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.