformation of 19. Sluder,A. E., Greenleaf, A. L. &Price, D. H. (1989)J.Biol. Chem. the arrested polymerase sufficiently to allow photolyase to 264, 8963-8969. repair the CPD. Translocation of the polymerase is not a 20. Guo, H. & Price, D. H. (1993) J. Biol. Chem. 268, 18762-18770. requirement for SII-mediated transcriptional bypass (28), and 21. Reines, D. (1991) J. Biol. Chem. 266, 10510-10517. it is unlikely that under the conditions presented here, the 22. Izban, M. G. & Luse, D. S. (1992) Genes Dev. 6, 1342-1356. 23. Wang, D. & Hawley, D. K. (1993) Proc. Natd. Acad. Sci. USA 90, polymerase translocated upstream ofthe CPD. Alternatively, 843-847. SII may induce a conformational change in the polymerase 24. Sawadogo, M., Sentenac, A. & Fromageot, P. (1980) J. Biol. Chem. that also requires interaction of a TRCF to enable access of 255, 12-15. the NER complex to the CPD. 25. Horikoshi, M., Sekimizu, K. & Natori, S. (1984) J. Biol. Chem. 259, SII may play a role in transcription-coupled repair, inde- 608-611. to the nascent tran- 26. Reinberg, D. & Roeder, R. G. (1987) J. Biol. Chem. 262, 3331-3337. pendent of damage recognition, prepare 27. Qian, X., Gozani, S. N., Yoon, H.-S., Jeon, C.-J., Agarwal, K. & script for the resumption ofelongation once the template has Weiss, M. A. (1993) Biochemistry 32, 9944-9959. been repaired. Current models for the elongation of RNA 28. Gu, W., Powell, W., Mote, J., Jr., & Reines, D. (1993) J. Biol. polymerases suggest that the catalytic site does not move in Chem. 268, 25604-25616. concert with the body of the polymerase (39). The catalytic 29. Eilat, D., Hochberg, M., Fischel, R. & Laskov, R. (1982) Proc. head moves forward as nucleotides are incorporated into the Natd. Acad. Sci. USA 79, 3818-3822. 30. Reines, D. & Mote, J., Jr. (1993) Proc. Natl. Acad. Sci. USA 90, growing RNA chain, but RNA polymerase translocation on 1917-1921. DNA is not necessarily coupled to RNA chain extension. To 31. Taylor, J.-S., Brockie, I. R. & O'Day, C. L. (1987) J. Am. Chem. account for this discontinuity, the catalytic head may extend Soc. 109, 6735-6742. and "snap" back within the body of the polymerase (40). It 32. Green, C. L., Loechler, E. L., Fowler, K. W. & Essigmann, J. M. is thought that when the polymerase is arrested, the catalytic (1984) Proc. Natd. Acad. Sci. USA 81, 13-17. head snaps back without concomitant forward movement of 33. Tsuboi, A., Conger, K., Garrett, K. P., Conaway, R. C., Conaway, J. W. & Arai, N. (1992) Nucleic Acids Res. 20, 3250. the polymerase. The end result is a dissociation ofthe 3' end 34. Sauerbier, W. (1976) Adv. Radiat. Biol. 6, 49-106. of the transcript from the catalytic head of the enzyme. 35. Sauerbier, W. & Hercules, K. (1978) Annu. Rev. Genet. 12, 329- Nascent transcript cleavage, induced by the GreA and GreB 363. proteins ofE. coli (40) or SII ofeukaryotes (41), may facilitate 36. Huang, J. C., Svoboda, D. L., Reardon, J. T. & Sancar, A. (1992) elongation by creating a new 3' end that is in contact with the Proc. Natd. Acad. Sci. USA 89, 3664-3668. site of It is possible that in CPD- 37. Kerppola, T. K. & Kane, C. M. (1990) Biochemistry 29, 269-278. catalytic the enzyme. 38. Mote, J., Jr., Ghanouni, P. & Reines, D. (1994) J. Mol. Biol. 238, induced arrest, the 3' end of the transcript dissociates ftom 725-737. the catalytic head of RNA pol II. SIT-dependent transcript 39. Chamberlin, M. J. (1994) Harvey Lect. 88, 1-21. cleavage could restore the 3' end of the transcript to the 40. Borukhov, S., Sagitov, V. & Goldfarb, A. (1993) Cell 72, 459-466. catalytic head of the polymerase and enable resumption of 41. Izban, M. G. & Luse, D. S. (1993) J. Biol. Chem. 268,12874-12885. Downloaded by guest on September 30, 2021