TATA-binding protein promotes the selective formation of UV-induced (6-4)-photoproducts and modulates DNA repair in the TATA box
1999; Springer Nature; Volume: 18; Issue: 2 Linguagem: Inglês
10.1093/emboj/18.2.433
ISSN1460-2075
Autores Tópico(s)Light effects on plants
ResumoArticle15 January 1999free access TATA-binding protein promotes the selective formation of UV-induced (6-4)-photoproducts and modulates DNA repair in the TATA box Abdelilah Aboussekhra Abdelilah Aboussekhra Search for more papers by this author Fritz Thoma Corresponding Author Fritz Thoma Institut für Zellbiologie, ETH-Zürich, Hönggerberg, CH-8093 Zürich, Switzerland Search for more papers by this author Abdelilah Aboussekhra Abdelilah Aboussekhra Search for more papers by this author Fritz Thoma Corresponding Author Fritz Thoma Institut für Zellbiologie, ETH-Zürich, Hönggerberg, CH-8093 Zürich, Switzerland Search for more papers by this author Author Information Abdelilah Aboussekhra2 and Fritz Thoma 1 1Institut für Zellbiologie, ETH-Zürich, Hönggerberg, CH-8093 Zürich, Switzerland 2King Faisal Specialist Hospital and Research Center, Department of Biological and Medical Research, MBC 03 PO Box 3354, Riyadh, 11211 Saudi Arabia *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:433-443https://doi.org/10.1093/emboj/18.2.433 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info DNA-damage formation and repair are coupled to the structure and accessibility of DNA in chromatin. DNA damage may compromise protein binding, thereby affecting function. We have studied the effect of TATA-binding protein (TBP) on damage formation by ultraviolet light and on DNA repair by photolyase and nucleotide excision repair in yeast and in vitro. In vivo, selective and enhanced formation of (6-4)-photoproducts (6-4PPs) was found within the TATA boxes of the active SNR6 and GAL10 genes, engaged in transcription initiation by RNA polymerase III and RNA polymerase II, respectively. Cyclobutane pyrimidine dimers (CPDs) were generated at the edge and outside of the TATA boxes, and in the inactive promoters. The same selective and enhanced 6-4PP formation was observed in a TBP–TATA complex in vitro at sites where crystal structures revealed bent DNA. We conclude that similar DNA distortions occur in vivo when TBP is part of the initiation complexes. Repair analysis by photolyase revealed inhibition of CPD repair at the edge of the TATA box in the active SNR6 promoter in vitro, but not in the GAL10 TATA box or in the inactive SNR6 promoter. Nucleotide excision repair was not inhibited, but preferentially repaired the 6-4PPs. We conclude that TBP can remain bound to damaged promoters and that nucleotide excision repair is the predominant pathway to remove UV damage in active TATA boxes. Introduction DNA is continuously damaged by intra- and extracellular DNA-damaging agents, which, unless repaired, may lead to mutations, cell death and cancer (Lindahl, 1993). To ensure efficient repair and maintenance of the genomic integrity, repair processes are integrated in a network with transcription, gene expression, replication and cell cycle control. Since DNA lesions are generated all over the genome, including active and inactive genes as well as in DNA elements necessary for regulation of gene expression or replication, understanding of DNA repair processes requires investigations of protein–DNA interactions, DNA-damage formation and repair at specific sites. Here we study how a key protein involved in transcription initiation by all nuclear RNA polymerases, the TATA-binding protein (TBP), affects formation of DNA damage by ultraviolet (UV) light and repair of UV lesions by photolyase and nucleotide excision repair (NER). UV light introduces two major stable forms of mutagenic photoproducts, cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). CPDs and 6-4PPs distort DNA structure (Wang and Taylor, 1991; Kim and Choi, 1995). The yields and type of damage depend on the sequence and structure of DNA (Lippke et al., 1981; Brash and Haseltine, 1982; Lyamichev et al., 1990, 1991), DNA-bending (Pehrson and Cohen, 1992) and folding of DNA into nucleosomes (Gale et al., 1987; Pehrson, 1989; Gale and Smerdon, 1990; Brown et al., 1993; Suquet and Smerdon, 1993; Schieferstein and Thoma, 1996, 1998; Mann et al., 1997; for reviews see Sage, 1993; Tornaletti and Pfeifer, 1996; Smerdon and Thoma, 1998). A modulation of damage formation was observed in vivo at binding sites of sequence-specific transcription factors suggesting that those factors alone or in combination with other chromatin proteins modulated DNA structure (e.g. Becker and Wang, 1984; Selleck and Majors, 1987b, 1988; Becker et al., 1989; Pfeifer et al., 1992; Gao et al., 1994; Tornaletti and Pfeifer, 1995). DNA lesions affect gene expression by blockage of elongating RNA polymerases in vitro (reviewed in Selby and Saucar, 1994; Donahue et al., 1994; Livingstone-Zatchej et al., 1997; Suter et al., 1997; Aboussekhra and Thoma, 1998). Alternatively, DNA lesions could affect transcription initiation and regulation by compromising binding of transcription factors or set up of the initiation complex. In support of this hypothesis, in vitro experiments with cellular extracts (Tommasi et al., 1996) and TFIIIA binding to 5S rDNA (Liu et al., 1997) showed that DNA damage prevents binding of specific transcription factors. Whether and how factor binding is compromised by UV-induced DNA lesions in vivo is unknown, and we do not know whether or how transcription initiation complexes affect and are affected by damage formation. CPDs and 6-4PPs can be repaired by photoreactivation and nucleotide excision repair. During photoreactivation, a damage-specific enzyme [CPD-photolyase or (6-4)-photolyase] binds to the photoproduct and reverts the damage in a light-dependent reaction, restoring the bases to their native form. CPD-photolyases were isolated from many organisms including Escherichia coli and Saccharomyces cerevisiae, while (6-4)-photolyases were found in Drosophila, Xenopus laevis and rattle snakes (reviewed in Yasui et al., 1994; Sancar, 1996b). In contrast to photoreactivation, NER is a highly conserved multistep mechanism which repairs a broad range of DNA damage including CPDs and 6-4PPs. During NER, the lesions are recognized and excised as a fragment of DNA, and the resulting gap is filled in by a DNA polymerase (reviewed in Sancar, 1996a; Wood, 1996). NER repairs 6-4PPs more rapidly than CPDs (Mccready and Cox, 1993; Szymkowski et al., 1993; Galloway et al., 1994; Suquet et al., 1995), which may result from a enhanced recognition of 6-4PPs due to greater DNA distortions. Like DNA-damage formation, both DNA repair mechanisms are modulated by chromatin structure and transcription. Photoreactivation is fast in nucleosome free regions and slow in positioned nucleosomes in vivo (Suter et al., 1997) and inefficient in reconstituted mononucleosomes in vitro (Schieferstein and Thoma, 1998). Moreover, photoreactivation is slow on the transcribed strand of genes transcribed by RNA polymerase II (RNAPII) and RNA polymerase III (RNAPIII), suggesting that polymerases stalled at DNA lesions inhibit accessibility of CPDs to photolyase (Livingstone-Zatchej et al., 1997; Suter et al., 1997; Aboussekhra and Thoma, 1998). NER is also affected at sites which are known to interact with transcription factors (Gao et al., 1994; Tu et al., 1996) or positioned nucleosomes (Wellinger and Thoma, 1997). NER shows rapid repair of the transcribed strand in RNAPII-transcribed genes (for a review see Friedberg, 1996), but not in RNAPI and RNAPIII genes (Christians and Hanawalt, 1993; Fritz and Smerdon, 1995; Dammann and Pfeifer, 1997; Aboussekhra and Thoma, 1998). This NER strand bias in RNAPII genes is possibly linked to the dual role of the general transcription factor TFIIH in transcription initiation and DNA repair (for a review see Friedberg, 1996). Fast NER at the start of transcription may indicate that recruitment of TFIIH for transcription initiation leads to an increased local concentration of repair factors (Tu et al., 1996; Teng et al., 1997). Among the transcription factors which might have a direct effect on DNA damage formation and repair is TBP. This protein is an essential component for transcription initiation by all three nuclear RNA polymerases (Hernandez, 1993). In RNAPII genes, TBP is part of the general transcription factor TFIID, binds to the TATA box and recruits the other general factors and RNAPII (reviewed in Orphanides et al., 1996). In RNAPIII genes, TBP is part of TFIIIB and is required for transcription of the TATA-less genes as well as for the yeast SNR6 gene which contains a TATA box (reviewed in Geiduscheck and Kassavatis, 1995). Crystal studies have shown that TBP makes contact with 8 bp of the TATA elements and dramatically alters DNA structure. The DNA is untwisted, sharply bent towards the major groove and exposes a wide, shallow minor groove to which TBP is bound (Kim et al., 1993a,b; reviewed in Patikoglou and Burley, 1997). The highest level of structural analysis was achieved with the ternary complexes of TBP–TFIIA–DNA (Geiger et al., 1996; Tan et al., 1996) and TBP–TFIIB–DNA (Nikolov et al., 1995). But an important question is whether similar distortions occur in the living cell when TBP is part of the initiation complex and whether the distortions are similar in the initiation complex of RNAPII and RNAPIII genes. A first indication for an altered DNA structure in vivo was the observation of enhanced UV photoproduct formation in the TATA elements of the transcriptionally active GAL1 and GAL10 genes in yeast (Selleck and Majors, 1987a,b). However, the nature of the photo-products was not identified and it remained open whether TBP was involved and sufficient for enhanced damage formation. To address this question, we have investigated UV-damage formation in yeast in the TATA boxes of a gene transcribed by RNAPIII, SNR6, and in a gene transcribed by RNAPII, GAL10. Since TBP is the only common factor in RNAPII and RNAPIII initiation complexes, this genetic approach allows identification of the effect of TBP on the TATA box structure in vivo. The studies are complemented by studies of the TBP–TATA box complex in vitro. We demonstrate that TBP is sufficient in vivo and in vitro to generate a specific DNA lesion (6-4PP) within the bent part of the TATA box, indicating that the same DNA structure occurs in living cells and in vitro. A recent study suggested that TFIID–TBP might be involved in DNA-repair processes. It was shown that TBP–TFIID binds selectively to cisplatin- or UV-damaged DNA. Cisplatin-treated or UV-irradiated DNA could be used as a competing binding site which may lure TBP–TFIID away from its normal promoter sequence (Vichi et al., 1997). This study brings up a set of important questions. What happens at the TATA box? Do TBP–TFIID proteins remain bound after damage formation in the TATA box, or are they displaced? How are DNA lesions repaired at the TATA box? Does TBP affect repair at the TATA box? Here, we use photolyase in vitro and in yeast to demonstrate that TBP and proteins of the RNAPIII initiation complex can remain bound to a damaged promoter element of the SNR6 gene and inhibit photoreactivation. In contrast, NER is not inhibited indicating differential roles of NER and PR at this promoter element. Moreover, neither photoreactivation nor NER are inhibited in the GAL10 gene, indicating differential stability of RNAPIII and RNAPII initiation complexes in vivo. Results TBP enhances pyrimidine dimer (PD) yields in the TATA box in vivo The SNR6 gene has a TATA box at position −30, an A-box within the transcribed region and an essential B-box located downstream of the termination signal (Brow and Guthrie, 1990). TFIIIC binds to A- and B-boxes and recruits TBP as part of TFIIIB to the TATA box (Burnol et al., 1993; Gerlach et al., 1995). TFIIIB protects ∼40 bp in footprinting experiments in vitro (Gerlach et al., 1995; Colbert et al., 1998) (Figure 1E). At this position, a similar sized footprint was observed in vivo (Marsolier et al., 1995), indicating that it was generated by the TBP–TFIIIB complex. We therefore refer to the TBP–TFIIIB complex, although realizing that additional proteins may be involved. The footprint is missing and the TATA region is accessible to nucleases in a transcriptionally silent mutant which has a 2 bp deletion in the B-box (snr6Δ2) (Marsolier et al., 1995). The protected TATA-box region contains three pyrimidine clusters (PCs): PC −21 in the bottom strand, which includes the TBP binding site, and PC −18 and PC −41 localized at both sides of the TATA box on the top strand. Figure 1.UV-damage formation and photoreactivation in the SNR6-TATA box region. Liquid cultures of AAY1 (SNR6, rad1Δ) and AAY2 (snr6Δ2, rad1Δ) were UV-irradiated (UV+, lanes 1–5) reincubated under yellow light (dark repair, lane 5) or photoreactivating light (photorepair, lanes 2–4) for the indicated repair times and DNA damage (CPDs and 6-4PPs) was analysed by a Taq-polymerase blockage assay using primer extension. (A) Primer extension products in the bottom strand. (B) Primer extension products in the top strand. The bands do not resolve individual PDs, since the primers had to be chosen more than 350 bp upstream and downstream of the TATA element (see Materials and methods) (Marsolier et al., 1995). (−21A)TTT, (−21B)TTT, (−41)TTTT, (−18)TTTTTTT indicate the position of the PCs. Lane 6 is DNA of non-irradiated cells. Asterisks indicate non-specific Taq polymerase arrests. Dots refer to sites which are repaired rapidly. T, C, A and G are sequencing lanes. Bottom panels are enlargements of the TATA-box region of lanes 1–5. (C) Quantitative analysis of PD yields in the wild-type SNR6 (wt, dark bars) and in the mutant snr6Δ2 (Δ2, white bars). −21, −41 and −18 refer to the PCs. Averages and standard deviations of three experiments are shown. (D) The fraction of PDs (%) removed after 1 h photoreactivation (photorepair). (E) The sequence of the SNR6 promoter, the region protected from DNase I digestion in vitro with purified TFIIIB (bars) (Gerlach et al., 1995), the region protected from micrococcal nuclease digestion in vivo (in dashed box) (Marsolier et al., 1995). This region is accessible to nuclease in the snr6Δ2 mutant. The dark box refers to the TATA element. −21A, −21B, −41 and −18 show the PCs. Download figure Download PowerPoint To investigate the effect of the TATA binding complex on UV-damage formation, the S.cerevisiae cells AAY1 (SNR6, rad1Δ) and AAY2 (snr6Δ2, rad1Δ) were UV-irradiated and the yields of PDs were analysed by primer extension. Figure 1A reveals two strong bands at position PC −21A and PC −21B in the wild-type SNR6 promoter (lanes 1, SNR6). A comparison with the other PD sites on the same strand, indicates that PC −21 is a hot spot for PD formation. Two bands were also generated in the snr6Δ2 mutant (Figure 1A, lane 1, snr6Δ2), but compared with other sites on the same strand, they were less prominent. A quantitative comparison showed that PD yields in PC −21 (TTTATTT) are ∼2-fold higher in the wild-type gene than in the mutant snr6Δ2 (Figure 1C). In PC −18 and PC −41, neither an enhancement nor a difference in PD yields was observed between the mutant and wild-type gene (Figure 1B and C). These results show that the enhancement of the PD yield is restricted to the TBP binding site (PC −21) and correlates with the presence of the TBP–TFIIIB complex. Previous work reported enhanced yields of photoproducts in the TATA box of the transcribed GAL1 and GAL10 genes (Selleck and Majors, 1987a). To investigate this observation in detail, AMY3 (rad1Δ) cells were grown either in glucose or galactose, then UV-irradiated and the photoproducts were mapped in the GAL10 promoter. The presumed GAL10 TATA region (Selleck and Majors, 1987a) contains two pyrimidine clusters PC −109 (5′CTT), which belongs to the canonical TATA box, and a flanking PC −104 (5′TCTT) (Figure 2B). UV-irradiation generated two bands in PC −109 (Figure 2A, lane 1). The upper thymine–cytosine dimer is induced with a lower yield than the lower thymine–thymine dimer. The overall yields in PC −109 were 2-fold higher in galactose than in glucose (Figure 2A, lane 1, galactose; Figure 2C, lane 1, glucose). Although the sequence of PC −104 and PC −109 are similar, the enhancement of PD yields is restricted to the PC −109. This result, together with previous observations (Selleck and Majors, 1987a,b), strongly suggest that TBP–TFIID enhances PD formation in its binding site (PC −109). The enhancement of PD formation in the GAL10 TATA box occurs at a similar site as in the SNR6 TATA box (PC −21) (Figure 1A), which indicates that enhanced photoproduct formation is an intrinsic property of TBP binding and seems to be independent of the promoter and TBP-associated factors. Figure 2.UV-damage formation and photoreactivation in the GAL10 TATA box region. Liquid cultures of AMY3 (rad1Δ) were UV-irradiated with a dose of 150 J/m2 and treated as in Figure 1. (A) Primer extension products. UV-irradiated DNA (lanes 1–5), non-irradiated DNA (lane 6), DNA sequencing (lanes T, C, A and G). (−104)TCTT and (−109)CTT indicate the PCs. The arrow indicates the position of the PDs formed in the TATA box. Bottom panels are enlargements of the (−109)CTT region. (B) The GAL10 TATA box region. The pyrimidine clusters are indicated, (−104)TCTT, (−109)CTT (white boxes), the TATA element (dark box) (Selleck and Majors, 1987b). Transcription initiation is further downstream (arrow). (C) Quantification of the fraction of molecules containing a PD in PC −104 and PC −109 (data from lane 1, glucose, and lane 1 galactose). (D) The fraction of molecules containing a PD in PC −104 and PC −109 after 0 and 60 min of repair by photolyase (data from lanes 1 and 4, glucose and galactose). Download figure Download PowerPoint TBP promotes the selective formation of 6-4PPs within the TATA box in vivo Since Taq polymerase is efficiently blocked at CPDs and 6-4PPs, the primer extension assay used above detected both classes of UV lesions (Wellinger and Thoma, 1996). To analyse the nature of the photoproducts formed in the SNR6 and GAL10 TATA elements, damaged DNA extracted from irradiated cells was treated with E.coli CPD-photolyase in vitro, which selectively removes the CPDs. Primer extension was then used to detect the presence of the remaining PDs, the 6-4PPs. In the TATA element and in the flanking region (Figure 3A, dots) of the snr6Δ2 mutant, the bands disappeared after photoreactivation, indicating that most of the PDs were CPDs (Figure 3A, lanes 4 and 5). However, when DNA from SNR6 wild-type cells was analysed, the bands in PC −21A and in the flanking region (Figure 3A, dot) disappeared, demonstrating that these PD were CPDs. In PC −21B, however, a signal persisted, indicating the presence of 6-4PPs. Hence, compared with the PC −21A and the flanking site, a large fraction of 6-4PPs can be detected in PC −21B only in the wild-type cell, when TBP is present. Figure 3.Formation of 6-4PPs in the TATA box of transcribed SNR6 and GAL10 genes. (A) Photoproducts formed in the SNR6 TATA box. FTY113 (SNR6) and FTY115 (snr6Δ2) were irradiated with 200 J/m2 (UV+) and the DNA was purified. DNA damage (CPDs and 6-4PP) is shown in lanes 1 and 4. An aliquot of damaged DNA was treated with E.coli photolyase to remove CPDs (photorepair+). The remaining photoproducts (6-4PP) are shown in lanes 2 and 5. The PCs are indicated (−21A)TTT, (−21B)TTT (as in Figure 1). Dots indicate bands outside the TATA box which are removed by photolyase. Stars refer to additional Taq polymerase blocks after photoreactivation. (B) Photoproducts formed in the GAL10 TATA box. W303-1a cultures grown in galactose or glucose were irradiated with 150 J/m2 (UV+). DNA was analysed as in (A). (−104)TCTT and (−109)CTT are indicated (as in Figure 2). Download figure Download PowerPoint A similar result was obtained by analysis of photodimers in the GAL10 TATA box. PDs induced in transcriptionally inactive state were repaired in vitro by photolyase and therefore are CPDs (Figure 3B, lanes 1 and 2, glucose). From the PDs induced in the transcribed state, PDs located in PC −104 were repaired by photolyase and represent CPDs. The signal in PC −109, however, was resistant to photoreactivation (Figure 3B, lanes 3 and 4, galactose) indicating the presence of a large fraction of 6-4PPs in the active GAL10 TATA box. These results show that the TBP-associated complexes in RNAPII and RNAPIII transcription systems promote a selective formation of 6-4PPs in their respective TATA boxes. Since TBP is the only protein shared between the RNAPIII and RNAPII initiation complex, these results suggest that TBP is responsible for 6-4PP formation. If TBP promotes the formation of 6-4PPs, the fraction of 6-4PPs formed in the TATA box would indicate the fraction of promoters loaded with TBP. Quantification of the photoreactivated samples, however, was limited by formation of additional stops for Taq polymerase (Figure 3A, stars) and a signal enhancement due to CPD removal between primer and 6-4PPs can not be excluded, although on average only 0.3 CPDs/kb were formed. Our estimations of the fraction of 6-4PPs in −21B yield a maximum of >90% and a minimum of 35%. This fraction was larger than in the immediately flanking regions (−21A; Figure 3A, dots). The high levels of 6-4PPs are consistent with the strong footprint observed over the TATA box (Marsolier et al., 1995). TBP promotes the selective formation of 6-4PPs and increases PD yields in vitro To directly test whether TBP alone is responsible for enhanced PD yields and preferential formation of 6-4PPs, the effects of TBP binding to DNA from SNR6 promoter (−60 to +1) was studied in vitro (Figure 4). The evolutionary conserved C-terminal domain of the S.cerevisiae TBP (cyTBP) (Tan et al., 1996) which contains the DNA binding specificity was used. Free DNA labelled at one end, and DNA complexed with TBP were UV-irradiated with 1 kJ/m2. DNA was purified and pyrimidine dimers were analysed using either T4 endonuclease V (T4-endoV), which cuts specifically at CPDs, or the Neurospora crassa UV-induced dimer endonuclease (UVDE), which cuts both at CPDs and 6-4PPs (Yajima et al., 1995). Analysis of PD-formation in both PC −18 and PC −41 revealed the same CPD patterns in presence or absence of TBP (Figure 4, lanes 2 and 5). Hence, TBP had no detectable effect on CPD formation on both flanking regions PC −18 and PC −41. Figure 4.Formation and photorepair of pyrimidine dimers in the SNR6 promoter region in vitro. Top and bottom strands were annealed, and the DNA was radioactively labelled at one end. Aliquots were complexed with ycTBP (+TBP), irradiated with UV light (UV+), and incubated with E.coli photolyase in presence of photoreactivating light (Photorepair+). The DNA was repurified and cut with T4-endoV at CPDs (T4-endoV+) or with UVDE at CPDs and 6-4PPs (UVDE+). The pyrimidine clusters are indicated (as in Figure 1). (Note that the signals of in −21A and −21B of lane 11 were slightly weaker than in lane 9, indicating that CPDs were not completely cut by UVDE. Longer incubations were avoided due to an additional nicking activity.) Download figure Download PowerPoint In contrast, analysis of the bottom strand revealed specific effects of TBP on damage formation in the TATA-element. Irradiation of free DNA generated similar yields of CPDs in PC −21A (8%) and PC −21B (9%), detected by both T4-endoV cleavage and by UVDE cleavage [6% (PC −21A), 5% (PC −21B); Figure 4, lanes 9 and 11]. In presence of TBP, however, the yield of photoproducts (CPDs and 6-4PPs) was about three times higher in PC −21B (18%) and slightly higher in PC −21A (8%; Figure 4, lane 15). This demonstrates that TBP enhances photoproduct formation. The enhanced signal in PC −21B compared with PC −21A (Figure 4, lane 15) and similar signals in PC −21B and PC −21A in absence of TBP (lane 11) clearly support the formation of 6-4PPs in PC −21B in presence of TBP. A comparison of PC −21B in lane 13 and 15 reveals low levels of CPDs but high levels of both photoproducts (CPDs and 6-4PP). The CPDs in PC −21B probably reflect the fraction of DNA not (or not properly) bound to TBP. In summary, both enhancement and selective formation of 6-4PPs in the TATA element reproduced the observations made in vivo and show that binding of TBP to DNA is sufficient to enhance PD formation and restrict the photodimer formation within the TATA box to the 6-4PPs class. The 6-4PP formation in vivo and in vitro occurs at the sites where crystal structure studies revealed bent DNA. Hence, these UV photofootprints provide direct evidence that these structural distortions in the complex are responsible for the preferential formation of 6-4PPs. Moreover, TBP in RNAPII and RNAPIII initiation complexes in vivo generates DNA distortions very similar to those observed in vitro. TBP remains bound after damage induction and inhibits CPD repair by photolyase in vitro A central question is whether the proteins remain bound after damage induction and how they affect repair. To address this question in vitro, free DNA and DNA–TBP complexes were UV-irradiated and treated with E.coli CPD photolyase in vitro. The remaining photoproducts were analysed by T4-endoV and UVDE digestion. CPDs in free DNA were repaired within 5 min (Figure 4, lanes 10 and 12). In presence of TBP, the CPDs in PC −21B were efficiently photorepaired, and indeed may reflect a fraction of DNA not bound to TBP. However, CPDs in PC −21A were resistant to repair (Figure 4, lane 14). This demonstrates that TBP remains bound to DNA, which is damaged at the edge of the TATA box in PC −21A, and inhibits photorepair, possibly by preventing access of CPDs to photolyase. In contrast, TBP binding did not affect photorepair in PC −18 and PC −41 (Figure 4, lane 6). This shows that TBP-mediated inhibition of photolyase is TATA-box specific. TBP complexes can remain bound to the damaged SNR6 TATA box and inhibit photorepair of CPDs in vivo DNA-repair by photolyase is a major pathway for CPD repair in many organisms including the yeast S.cerevisiae. The action of photolyase was shown to be restricted by folding of DNA into nucleosomes and by RNA polymerases blocked at CPDs, but photoreactivation was very efficient in open promoters which are not folded in nucleosomes (Livingstone-Zatchej et al., 1997; Suter et al., 1997). Here we analysed whether the TBP containing initiation complexes remain bound after damage induction in vivo and how photolyase can repair damaged promoter elements. We used yeast strains deficient in NER: AAY1 (SNR6, rad1Δ) and AAY2 (snr6Δ2, rad1Δ). After damage induction, yeast cultures were exposed to photoreactivating light and the remaining PDs were analysed (Figure 1). In the snr6Δ2 mutant, in the absence of the TFIIIB footprint, ∼70% of the lesions were photorepaired in PC −21 (−21A and −21B) within 1 h (Figure 1A and D). The repair efficiency was similar to that of other sites on the same strand (Figure 1A). However, in the wild-type SNR6 promoter, repair of CPDs by photolyase was strongly inhibited in PC −21. In 1 h, only ∼5% of the lesions were photorepaired (both bands; Figure 1D, −21wt). The enlargement in Figure 1A, SNR6, shows no decrease in signal intensity in PC −21A with increasing repair time. (The 6-4PPs in PC −21B can not be repaired by the photolyase of S.cerevisiae.) Other sites in the flanking region were efficiently repaired (Figure 1A, dots). The lack of photorepair in PC −21A of the wild-type SNR6 strongly suggests that the TBP–TFIIIB complex remains bound after damage induction and inhibits access of photolyase to CPDs. Photorepair of CPDs in PC −18 and PC −41 was also reduced in wild-type promoter, but <2-fold compared with the mutant promoter (Figure 1B and D). This indicates that the TBP–TFIIIB complex inhibits DNA repair by photolyase within the SNR6 promoter and that this inhibition is most tightly restricted to the TBP cognate site. This is direct evidence that transcription factors can inhibit the accessibility of DNA damage to repair enzymes and prevent activity of a major repair pathway. In the GAL10 promoter the situation is distinct. In the inactive promoter (Figure 2A, glucose) photorepair was efficient at all sites (Figure 2A, compare lanes 2–4 with lanes 1 and 5; Figure 2D). In the active promoter, DNA damage in PC −109 can not be photorepaired, since it consists of 6-4PPs (Figure 2A and D, galactose). However, CPDs in PC −104 were efficiently repaired and as efficient as many other sites on the same strand (Figure 2A, dots). Hence, photorepair of CPDs in PC −104 is not inhibited. Apparently photolyase finds access to CPDs suggesting that the TBP initiation complex was displaced by UV damage. This is in contrast to the SNR6 promoter, and may indicate differential stability of the RNAPII and RNAPIII initiation complexes. Preferential nucleotide excision repair of 6-4PPs in the SNR6 and GAL10 TATA boxes NER is the second repair pathway for UV-lesions. It removes 6-4PPs as well as CPDs and it shares proteins of the general transcription factor TFIIH with the RNAPII transcription system. Since TBP is involved in recruiting TFIIH to the promoter, it is attractive to speculate that TBP might also play a role in TATA box repair. Primer extension technique was used to investigate the effect of the TBP-associated complex on NER. For the SNR6 gene FTY113 (SNR6, RAD1) and FTY115 (snr6Δ2, RAD1) cells were UV-irradiated and re-incubated in the dark for different repair times (Figure 5A shows an autoradiograph). In comparison with photoreactivation, NER is relatively inefficie
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