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Defective Kin28, a subunit of yeast TFIIH, impairs transcription-coupled but not global genome nucleotide excision repair
Mutation Research 409 Ž1998. 181–188
Defective Kin28, a subunit of yeast TFIIH, impairs transcription-coupled but not global genome nucleotide excision repair Marcel Tijsterman 1, Judith G. Tasseron-de Jong, Richard A. Verhage, Jaap Brouwer ) Medical Genetic Centre, Department of Molecular Genetics, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden UniÕersity, PO Box 9502, 2300 RA Leiden, Netherlands Received 31 August 1998; revised 13 October 1998; accepted 26 October 1998
Abstract The essential Saccharomyces cereÕisiae KIN28 gene encodes a subunit of general transcription factor TFIIH, a multiprotein complex required for RNA polymerase II transcription initiation and nucleotide excision repair ŽNER.. Kin28 is implicated in the transition from transcription initiation to transcription elongation by phosphorylation of the carboxy-terminal domain ŽCTD. of the largest subunit of the RNA polymerase II complex. Here, we explore the possibility that Kin28 like the other subunits of TFIIH is involved in NER in vivo, using yeast cells carrying either a wildtype or a thermosensitive KIN28 allele. The removal of UV induced cyclobutane pyrimidine dimers ŽCPDs. was monitored at base resolution from both strands of the RNA polymerase II transcribed genes RPB2 and URA3. Cells carrying the thermosensitive KIN28 allele display a transcription-coupled repair ŽTCR. defect at the non-permissive temperature, which was most pronounced directly downstream of transcription initiation, probably as an indirect result of a general decrease in the level of RNA polymerase II transcription. The fact that CPD removal in non-transcribed DNA is completely unaffected in these cells indicates that Kin28 is not essential for general NER in vivo, providing the first example of a TFIIH subunit that is required for TCR but not for NER in general. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Kin28; CPD; Nucleotide excision repair; Transcription-coupled repair; Transcription; Yeast
1. Introduction Over the last decade, two direct links between the processes of RNA polymerase II transcription and )
Corresponding author. Tel.: q31-071-5274755; Fax: q31071-5274537; E-mail: [email protected] 1 Present address: Division of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.
nucleotide excision repair have been identified. Firstly, DNA damage situated in the transcribed strand of RNA polymerase II genes is repaired faster than damage present in non-transcribed DNA w1x, a phenomenon designated transcription-coupled repair ŽTCR.. Secondly, in vitro reconstitution of both NER and RNA polymerase II transcription requires the multiprotein complex TFIIH w2–4x. When known NER proteins were identified as subunits of TFIIH w5–7x it was proposed that the TCR phenomenon
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resulted directly from the dual function of TFIIH in both processes by providing a rapid deposition of NER proteins in the immediate vicinity of transcription blocking DNA lesions w8x. This hypothesis, based on the assumption that TFIIH is a component of the elongating transcription machinery, has become less attractive in the light of biochemical data which revealed that all general transcription factors dissociate from the transcription complex at or soon after promoter clearance leaving the elongating transcription machinery deprived of TFIIH w9,10x. TFIIH in yeast comprises nine subunits, four of which have enzymatic activity. An ATPaserhelicase activity resides in Rad25ŽSsl2. and in Rad3 w11,12x, while Kin28 and Ccl 1 constitute a cyclin-dependent kinasercyclin pair w13,14x, with specificity for the carboxyl-terminal repeat domain ŽCTD. of RNA polymerase II w15,16x. The other subunits have no apparent intrinsic catalytic activity but all are essential for yeast viability w17–19x. In yeast, this holocomplex can be resolved in two subcomplexes; core-TFIIH, which includes the subunits: Rad25, Rad3, Ssl1, Tfb1, Tfb2, Tfb3 and Tfb4 w6,19,20x, and a kinase complex ŽTFIIK., comprising the kinase and cyclin subunits w14x. Whereas core-TFIIH, together with other NER proteins, is sufficient to reconstitute NER in vitro, holo-TFIIH is required for transcription initiation w21x. An essential role of TFIIH in the latter process in vivo is supported by extensive genetic studies using conditional mutations in the yeast genes RAD25, RAD3, TFB1 and SSL1 w19,22–24x. Additionally, it was shown that TFIIK subunit Kin28 is involved in RNA polymerase II transcription in vivo. Thermosensitive kin28 mutants display dramatically reduced transcription levels at the non-permissive temperature as well as a decreased ability to phosphorylate the CTD of RNA polymerase II w15,16x. Although TFIIK is not required to reconstitute NER in vitro w25x, a direct involvement of holo-TFIIH in NER in vivo has been postulated because microinjection of antibodies against the MO15 protein, the human homolog of Kin28, into normal human fibroblasts resulted in a decrease in NER in these cells w26x. A discrepancy between the requirements for in vivo vs. in vitro NER has been observed for a number of yeast proteins, e.g., Mms19, Rad7 and Rad16 are not essential for NER in a reconstituted in vitro NER system w27x but their
absence in vivo leads to a distinctive repair deficient phenotype w28–30x. In addition, transcription-coupled repair can not be reconstituted in vitro Žyet. and therefore the identification of factors involved in this subpathway of NER depends solely on in vivo analysis. Here, we evaluated the participation of holo-TFIIH in NER by analysing repair in yeast strains carrying a thermosensitive kin28 allele w16x. We used nucleotide resolution analysis to address the influence of defective Kin28 on the removal of UV-induced cyclobutane pyrimidine dimers ŽCPDs. from the RNA polymerase II transcribed genes RPB2 and URA3. We found that whereas functional Kin28 is not required for NER of non-transcribed DNA, transcription-coupled repair is severely affected at the nonpermissive temperature.
2. Materials and methods 2.1. Strains The yeast strains used were W303; 23-2.5A Ž MATa ade2-1 ade3-22 can1-100 his3-11,15 ura3-1 kin28 D< LEU2 wYCplac22-KIN28 x. and W303; 261.2A Ž MATa ade2-1 ade3-22 can1-100 his3-11,15 ura3-1 kin28 D< LEU2 wYCPlac22-kin28-ts16 x. of which the construction has been described Žgift of G. Laff and M.J. Solomon w16x.. These strains were rendered rad7D by one-step gene replacement. The rpb1-1 strain used for northern analysis was Y262 Ž MATa ura3-52 his4-359 rpb1-1.Žgift of R.A. Young.. All strains were kept on selective YNB Ž0.67% yeast nitrogen base, 2% glucose, 2% bacto agar. supplemented with the appropriate nutrients. Cells were grown in complete medium ŽYEPD: 1% yeast extract, 2% bacto peptone, 2% glucose. at 288C under vigorous shaking. 2.2. CPD repair analysis Cells diluted in chilled phosphate-buffered saline were irradiated with 254 nm UV light ŽPhilips T U V 30 W. with 70 Jrm2 , collected by centrifugation, resuspended in complete medium and incubated for various times in the dark prior to DNA isolation. DNA samples Ž25 mg. were digested with appropri-
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ate restriction endonucleases, precipitated and RPB2 or URA3 fragments were isolated and end-labeled as described previously w31x using fragment-specific oligonucleotides Žsequences available upon request.. CPDs were identified using T4endoV. DNA samples were divided in two equal parts. One was incubated with T4endoV, the other was mock treated. Samples were subjected to spin column chromatography and lyophilized to small volumes. Approximately equal amounts of radioactivity were loaded on 6% denaturing acrylamide gels. After drying, autoradiograms were prepared from the gels.
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3. Results A kin28 D mutant can not be constructed because the KIN28 geneproduct is essential for yeast viability. We therefore used isogenic haploid strains which contain a disrupted chromosomal KIN28 gene complemented with either a wildtype Ž KIN28-wt . or a thermosensitive Ž kin28-ts16 . allele located on a plasmid. Both strains have been characterized in detail w16x revealing that loss of Kin28 function leads to a general decrease in the steady state level of mRNA. In agreement with these data, a decline in the steady
Fig. 1. The influence of Kin28 on repair of UV-induced CPDs along the transcription initiation site of the RPB2 locus. Cells carrying a wildtype-KIN28 Ž KIN28-wt . or a thermosensitive allele Ž kin28-ts16 . were grown at 238C, incubated for 2 h at 238C ŽA q C. or at 378C ŽB q D., irradiated and incubated in YEPD for the indicated times keeping the temperature constant during this procedure. Data are for the template strand nt. y40 to q75 with respect to the transcription initiation site indicated by the large arrow. Samples mock-treated and treated with the dimer-specific enzyme T4 endonuclease V are denoted y and q, respectively.
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state mRNA levels was observed upon shifting to the restrictive temperature, for both RNA polymerase II transcribed genes URA3 and RPB2 Ždata not shown., which we use in this study to analyse NER in vivo. To analyse the effect of defective Kin28 on NER in vivo, the following experimental setup was chosen. KIN28-wt and kin28-ts16 cells were grown at 238C to A 600 s 1.0–1.2. Then, cultures were either shifted to the restrictive Ž378C. or left at the permissive Ž238C. temperature for 2 h. Subsequently, cells were collected by centrifugation, resuspended in phosphate-buffered saline and irradiated with 70 Jrm2 keeping the temperature constant during this procedure. Repair was analysed by determination of the CPD load in DNA isolated directly after irradiation and at several post-incubation timepoints. We first analysed the repair of the template strand of the RPB2 locus around the start site of transcription. Since CPDs located downstream of transcription initiation are removed by the transcription-coupled repair pathway whereas removal of lesions positioned upstream of this site depends on global genome repair w31x, this choice of target allows the study of both modes of repair directly on a single DNA stretch. In wildtype Ž KIN28 . cells CPDs in transcribed as well as in non-transcribed DNA Župstream of transcription initiation and in the non-transcribed strand. are removed more efficiently at 378C compared to 238C Žsee Fig. 1B and A.. The effect of defective Kin28 is therefore analysed by comparing the kin28ts mutant with the wildtype strain at the restrictive temperature. Fig. 1 shows that at the non-permissive temperature, lesions upstream of the transcription initiation site are repaired equally efficient in kin28-ts cells and in KIN28-wt cells Žcompare panel D with panel B. indicating that general NER Žglobal genome repair. does not depend on functional Kin28. In addition, repair of the non-trancribed strand is not affected by a thermoconditionally defective Kin28 Ždata not shown.. However, transcription-coupled repair is affected as a reduction is observed in the rate with which CPDs are removed from downstream sequences. Albeit more slowly, CPDs are still removed from the template strand in kin28-ts cells at the non-permissive temperature. This residual repair might result from general Žtranscription-independent. repair operating on lesions in this strand, as this
Fig. 2. The influence of Kin28 on transcription-coupled repair specifically. Isogenic rad7 strains carrying ŽA. a wildtype-KIN28 Ž KIN28-wt . or ŽB. a thermosensitive allele Ž kin28-ts16 . were grown at 238C, incubated for 2 h at 378C, irradiated and allowed repair for the indicated times keeping the temperature constant during this procedure. Data are for the RPB2 template strand nt. y40 to q200 with respect to the transcription initiation site indicated by the large arrow. Samples mock-treated and treated with the dimer-specific enzyme T4 endonuclease V are denoted y and q, respectively.
M. Tijsterman et al.r Mutation Research 409 (1998) 181–188
mode of repair is fully functional at the elevated temperature. Therefore, we repeated the analysis in a rad7 background. Because rad7D mutants are disturbed in global genome repair, and consequently are
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unable to remove CPDs from non-transcribed DNA w30x, TCR can be studied exclusively. Fig. 2 shows NER in KIN28-wt,rad7 cells and in kin28-ts16,rad7 cells, both at the non-permissive temperature. As we
Fig. 3. Repair of UV-induced CPDs at single nucleotide resolution along the transcription initiation site of the URA3 locus in strains carrying a wildtype-KIN28 Ž KIN28-wt . or a thermosensitive allele Ž kin28-ts16 .. Cells grown at 238C were preincubated at 378C for 2 h, irradiated and allowed repair for the indicated times at this temperature. Data are for the template strand nt. y80 to q130. The large arrow indicates the transcription–initiation site Žq1. and the direction of transcription. Samples mock-treated and treated with the dimer-specific enzyme T4 endonuclease V are denoted y and q, respectively. The asterisk indicates a UV-independent background signal.
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have described previously w31x, and can be concluded again from Fig. 2A, lesions upstream of transcription are not repaired at all in rad7 mutants but CPDs in transcribed DNA are still repaired efficiently. When in addition, cells are mutated in KIN28, repair of the latter class of lesions is affected demonstrating that efficient TCR requires functional Kin28. Notably, a low level of TCR is still observed in this genetic background at the non-permissive temperature. Because the decline of mRNA levels in kin28ts16 cells when shifted to 378C was not as profound as observed in a strain with a thermosensitive mutation Ž rpb1-1. in one of the RNA polymerase II subunits Ždata not shown., we suggest that the transcription level of the RPB2 locus is not completely zero in these mutants, resulting in the observed residual TCR. Upon close examination of the effect of defective Kin28 on the repair rate of individual dimer sites in the transcribed strand, we observed that some lesions were more affected than others. Especially CPDs induced at nt. q23 to q27 in the RPB2 transcribed strand are hardly repaired as they are still detected after 40 min Žsee Fig. 1, panel D. whereas neighbouring sequences, both upstream and downstream are almost completely repaired within this time. We subsequently analysed the URA3 locus because this gene contains more putative dimer sites in the immediate vicinity of the transcription initiation site. Fig. 3 shows, in line with the RPB2 data that TCR is affected in the kin28-ts strain by the elevated temperature and that the reduction in NER efficiency is most pronounced directly downstream of transcription initiation.
4. Discussion Here, we show that yeast Kin28 is not involved in general NER in vivo. UV-induced lesions are removed from non-transcribed DNA in kin28-ts cells at the non-permissive temperature as efficiently as in cells carrying a functional KIN28 allele. Similar experiments have revealed that core-TFIIH subunits are essential for this mode of NER w32,33x. Together, these data indicate that TFIIH operates in NER in vivo independently of TFIIK. A distinction between core- and holo-TFIIH based on these repair pheno-
types is in accordance with recent biochemical studies which show that core-TFIIH, together with other repair proteins, is sufficient to reconstitute damage dependent incision on naked plasmid DNA w25x. Also, cell-free extracts made of kin28-ts16 cells are capable of performing NER in vitro Žour unpublished observations.. Still, NER is affected in kin28-ts cells albeit specifically of lesions in transcribed DNA. Although we can not formally exclude that Kin28 also functions at the site of base damage to restart stalled transcription, we explain the observed TCR defect as a secondary effect of the general decrease in the level of RNA polymerase II transcription ww15,16x, data not shownx, which results directly from inactivating Kin28. Lesions are not repaired efficiently from transcribed DNA in these cells because they are not rapidly encountered by the elongating transcription machinery which therefore can not exert its damage signalling function in TCR. Interestingly, the most profound reduction in the repair rate is observed directly downstream of transcription initiation in both genes analysed, which might result from the presence of a DNA-binding protein or protein complex that reduces the accessibility of repair proteins towards DNA lesions. Nucleosomes are known modulators of repair efficiency w34,35x and we can not exclude the possibility that a positioned nucleosome, known to be present around the transcription initiation site in the URA3 locus w36x, is the cause of the observed local NER minimum in this study. The observation, however, that this NER impediment is confined to the promoter proximal region only and is not observed at downstream core-nucleosomal sequences Ždata not shown. argues against such an explanation. A more plausible cause for a NER obstructing DNA binding protein resides in the proposed function of Kin28 in triggering the release of RNAPII paused close behind the transcription start site ww37,38x and references thereinx. Under conditions where progression to transcription elongation is blocked, such a paused RNAPII complex could interfere with NER. Although stalled RNA polymerase II complex did not appear to inhibit repair in vitro w39x, a number of observations strongly suggest that transcription can interfere with repair in vivo: Ži. Sequences transcribed by RNA polymerase III are repaired less efficient compared to the non-transcribed
M. Tijsterman et al.r Mutation Research 409 (1998) 181–188
strand w40x. Žii. Enhanced levels of transcription in RNA polymerase II transcribed genes lead to reduction in the level of CPD repair in such genes by photoreactivating enzyme w41x. Žiii. In the absence of Rad26, repair of RNA polymerase II transcribed DNA sequences at internucleosomal regions is slower compared to lesions in the opposite strand suggesting that blocked RNAPII impedes NER ŽM. Tijsterman and J. Brouwer, submitted.. In summary, we used yeast strains carrying a thermosensitive KIN28 allele Ž kin28-ts16 . to investigate the influence of the encoded geneproduct on NER in general, and on transcription-coupled repair. The removal of UV-induced CPDs was monitored at base resolution from both strands of the RNA polymerase II transcribed genes RPB2 and URA3 including the promoter proximal sequences. We show, in agreement with biochemical analysis, that core-TFIIH is sufficient for NER in vivo and in addition that mutations in affecters of RNAPII transcription can indirectly lead to a TCR defective phenotype.
Acknowledgements We thank Geoffry Laff and Dr. M.J. Solomon for generously providing the yeast strains W303; 23-2.5A and 26-1.2A. This work was supported by grants from the J.A. Cohen Institute for Radiopathology and Radiation Protection.
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Defective Kin28, a subunit of yeast TFIIH, impairs transcription-coupled but not global genome nucleotide excision repair Marcel Tijsterman 1, Judith G. Tasseron-de Jong, Richard A. Verhage, Jaap Brouwer ) Medical Genetic Centre, Department of Molecular Genetics, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden UniÕersity, PO Box 9502, 2300 RA Leiden, Netherlands Received 31 August 1998; revised 13 October 1998; accepted 26 October 1998
Abstract The essential Saccharomyces cereÕisiae KIN28 gene encodes a subunit of general transcription factor TFIIH, a multiprotein complex required for RNA polymerase II transcription initiation and nucleotide excision repair ŽNER.. Kin28 is implicated in the transition from transcription initiation to transcription elongation by phosphorylation of the carboxy-terminal domain ŽCTD. of the largest subunit of the RNA polymerase II complex. Here, we explore the possibility that Kin28 like the other subunits of TFIIH is involved in NER in vivo, using yeast cells carrying either a wildtype or a thermosensitive KIN28 allele. The removal of UV induced cyclobutane pyrimidine dimers ŽCPDs. was monitored at base resolution from both strands of the RNA polymerase II transcribed genes RPB2 and URA3. Cells carrying the thermosensitive KIN28 allele display a transcription-coupled repair ŽTCR. defect at the non-permissive temperature, which was most pronounced directly downstream of transcription initiation, probably as an indirect result of a general decrease in the level of RNA polymerase II transcription. The fact that CPD removal in non-transcribed DNA is completely unaffected in these cells indicates that Kin28 is not essential for general NER in vivo, providing the first example of a TFIIH subunit that is required for TCR but not for NER in general. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Kin28; CPD; Nucleotide excision repair; Transcription-coupled repair; Transcription; Yeast
1. Introduction Over the last decade, two direct links between the processes of RNA polymerase II transcription and )
Corresponding author. Tel.: q31-071-5274755; Fax: q31071-5274537; E-mail: [email protected] 1 Present address: Division of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.
nucleotide excision repair have been identified. Firstly, DNA damage situated in the transcribed strand of RNA polymerase II genes is repaired faster than damage present in non-transcribed DNA w1x, a phenomenon designated transcription-coupled repair ŽTCR.. Secondly, in vitro reconstitution of both NER and RNA polymerase II transcription requires the multiprotein complex TFIIH w2–4x. When known NER proteins were identified as subunits of TFIIH w5–7x it was proposed that the TCR phenomenon
0921-8777r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 8 7 7 7 Ž 9 8 . 0 0 0 6 0 - 3
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resulted directly from the dual function of TFIIH in both processes by providing a rapid deposition of NER proteins in the immediate vicinity of transcription blocking DNA lesions w8x. This hypothesis, based on the assumption that TFIIH is a component of the elongating transcription machinery, has become less attractive in the light of biochemical data which revealed that all general transcription factors dissociate from the transcription complex at or soon after promoter clearance leaving the elongating transcription machinery deprived of TFIIH w9,10x. TFIIH in yeast comprises nine subunits, four of which have enzymatic activity. An ATPaserhelicase activity resides in Rad25ŽSsl2. and in Rad3 w11,12x, while Kin28 and Ccl 1 constitute a cyclin-dependent kinasercyclin pair w13,14x, with specificity for the carboxyl-terminal repeat domain ŽCTD. of RNA polymerase II w15,16x. The other subunits have no apparent intrinsic catalytic activity but all are essential for yeast viability w17–19x. In yeast, this holocomplex can be resolved in two subcomplexes; core-TFIIH, which includes the subunits: Rad25, Rad3, Ssl1, Tfb1, Tfb2, Tfb3 and Tfb4 w6,19,20x, and a kinase complex ŽTFIIK., comprising the kinase and cyclin subunits w14x. Whereas core-TFIIH, together with other NER proteins, is sufficient to reconstitute NER in vitro, holo-TFIIH is required for transcription initiation w21x. An essential role of TFIIH in the latter process in vivo is supported by extensive genetic studies using conditional mutations in the yeast genes RAD25, RAD3, TFB1 and SSL1 w19,22–24x. Additionally, it was shown that TFIIK subunit Kin28 is involved in RNA polymerase II transcription in vivo. Thermosensitive kin28 mutants display dramatically reduced transcription levels at the non-permissive temperature as well as a decreased ability to phosphorylate the CTD of RNA polymerase II w15,16x. Although TFIIK is not required to reconstitute NER in vitro w25x, a direct involvement of holo-TFIIH in NER in vivo has been postulated because microinjection of antibodies against the MO15 protein, the human homolog of Kin28, into normal human fibroblasts resulted in a decrease in NER in these cells w26x. A discrepancy between the requirements for in vivo vs. in vitro NER has been observed for a number of yeast proteins, e.g., Mms19, Rad7 and Rad16 are not essential for NER in a reconstituted in vitro NER system w27x but their
absence in vivo leads to a distinctive repair deficient phenotype w28–30x. In addition, transcription-coupled repair can not be reconstituted in vitro Žyet. and therefore the identification of factors involved in this subpathway of NER depends solely on in vivo analysis. Here, we evaluated the participation of holo-TFIIH in NER by analysing repair in yeast strains carrying a thermosensitive kin28 allele w16x. We used nucleotide resolution analysis to address the influence of defective Kin28 on the removal of UV-induced cyclobutane pyrimidine dimers ŽCPDs. from the RNA polymerase II transcribed genes RPB2 and URA3. We found that whereas functional Kin28 is not required for NER of non-transcribed DNA, transcription-coupled repair is severely affected at the nonpermissive temperature.
2. Materials and methods 2.1. Strains The yeast strains used were W303; 23-2.5A Ž MATa ade2-1 ade3-22 can1-100 his3-11,15 ura3-1 kin28 D< LEU2 wYCplac22-KIN28 x. and W303; 261.2A Ž MATa ade2-1 ade3-22 can1-100 his3-11,15 ura3-1 kin28 D< LEU2 wYCPlac22-kin28-ts16 x. of which the construction has been described Žgift of G. Laff and M.J. Solomon w16x.. These strains were rendered rad7D by one-step gene replacement. The rpb1-1 strain used for northern analysis was Y262 Ž MATa ura3-52 his4-359 rpb1-1.Žgift of R.A. Young.. All strains were kept on selective YNB Ž0.67% yeast nitrogen base, 2% glucose, 2% bacto agar. supplemented with the appropriate nutrients. Cells were grown in complete medium ŽYEPD: 1% yeast extract, 2% bacto peptone, 2% glucose. at 288C under vigorous shaking. 2.2. CPD repair analysis Cells diluted in chilled phosphate-buffered saline were irradiated with 254 nm UV light ŽPhilips T U V 30 W. with 70 Jrm2 , collected by centrifugation, resuspended in complete medium and incubated for various times in the dark prior to DNA isolation. DNA samples Ž25 mg. were digested with appropri-
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ate restriction endonucleases, precipitated and RPB2 or URA3 fragments were isolated and end-labeled as described previously w31x using fragment-specific oligonucleotides Žsequences available upon request.. CPDs were identified using T4endoV. DNA samples were divided in two equal parts. One was incubated with T4endoV, the other was mock treated. Samples were subjected to spin column chromatography and lyophilized to small volumes. Approximately equal amounts of radioactivity were loaded on 6% denaturing acrylamide gels. After drying, autoradiograms were prepared from the gels.
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3. Results A kin28 D mutant can not be constructed because the KIN28 geneproduct is essential for yeast viability. We therefore used isogenic haploid strains which contain a disrupted chromosomal KIN28 gene complemented with either a wildtype Ž KIN28-wt . or a thermosensitive Ž kin28-ts16 . allele located on a plasmid. Both strains have been characterized in detail w16x revealing that loss of Kin28 function leads to a general decrease in the steady state level of mRNA. In agreement with these data, a decline in the steady
Fig. 1. The influence of Kin28 on repair of UV-induced CPDs along the transcription initiation site of the RPB2 locus. Cells carrying a wildtype-KIN28 Ž KIN28-wt . or a thermosensitive allele Ž kin28-ts16 . were grown at 238C, incubated for 2 h at 238C ŽA q C. or at 378C ŽB q D., irradiated and incubated in YEPD for the indicated times keeping the temperature constant during this procedure. Data are for the template strand nt. y40 to q75 with respect to the transcription initiation site indicated by the large arrow. Samples mock-treated and treated with the dimer-specific enzyme T4 endonuclease V are denoted y and q, respectively.
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state mRNA levels was observed upon shifting to the restrictive temperature, for both RNA polymerase II transcribed genes URA3 and RPB2 Ždata not shown., which we use in this study to analyse NER in vivo. To analyse the effect of defective Kin28 on NER in vivo, the following experimental setup was chosen. KIN28-wt and kin28-ts16 cells were grown at 238C to A 600 s 1.0–1.2. Then, cultures were either shifted to the restrictive Ž378C. or left at the permissive Ž238C. temperature for 2 h. Subsequently, cells were collected by centrifugation, resuspended in phosphate-buffered saline and irradiated with 70 Jrm2 keeping the temperature constant during this procedure. Repair was analysed by determination of the CPD load in DNA isolated directly after irradiation and at several post-incubation timepoints. We first analysed the repair of the template strand of the RPB2 locus around the start site of transcription. Since CPDs located downstream of transcription initiation are removed by the transcription-coupled repair pathway whereas removal of lesions positioned upstream of this site depends on global genome repair w31x, this choice of target allows the study of both modes of repair directly on a single DNA stretch. In wildtype Ž KIN28 . cells CPDs in transcribed as well as in non-transcribed DNA Župstream of transcription initiation and in the non-transcribed strand. are removed more efficiently at 378C compared to 238C Žsee Fig. 1B and A.. The effect of defective Kin28 is therefore analysed by comparing the kin28ts mutant with the wildtype strain at the restrictive temperature. Fig. 1 shows that at the non-permissive temperature, lesions upstream of the transcription initiation site are repaired equally efficient in kin28-ts cells and in KIN28-wt cells Žcompare panel D with panel B. indicating that general NER Žglobal genome repair. does not depend on functional Kin28. In addition, repair of the non-trancribed strand is not affected by a thermoconditionally defective Kin28 Ždata not shown.. However, transcription-coupled repair is affected as a reduction is observed in the rate with which CPDs are removed from downstream sequences. Albeit more slowly, CPDs are still removed from the template strand in kin28-ts cells at the non-permissive temperature. This residual repair might result from general Žtranscription-independent. repair operating on lesions in this strand, as this
Fig. 2. The influence of Kin28 on transcription-coupled repair specifically. Isogenic rad7 strains carrying ŽA. a wildtype-KIN28 Ž KIN28-wt . or ŽB. a thermosensitive allele Ž kin28-ts16 . were grown at 238C, incubated for 2 h at 378C, irradiated and allowed repair for the indicated times keeping the temperature constant during this procedure. Data are for the RPB2 template strand nt. y40 to q200 with respect to the transcription initiation site indicated by the large arrow. Samples mock-treated and treated with the dimer-specific enzyme T4 endonuclease V are denoted y and q, respectively.
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mode of repair is fully functional at the elevated temperature. Therefore, we repeated the analysis in a rad7 background. Because rad7D mutants are disturbed in global genome repair, and consequently are
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unable to remove CPDs from non-transcribed DNA w30x, TCR can be studied exclusively. Fig. 2 shows NER in KIN28-wt,rad7 cells and in kin28-ts16,rad7 cells, both at the non-permissive temperature. As we
Fig. 3. Repair of UV-induced CPDs at single nucleotide resolution along the transcription initiation site of the URA3 locus in strains carrying a wildtype-KIN28 Ž KIN28-wt . or a thermosensitive allele Ž kin28-ts16 .. Cells grown at 238C were preincubated at 378C for 2 h, irradiated and allowed repair for the indicated times at this temperature. Data are for the template strand nt. y80 to q130. The large arrow indicates the transcription–initiation site Žq1. and the direction of transcription. Samples mock-treated and treated with the dimer-specific enzyme T4 endonuclease V are denoted y and q, respectively. The asterisk indicates a UV-independent background signal.
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have described previously w31x, and can be concluded again from Fig. 2A, lesions upstream of transcription are not repaired at all in rad7 mutants but CPDs in transcribed DNA are still repaired efficiently. When in addition, cells are mutated in KIN28, repair of the latter class of lesions is affected demonstrating that efficient TCR requires functional Kin28. Notably, a low level of TCR is still observed in this genetic background at the non-permissive temperature. Because the decline of mRNA levels in kin28ts16 cells when shifted to 378C was not as profound as observed in a strain with a thermosensitive mutation Ž rpb1-1. in one of the RNA polymerase II subunits Ždata not shown., we suggest that the transcription level of the RPB2 locus is not completely zero in these mutants, resulting in the observed residual TCR. Upon close examination of the effect of defective Kin28 on the repair rate of individual dimer sites in the transcribed strand, we observed that some lesions were more affected than others. Especially CPDs induced at nt. q23 to q27 in the RPB2 transcribed strand are hardly repaired as they are still detected after 40 min Žsee Fig. 1, panel D. whereas neighbouring sequences, both upstream and downstream are almost completely repaired within this time. We subsequently analysed the URA3 locus because this gene contains more putative dimer sites in the immediate vicinity of the transcription initiation site. Fig. 3 shows, in line with the RPB2 data that TCR is affected in the kin28-ts strain by the elevated temperature and that the reduction in NER efficiency is most pronounced directly downstream of transcription initiation.
4. Discussion Here, we show that yeast Kin28 is not involved in general NER in vivo. UV-induced lesions are removed from non-transcribed DNA in kin28-ts cells at the non-permissive temperature as efficiently as in cells carrying a functional KIN28 allele. Similar experiments have revealed that core-TFIIH subunits are essential for this mode of NER w32,33x. Together, these data indicate that TFIIH operates in NER in vivo independently of TFIIK. A distinction between core- and holo-TFIIH based on these repair pheno-
types is in accordance with recent biochemical studies which show that core-TFIIH, together with other repair proteins, is sufficient to reconstitute damage dependent incision on naked plasmid DNA w25x. Also, cell-free extracts made of kin28-ts16 cells are capable of performing NER in vitro Žour unpublished observations.. Still, NER is affected in kin28-ts cells albeit specifically of lesions in transcribed DNA. Although we can not formally exclude that Kin28 also functions at the site of base damage to restart stalled transcription, we explain the observed TCR defect as a secondary effect of the general decrease in the level of RNA polymerase II transcription ww15,16x, data not shownx, which results directly from inactivating Kin28. Lesions are not repaired efficiently from transcribed DNA in these cells because they are not rapidly encountered by the elongating transcription machinery which therefore can not exert its damage signalling function in TCR. Interestingly, the most profound reduction in the repair rate is observed directly downstream of transcription initiation in both genes analysed, which might result from the presence of a DNA-binding protein or protein complex that reduces the accessibility of repair proteins towards DNA lesions. Nucleosomes are known modulators of repair efficiency w34,35x and we can not exclude the possibility that a positioned nucleosome, known to be present around the transcription initiation site in the URA3 locus w36x, is the cause of the observed local NER minimum in this study. The observation, however, that this NER impediment is confined to the promoter proximal region only and is not observed at downstream core-nucleosomal sequences Ždata not shown. argues against such an explanation. A more plausible cause for a NER obstructing DNA binding protein resides in the proposed function of Kin28 in triggering the release of RNAPII paused close behind the transcription start site ww37,38x and references thereinx. Under conditions where progression to transcription elongation is blocked, such a paused RNAPII complex could interfere with NER. Although stalled RNA polymerase II complex did not appear to inhibit repair in vitro w39x, a number of observations strongly suggest that transcription can interfere with repair in vivo: Ži. Sequences transcribed by RNA polymerase III are repaired less efficient compared to the non-transcribed
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strand w40x. Žii. Enhanced levels of transcription in RNA polymerase II transcribed genes lead to reduction in the level of CPD repair in such genes by photoreactivating enzyme w41x. Žiii. In the absence of Rad26, repair of RNA polymerase II transcribed DNA sequences at internucleosomal regions is slower compared to lesions in the opposite strand suggesting that blocked RNAPII impedes NER ŽM. Tijsterman and J. Brouwer, submitted.. In summary, we used yeast strains carrying a thermosensitive KIN28 allele Ž kin28-ts16 . to investigate the influence of the encoded geneproduct on NER in general, and on transcription-coupled repair. The removal of UV-induced CPDs was monitored at base resolution from both strands of the RNA polymerase II transcribed genes RPB2 and URA3 including the promoter proximal sequences. We show, in agreement with biochemical analysis, that core-TFIIH is sufficient for NER in vivo and in addition that mutations in affecters of RNAPII transcription can indirectly lead to a TCR defective phenotype.
Acknowledgements We thank Geoffry Laff and Dr. M.J. Solomon for generously providing the yeast strains W303; 23-2.5A and 26-1.2A. This work was supported by grants from the J.A. Cohen Institute for Radiopathology and Radiation Protection.
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