The Defect in Transcription-Coupled Repair Displayed by a Saccharomyces cerevisiae rad26 Mutant Is Dependent on Carbon Source and Is Not Associated With a Lack of Transcription

Corresponding author: Kevin Sweder, Laboratory for Cancer Research, College of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Rd., Piscataway, NJ 08854-8020., sweder{at}rci.rutgers.edu (E-mail)

Communicating editor: G. R. SMITH

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Nucleotide excision repair (NER) is an evolutionarily conserved pathway that removes DNA damage induced by ultraviolet irradiation and various chemical agents that cause bulky adducts. Two subpathways within NER remove damage from the genome overall or the transcribed strands of transcribing genes (TCR). TCR is a faster repair process than overall genomic repair and has been thought to require the RAD26 gene in Saccharomyces cerevisiae. Rad26 is a member of the SWI/SNF family of proteins that either disrupt chromatin or facilitate interactions between the RNA Pol II and transcription activators. SWI/SNF proteins are required for the expression or repression of a diverse set of genes, many of which are differentially transcribed in response to particular carbon sources. The remodeling of chromatin by Rad26 could affect transcription and/or TCR following formation of DNA damage and other stress-inducing conditions. We speculate that another factor(s) can substitute for Rad26 under particular growth conditions. We therefore measured the level of repair and transcription in two different carbon sources and found that the defect in the rad26 mutant for TCR was dependent on the type of carbon source. Furthermore, TCR did not correlate with transcription rate, suggesting that disruption of RAD26 leads to a specific defect in DNA repair and not transcription.

NUCLEOTIDE excision repair (NER) is an evolutionarily conserved pathway that removes DNA damage induced by ultraviolet (UV) irradiation and various chemical agents (ABOUSSEKHRA et al. 1995 ; FRIEDBERG et al. 1995 ; PRAKASH and PRAKASH 2000 ). NER requires a core of proteins that remove damaged DNA and synthesize new DNA and a set of proteins that give specificity to the NER machinery for either the global genomic repair (GGR) or actively transcribed genes (TCR). Repair of the transcribed strand of active genes is faster than repair of the nontranscribed strand and the genome overall (SMERDON et al. 1990 ; LEADON and LAWRENCE 1992 ; SWEDER and HANAWALT 1992 ), hinting that either RNA polymerase II (RNA Pol II) or a concomitant transcription-enabling process (i.e., chromatin remodeling) acts in facilitating the faster repair.

In Saccharomyces cerevisiae, a characteristic phenotype of rad26 mutant cells is a deficiency in the preferential repair of the transcribed strand of active genes (VAN GOOL et al. 1994 ). A similar deficiency in repair is found in Cockayne's syndrome (CS), a rare autosomal recessive disorder in humans (VENEMA et al. 1990 ; VAN HOFFEN et al. 1993 ). The clinical manifestations of CS include neurodegeneration, neuronal dysmyelination, dwarfism, and subcutaneous fat deficiency, in addition to an increased sensitivity to ultraviolet radiation in situ and in cultured cells (NANCE and BERRY 1992 ).

Based on sequence homology, Rad26 is akin to the SWI2/SNF2 family of proteins (EISEN et al. 1995 ), whose members have been implicated in transcriptional activation of a diverse set of genes (i.e., SUC2, Ty elements, ADH2, INO1, GAL1, GAL10, and HO; NEIGEBORN and CARLSON 1984 ; NEIGEBORN et al. 1986 ; HAPPEL et al. 1991 ; PETERSON and HERSKOWITZ 1992 ; WINSTON and CARLSON 1992 ; PETERSON and TAMKUN 1995 ). Two models have been proposed to explain the function of the SWI/SNF proteins. In one model, SWI/SNF actively disrupts chromatin structure to allow increased access of the transcription machinery. In the other model, SWI/SNF mediates interactions between the RNA Pol II complex and activators, to increase binding and transcription (KINGSTON et al. 1996 ). The SWI/SNF complex and another, related remodeling complex, RSC (remodel the structure of chromatin), also repress expression of mammalian c-Fos and yeast CHA1 genes, respectively (MOREIRA and HOLMBERG 1999 ; MURPHY et al. 1999 ). Kingston and colleagues proposed that SWI/SNF catalyzes the transition between open and closed chromatin states to initiate the activation or repression of the targeted genes (KINGSTON and NARLIKAR 1999 ). We hypothesize that Rad26, as a member of this family of remodeling proteins and a DNA-dependent ATPase, is active in TCR by displacing the transcription machinery from damaged DNA in chromatin. Alternatively, Rad26 could be targeted to damaged chromatin in association with a remodeling complex, which may destabilize nucleosome-DNA interactions or displace the nucleosome, making DNA damage accessible to repair proteins.

During the course of other investigations with the rad26 mutant, we found differences in TCR that appeared to be dependent upon the carbon source. While members of the SWI/SNF complex have an indirect role in modulating the expression of genes subject to catabolite repression (GANCEDO 1998 ), evidence that SWI/SNF responds to the presence of glucose is lacking (GANCEDO 1998 ). Rad26 may behave similarly to other SWI/SNF proteins and modulate repair differentially in response to alternative carbon sources. We explored further the role of Rad26 in transcription and/or TCR following UV irradiation, in the presence of different carbon sources. We measured the level of repair and transcription of two genes transcribed by RNA Pol II in wild-type and rad26 mutant strains grown in glucose- or galactose-containing media. We report here that the defect in TCR displayed by rad26 mutants is dependent on carbon source and is not associated with a lack of transcription. In addition, we measured the growth and survival of wild-type and rad26 strains in glucose- and galactose-containing media before and after UV irradiation and found that neither survival nor growth were significantly affected by the carbon source used.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Media, plasmids, and strains:
All media were prepared as described by ADAMS et al. 1998 . Synthetic complete raffinose (SCRaf) medium is 0.67% Bacto-yeast nitrogen base without amino acids, 2% raffinose, and 0.2% drop-out mix lacking uracil (ADAMS et al. 1998 ). Uracil was added to 20 µg/ml (final concentration). YPGal/Raf is 1% yeast extract, 2% yeast peptone, 1% raffinose, and 2% galactose. YPGlu/Raf is 1% yeast extract, 2% yeast peptone, 1% raffinose, and 2% glucose. YEP-Glycerol is 1% yeast extract, 2% yeast peptone, 3% (v/v) glycerol. Overnight yeast cultures were grown in raffinose-containing medium; glucose or galactose was added to a final concentration of 2% as required. Agar (2%) was added to media for plates. Yeast strains used in this study were W303-1B (MAT ho ade2-1 trp1-1 leu2-3, 112 can1-100 his3-11,15 ura3-1) and MGSC102 (same as W303-1B except rad26::HIS3; kindly provided by Alain van Gool). Plasmid pKS212, as described by SWEDER et al. 1996 is a Bluescript vector (Stratagene, La Jolla, CA) into which the internal 1.0-kb EcoRI-XhoI fragment from RPB2 was inserted (SWEDER and HANAWALT 1992 ). RPB2 encodes the second largest subunit of RNA Pol II and is actively transcribed during exponential growth (HOLSTEGE et al. 1998 ). Plasmid pKS212 was linearized by cleaving with XhoI or EcoRI and was incubated with T7 RNA polymerase or T3 RNA polymerase, respectively, rNTPs, and [-³²P]CTP (Amersham, Piscataway, NJ) under conditions recommended by the manufacturer (Boehringer Mannheim, Indianapolis) to generate strand-specific RNA probes for RPB2. Plasmid pKS213 is a pGEM-T vector (Promega, Madison, WI) into which the entire 3.75-kb SSD1 gene was inserted. SSD1 is also transcribed during exponential growth, and its product, Ssd1, is an RNA-binding protein (WERNER-WASHBURNE et al. 1989 ; UESONO et al. 1997 ). Plasmid pKS213 was linearized by cleaving with PstI or NcoI and was incubated with T7 RNA polymerase or Sp6 RNA polymerase, respectively, rNTPs, and [-³²P]CTP (Amersham) under conditions recommended by the manufacturer (Boehringer Mannheim) to generate strand-specific RNA probes for SSD1.

Growth and survival after UV irradiation of yeast cells:
For repair experiments, yeast cultures were grown overnight in YPGal/Raf or SCRaf medium (5 ml). Cells were then inoculated into 50 ml of YPGal/Raf or SCRaf medium supplemented or not with either 2% glucose or 2% galactose and irradiated in logarithmic phase growth as previously described (SWEDER and HANAWALT 1994 ). All manipulations were performed under yellow light to preclude photoreactivation. Exponentially growing cultures were collected by centrifugation and resuspended in ice-cold phosphate-buffered saline (PBS) at 1 x 10⁷ cells/ml. Shaking cell suspensions (0.2 cm deep to ensure a uniform UV dose to all cells) were irradiated with predominantly 254 nm UV light at 1.33 J/m²/sec using an American Ultraviolet germicidal lamp. The cells were collected by centrifugation after irradiation and resuspended in their original growth medium at 30°. Aliquots were then taken at different time points and growth was measured by OD₆₀₀ and cell density by hemocytometer.

To determine growth rates for wild-type and rad26 strains, yeast cultures were grown overnight in SCRaf medium (5 ml). Cells were then inoculated into 25 ml of SCRaf medium supplemented or not with either 2% glucose or 2% galactose and irradiated in logarithmic phase growth. All manipulations were performed under yellow light to preclude photoreactivation. Cell number before and after UV irradiation was determined by hemocytometer and cell density by OD₆₀₀. The relationship between cell number and OD₆₀₀ is strain dependent and was determined empirically for each strain.

To measure survival, cells that were grown overnight in SCRaf medium (5 ml) were inoculated into 25 ml of SCRaf medium containing 2% glucose or 2% galactose and grown to logarithmic phase. Cell densities were determined with a hemocytometer, and 500–1000 cells were spread on agar plates (SCRaf) containing 2% galactose or 2% glucose and irradiated at 0, 30, 60, and 90 J/m². Control (unirradiated) and irradiated plates were incubated in the dark at 30° for 3 days to allow for growth and colonies were counted.

Isolation of yeast DNA:
Cell walls were digested with Zymolyase 100T (ICN Biochemicals) in zymolyase buffer (1 M sorbitol/0.25 M EDTA (pH 8.0)/20 mM 2-mercaptoethanol) at 37° for 30 min. Spheroplasts were collected by centrifugation and resuspended in 0.2 ml of zymolyase buffer (SWEDER and HANAWALT 1992 ). Spheroplasts were diluted with 2.8 ml of 0.05 M Tris-HCl (pH 8.5)/0.05 M EDTA and lysed by addition of 0.2 ml of 20% sarkosyl (NASMYTH and REED 1980 ). The mixture was chilled on ice for >10 min. Cellular debris and sarkosyl were precipitated by addition of 0.64 ml of 5 M potassium acetate, incubated at 4° overnight, and centrifuged at 4500 rpm in a Sorvall HL-6000 rotor at 4° for 25 min. After phenol extractions, supernatants containing chromosomal DNA were transferred to fresh tubes and precipitated by addition of two volumes of ice-cold 95% ethanol and washed once with ice-cold 70% ethanol (SAMBROOK et al. 1989 ). Samples were incubated in the presence of RNaseA (50 µg/ml) to digest RNA and the DNA was digested to completion with PvuI and PvuII. Restricted DNA was ethanol precipitated, washed, and resuspended in 10 mM Tris-HCl (pH 7.5)/1 mM EDTA (TE) and stored at 4°.

Strand-specific analysis of frequency of cyclobutane pyrimidine dimers:
The incidence of cyclobutane pyrimidine dimers (CPDs) in a particular restriction fragment was determined by methods previously described (BOHR et al. 1985 ; MELLON et al. 1987 ; SWEDER and HANAWALT 1994 ). Equal amounts of purified, restricted DNA were either mock treated or treated with T4 endonuclease V for 20 min at 37°. DNA was denatured and electrophoresed through alkaline agarose (0.5% w/v) gels and transferred to Hybond N⁺ membranes. Membranes were prehybridized for at least 2 hr and then hybridized with radioactive strand-specific RNA probes for RPB2 and SSD1. Autoradiographs were scanned using a Hewlett-Packard Scanjet II and the Deskscan application; signal intensities were quantified using National Institutes of Health Image 1.62.

Transcription run-on assay:
Transcription elongation complexes in permeabilized cells were detected by radiolabeling nascent transcripts essentially as described by WARNER 1991 . Cells were grown to early logarithmic phase at 30° in SCRaf medium containing 2% glucose or 2% galactose and irradiated with 254 nm light as described above. We determined cell densities using a hemocytometer and optical densities at 600 nm before UV irradiation and after collecting each subsequent aliquot. Following irradiation, 7.5 x 10⁷ cells were mixed with an equal volume of ice or returned to their original media for 30 and 90 min to enable repair. Following the repair incubations, cells were mixed with an equal volume of ice and collected by centrifugation at 4500 rpm in an Eppendorf 5804R centrifuge. Cells were resuspended in a buffer of 10 mM Tris, pH 7.5, 5 mM MgCl₂, and 100 mM NaCl and incubated on ice for 5 min. Cells were collected by centrifugation at 4500 rpm and resuspended in 0.95 ml of ice-cold water. Fifty microliters of sarcosine (10% w/v) were added and the cells were incubated on ice for an additional 15 min. Permeabilized cells were collected by centrifugation at 4500 rpm and resuspended in 120 µl of reaction buffer (50 mM Tris, pH 8.0, 100 mM KCl, 5 mM MgCl₂, 1 mM MnCl₂, 2 mM dithiothreitol, 250 mM ATP, 125 mM GTP, 125 mM CTP, and 1 µCi/µl [-³²P]UTP) and incubated at room temperature for 15 min. Cells were collected by centrifugation at 4500 rpm, resuspended in 200 µl zymolyase buffer and stored at -20°. Permeabilized cells were added directly to the hybridization solution.

To control for differences in loading between lanes, PCR products of RPB2 and SSD1 immobilized on Hybond N⁺ membranes were hybridized with in vitro-generated transcripts for RPB2 and SSD1. Equivalent hybridization signals were observed for all lanes. After stripping, membranes were exposed to X-ray film with an intensifying screen for 1 day to confirm that no signal was detected. The membranes were then hybridized with radioactively labeled nascent RNA transcripts (above) for 16 hr at 50°. Membranes were hybridized with RNA from either UV-irradiated wild-type parent or rad26 strains grown in SCRaf media containing 2% glucose or 2% galactose. X-ray film was exposed to membranes hybridized with wild-type and rad26 RNA for 7 or 10 days, respectively.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

TCR requires Rad26 in a carbon source-dependent manner:
To examine the role of Rad26 in TCR under different growth conditions, we determined the removal of CPDs from each strand of RPB2 in a rad26 mutant strain (defective in TCR) and in the wild-type (repair proficient) parent strain (W303-1B) grown in SCRaf/glucose or SCRaf/galactose. Fig 1A shows representative autoradiograms of membranes containing DNA from an experiment examining repair in strains grown in glucose-containing medium. Removal of CPDs from each strand of the RPB2 gene in wild-type (W303-1B) and rad26 (MGSC102) was determined from several experiments and is represented graphically in Fig 1B. When the parent strain was grown in glucose, damage from the transcribed strand of RPB2 was removed at a faster rate than the already proficient removal of damage from the nontranscribed strand, as expected for a repair-proficient strain. In the rad26 strain, repair of the transcribed strand was reduced to the level of repair of the nontranscribed strand in the parent strain. Repair of the transcribed strand is slightly higher than repair of the nontranscribed strand in the rad26 mutant. However, repair of both strands was slower relative to the repair of the transcribed strand of the parent strain. The lack of TCR in rad26 was previously reported (VAN GOOL et al. 1994 ; VERHAGE et al. 1996 ) and our results are in good agreement.

View larger version (50K): In this window In a new window Download PPT slide   Figure 1. (A) Autoradiograms illustrating removal of CPDs from each of the strands of the RPB2 gene in the wild-type parent strain (W303-1B) and a rad26 mutant (MGSC102) grown in the presence of glucose. Glucose was added (2% final concentration) to cultures in synthetic complete medium containing 2% raffinose. Six to eight hours later, exponentially growing cultures were UV irradiated with 60 J/m2 and incubated in growth medium for the times indicated. DNA purified from the cells was assayed using the CPD-specific T4 endonuclease V as described in MATERIALS AND METHODS. DNA was transferred to Hybond N+ membrane and hybridized with a radioactive RNA probe specific for the nontranscribed strand of the RPB2 gene and an autoradiogram was generated. The probe was stripped off and the immobilized DNA was then hybridized with an RNA probe specific for the transcribed strand. The autoradiograms show the 5.3-kb PvuI-PvuII restriction fragment. (B) Time course for removal of CPDs from each of the two strands of RPB2 in a rad26 mutant (MGSC102) and the wild-type parental strain (W303-1B) grown in the presence of glucose (as described in A). Repair was determined from the measured incidences of CPDs in each strand of the PvuI-PvuII restriction fragment of the RPB2 gene. Values represent the average of four experiments for rad26 and two experiments for the wild type. Transcribed strand, wild type, solid square; nontranscribed strand, wild type, open square; transcribed strand, rad26, solid circle; nontranscribed strand, rad26, open circle.

Surprising results were observed when repair rates were determined for wild-type and rad26 strains grown in galactose-containing medium. Fig 2A shows the results from an experiment examining repair in the same strains as above grown in the presence of galactose, instead of glucose. Removal of CPDs from each strand of the RPB2 gene in wild-type and rad26 strains is presented graphically in Fig 2B. Repair in the wild-type strain grown in galactose-containing medium was the same as the repair observed for the wild-type strain grown in the presence of glucose; i.e., there is faster repair of the transcribed strand relative to the nontranscribed strand. In contrast to what was observed for repair in cells grown in glucose, repair of the transcribed strand in the rad26 strain cultured in galactose-containing medium was highly efficient. Indeed, the rate of repair in the transcribed and nontranscribed strands in the rad26 mutant grown in galactose-containing medium was essentially the same as in the wild-type parent strain. Thus, the rad26 mutant displays a defect in TCR when the cells are grown in glucose-containing medium, but not in galactose-containing medium. In the presence of galactose, repair in the rad26 mutant is proficient for both the transcribed and nontranscribed strands of RPB2, although repair of the transcribed strand is faster than repair of the nontranscribed strand.

View larger version (48K): In this window In a new window Download PPT slide   Figure 2. (A) Autoradiograms illustrating removal of CPDs from each of the strands of the RPB2 gene in the wild-type parent strain (W303-1B) and a rad26 mutant (MGSC102) grown in the presence of galactose. As described in Fig 1A, except galactose was substituted for glucose. The autoradiograms show the 5.3-kb PvuI-PvuII restriction fragment. (B) Time course for removal of CPDs from each of the two strands of RPB2 in a rad26 mutant (MGSC102) and the wild-type parental strain (W303-1B) grown in the presence of galactose (as described in Fig 1A, except galactose was substituted for glucose). Repair was determined from the measured incidences of CPDs in each strand of the PvuI-PvuII restriction fragment of the RPB2 gene. Values represent the average of five experiments for rad26 and three experiments for the wild type. Transcribed strand, wild type, solid square; nontranscribed strand, wild type, open square; transcribed strand, rad26, solid circle; nontranscribed strand, rad26, open circle.

To determine if the level of repair in the rad26 mutant was in some way influenced by the synthetic complete medium used in our experiments, we also measured repair of RPB2 in the rad26 mutant grown in rich YPGal/Raf and YPGlu/Raf media (data not shown). We observed greater repair in rad26 cells grown in YPGal/Raf (as in Fig 2B) than in YPGlu/Raf. Thus, the differences that we observed are due exclusively to carbon source variation and not another nutrient or growth effect.

Carbon source-dependent TCR in a rad26 mutant is not restricted to RPB2:
To determine if the defect in TCR conferred by rad26 is restricted to the RPB2 gene or if it is a general carbon source-dependent defect in TCR, we examined repair of another gene transcribed by RNA Pol II. We determined the removal of CPDs from each strand of SSD1 in the wild-type and rad26 strains grown in glucose- and galactose-containing medium and observed similar results as in RPB2 (Fig 3). When glucose was used as the carbon source, TCR was nearly absent in the SSD1 gene in the rad26 mutant. Repair rates were determined for SSD1 in the wild-type and rad26 strains grown in galactose-containing medium, and TCR was present in both strains (Fig 4).

View larger version (18K): In this window In a new window Download PPT slide   Figure 3. Time course for removal of CPDs from each of the two strands of SSD1 in a rad26 mutant (MGSC102) and the wild-type parental strain (W303-1B) grown in the presence of glucose (as described in Fig 1A). Repair was determined from the measured incidences of CPDs in each strand of the PvuI-PvuII restriction fragment of the SSD1 gene. Values represent the average of four experiments for rad26 and two experiments for the wild type. Transcribed strand, wild type, solid square; nontranscribed strand, wild type, open square; transcribed strand, rad26, solid circle; nontranscribed strand, rad26, open circle.

View larger version (19K): In this window In a new window Download PPT slide   Figure 4. Time course for removal of CPDs from each of the two strands of SSD1 in a rad26 mutant (MGSC102) and the wild-type parental strain (W303-1B) grown in the presence of galactose (same as in Fig 1A except galactose was substituted for glucose). Repair was determined from the measured incidences of CPDs in each strand of the PvuI-PvuII restriction fragment of the SSD1 gene. Values represent the average of five experiments for rad26 and three experiments for the wild type.Transcribed strand, wild type, solid square; nontranscribed strand, wild type, open square; transcribed strand, rad26, solid circle; nontranscribed strand, rad26, open circle.

The ability of rad26 mutants to perform TCR is not restricted to galactose:
Is the efficient TCR we observed in rad26 mutants limited to galactose-containing medium? To answer this question, we measured repair in wild-type and rad26 strains grown in a different carbon source (raffinose). Repair of the transcribed strand of the RPB2 gene for the rad26 mutant grown in SCRaf is similar to that seen for repair of the RPB2 in the rad26 strain when grown in galactose (data not shown). Approximately 44% of CPDs were removed from the transcribed strand and 21% from the nontranscribed strand by 30 min following UV irradiation. We also explored the effect of a nonfermentable carbon source, glycerol, on repair in wild-type and rad26 strains. After cells were grown on glycerol and UV irradiated, we determined repair for the RPB2 gene for wild-type and rad26 strains and found it to be comparable to that seen for the rad26 mutant when grown in glucose (data not shown).

Transcription rate is not affected by the carbon source:
Given that transcription is required for TCR (LEADON and LAWRENCE 1992 ; SWEDER and HANAWALT 1992 ), the deficiency in TCR in rad26 could be due to defective transcription rather than defective TCR. The level of repair observed for the wild-type and rad26 strains could be influenced by the level of transcription of RPB2 and SSD1. Therefore, we determined the rate at which RPB2 and SSD1 were transcribed following UV irradiation. We used the transcript elongation assay described by WARNER 1991 with modifications. This assay measures the RNA polymerase density within a transcription unit by elongating nascent transcripts in permeabilized cells in the presence of radiolabeled UTP. The transcription elongation assay is not a measurement of the elongation complexes stalled at sites of DNA damage. Fig 5A–D, shows a time course for RPB2 and SSD1 mRNA synthesis following exposure to 60 J/m² of UV radiation. RPB2 is transcribed in rad26 and wild-type cells following UV irradiation in both glucose- and galactose-containing medium (Fig 5B and Table 1). SSD1 is also transcribed but at a much lower level than RPB2 is transcribed under the same experimental conditions for the parental wild-type strain and the rad26 mutant (Fig 5D). Results presented in Table 1 are the average of five (rad26) and six (wild type) experiments. There is considerable variability in experimental results, but there was no obvious correlation between transcription rate and TCR for RPB2 and SSD1. Thus, the lack of TCR observed for cells grown in glucose is not due to a lack of transcription.

View larger version (34K): In this window In a new window Download PPT slide   Figure 5. Time course for transcription of RPB2 and SSD1 in a rad26 mutant (MGSC102) and the wild-type parental strain (W303-1B). Cells were grown as described in MATERIALS AND METHODS. To control for loading, Hybond N+ nylon membranes containing immobilized PCR products for RPB2 and SSD1 were hybridized with radioactively labeled RPB2 and SSD1 RNA probes, respectively, and exposed to X-ray film. Membranes were then stripped and hybridized with radioactively labeled nascent RNA transcripts from permeabilized cells. (A) In vitro-transcribed RPB2 probe. (B) rad26 (left) and wild-type parent (right) RPB2 nascent transcripts. (C) In vitro-transcribed SSD1 probe. (D) rad26 (left) and wild-type parent (right) SSD1 nascent transcripts.

Cell growth and survival of wild type and rad26 after UV irradiation are unaffected by carbon source:
Since the rad26 mutant shows a repair defect when cells are grown in glucose-containing medium, but not in galactose-containing medium, UV survival and the recovery of cell growth after UV irradiation may be affected by carbon source as well. To determine if this was the case, we examined UV survival of the wild-type and rad26 strains in SCRaf/glucose or SCRaf/galactose and observed no significant difference. Our results are consistent with previously reported survival curves for the same rad26 mutant on rich glucose medium (VAN GOOL et al. 1994 ).

Growth rates in the presence of glucose or galactose were determined for wild-type and rad26 cells following UV irradiation. In parallel, we examined the growth rates for unirradiated wild-type and rad26 cells in glucose- or galactose-containing medium. Growth rate was measured over the first 2 hr (two experiments) or 2¹/₂ hr (one experiment) following UV irradiation. There were small differences in growth rates for the wild-type strain and the rad26 strain when grown in glucose- or galactose-containing medium in the absence of UV radiation. Following exposure to 60 J/m² of UV radiation, there was a small increase in the doubling time for both wild-type parent and the rad26 strain under all conditions as expected, but, again, no significant differences can be appreciated between rad26 and the wild-type strain. (Fig 6).

View larger version (17K): In this window In a new window Download PPT slide   Figure 6. Growth rates of a rad26 mutant (MGSC102) and the wild-type parental strain (W303-1B) in glucose- or galactose-containing medium. Exponentially growing cultures at 30° were either UV irradiated or not and then incubated in growth medium at 30°. UV irradiations were with 60 J/m2. Growth was monitored for 2 hr (two experiments) or 21/2 hr (one experiment) following UV irradiation by measuring the optical density of the cultures at 600 nm.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Disruption of RAD26 was shown to result in a defect in TCR (VAN GOOL et al. 1994 ). We also found that there was little TCR at two loci that are transcribed by RNA Pol II in a rad26 mutant grown in the presence of glucose. Surprisingly, we found that the defect in TCR in a rad26 mutant was dependent upon the carbon source, since rapid repair of the transcribed strands of both RPB2 and SSD1 was observed in the rad26 mutant grown in galactose-containing medium. This rapid repair was equivalent to the rate of repair of the transcribed strand of RPB2 and SSD1 in the wild-type strain, W303-1B, in either glucose- or galactose-containing medium. The increased repair observed in the rad26 mutant grown in galactose could be due to galactose-induced expression of NER proteins. However, in another wild-type background, the expression of genes encoding previously identified proteins of the NER pathway is not significantly affected in glucose or galactose medium (ROTH et al. 1998 ). Additionally, we also observed levels of TCR in the rad26 mutant grown in a different fermentable carbon source (raffinose) that are similar to the levels observed in galactose. We conclude that the absence of TCR in this rad26 mutant is not caused by a defect in the ability to perform TCR, but only a defect in TCR under particular growth conditions.

Previously, a role for Rad26, and its human homologue CSB, in TCR was proposed on the basis of its homology to members of the SWI2/SNF2 family of DNA-dependent ATPases (EISEN et al. 1995 ). Two activities have thus far been detected for SWI/SNF: (1) remodeling of chromatin by destabilizing the contacts between DNA and core histones while maintaining all components of the histone octamer intact (COTE et al. 1994 ; IMBALZANO et al. 1994 ; CAIRNS et al. 1996 ) and (2) physical transfer of histones along DNA or to other DNA molecules (LORCH et al. 1999 ). Both activities could make the damaged DNA more accessible to repair proteins. Rad26 may enable faster repair of chromatin by remodeling or displacing the stalled RNA pol II complex from the damaged DNA.

Elaborating on the previous model for the role of Rad26 in TCR (EISEN et al. 1995 ), it is possible that Rad26, as part of a remodeling complex, is targeted to or targets the remodeling activities of the complex to the stalled RNA Pol II or to damaged DNA. When we examined repair of the RPB2 and SSD1 genes, we noted an elevated repair of the transcribed strands of these genes when grown on galactose. Additionally, we observed an increase in repair of the nontranscribed strand of the RPB2 gene in cells grown in the presence of galactose. Thus, effect of increased repair is not limited to just TCR (at least for the RPB2 gene). We propose a model in which another protein substitutes for Rad26 in a given protein complex that is active in rad26 cells growing in the presence of galactose, but is repressed in the presence of glucose. Alternatively, a different remodeling complex independent of Rad26 may be active in rad26 cells growing in galactose-containing medium. This putative glucose-independent activity might be recruited to damaged DNA in rad26 cells in the absence of functional Rad26.

We measured the transcription rates of RPB2 and SSD1 in UV-irradiated rad26 and parent cells by a run-on assay in permeabilized cells. We observed that transcription occurred efficiently following UV irradiation in glucose- or galactose-containing media, although efficient TCR was detected in the rad26 mutant only when grown in galactose. Thus, despite the fact that RPB2 and SSD1 were transcribed in glucose-grown rad26, we did not observe TCR. Therefore, the defect in TCR in the rad26 mutant is due to a defect in repair, rather than a defect in transcription. This is the first report demonstrating that transcription and TCR are distinct events, although they may normally be associated.

Transcription per se is not a prerequisite for remodeling nucleosomes; rather, transcription may occur subsequent to nucleosome remodeling. Nucleosome modifications that are independent of transcription have been reported in yeast for the homothallic switching (HO) endonuclease and the SUC2 promoters (HIRSCHHORN et al. 1992 ; COSMA et al. 1999 ; KREBS et al. 1999 ). The sequential binding of activator heterodimer Swi4/Swi6 and activator Swi5 to the HO promoter followed by SWI/SNF and SAGA binding occur prior to transcription activation (COSMA et al. 1999 ; KREBS et al. 1999 ). We observed transcription of RPB2 and SSD1 under all growth conditions, yet there was no deficient TCR in rad26 cells growing in the presence of glucose. These results suggest that any remodeling process that permits repair to take place is independent of transcription or subsequent to transcription. We emphasize the fact that TCR occurs in the rad26 mutant grown in galactose, an observation that cannot rule out an association between the stalled RNA Pol II and the repair process. Thus, preferential repair of the transcribed strand could be the result of increased binding of remodeling complexes to regulatory elements, or a restriction of nucleosome movement by transcription factor binding to DNA in transcribing genes.

Our results and conditions differ from those of previous reports demonstrating TCR in the galactose-inducible GAL7 gene in the same rad26 mutant that we examined (VERHAGE et al. 1996 ). Verhage and co-workers observed that TCR was present in the GAL7 gene in cells grown in galactose and absent in cells grown in glucose. In addition, the GAL7 gene is induced 1000x in galactose-containing medium and actively repressed in the presence of glucose, not merely off. Active repression requires the inhibition of factor(s) prior to transcription, not simply the lack of transcription initiation. By looking at repair in RPB2 and SSD1, genes shown not to be induced by galactose (ROTH et al. 1998 ), we demonstrate here that TCR may be present or absent in the same gene undergoing transcription.

Our results have important implications for the repair defect in CS patients. It seems unlikely that a general transcription deficiency is responsible for the lack of TCR in CS cells. Paradoxically, it was shown that cells from individuals with CS, in addition to a defect in TCR, have lower transcription as compared to controls following UV irradiation (BALAJEE et al. 1997 ). Although transcription is required to enable TCR (LEADON and LAWRENCE 1992 ; SWEDER and HANAWALT 1992 ), we observed that transcription rate does not correlate with TCR in two different genetic backgrounds (LOMMEL et al. 2000 ; this report). Similarly, a defect in TCR of 8-oxoGuanine in CSB cells is not due to a lack of transcription of the lesion-containing sequence (LE PAGE et al. 2000 ). Thus, a reduction in TCR is not the result of a lack of transcription. We think that the lack of TCR in CS cells is the result of a repair defect and not a transcription defect. Furthermore, we extend our observations in yeast to suggest that the repair deficiency observed in CS following stress (i.e., UV irradiation and ionizing radiation) may be dependent on a developmental growth condition analogous to carbon source utilization in yeast.

	ACKNOWLEDGMENTS

We thank Drs. Kiran Madura, Lenore Neigeborn, Marian Carlson, and Michael Reagan for helpful comments and/or critical reading of this manuscript. M.B. was supported by a Minority Predoctoral Fellowship (1F31 CA 83357-01) from the National Institutes of Health. K.S.S. was supported by Public Health Service grant R29 GM53717 from the National Institutes of Health.

Manuscript received September 18, 2000; Accepted for publication April 23, 2001.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ABOUSSEKHRA, A., M. BIGGERSTAFF, M. K. SHIVJI, J. A. VILPO, and V. MONCOLLIN et al., 1995  Mammalian DNA nucleotide excision repair reconstituted with purified protein components. Cell 80:859-868[Medline].

ADAMS, A., D. E. GOTTSCHLING, C. A. KAISER and T. STEARNS, 1998 Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Plainview, NY.

BALAJEE, A. S., A. MAY, G. L. DIANOV, E. C. FRIEDBERG, and V. A. BOHR, 1997  Reduced RNA polymerase II transcription in intact and permeabilized Cockayne syndrome group B cells. Proc. Natl. Acad. Sci. USA 94:4306-4311[Abstract/Free Full Text].

BOHR, V. A., C. A. SMITH, D. S. OKUMOTO, and P. C. HANAWALT, 1985  DNA repair in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall. Cell 40:359-369[Medline].

CAIRNS, B. R., Y. LORCH, Y. LI, M. ZHANG, and L. LACOMIS et al., 1996  RSC, an essential, abundant chromatin-remodeling complex. Cell 87:1249-1260[Medline].

COSMA, M. P., T. TANAKA, and K. NASMYTH, 1999  Ordered recruitment of transcription and chromatin remodeling factors to a cell cycle- and developmentally regulated promoter. Cell 97:299-311[Medline].

CÔTÉ, J., J. QUINN, J. L. WORKMAN, and C. L. PETERSON, 1994  Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex. Science 265:53-60[Abstract/Free Full Text].

EISEN, J. A., K. S. SWEDER, and P. C. HANAWALT, 1995  Evolution of the SNF2 family of proteins: subfamilies with distinct sequences and functions. Nucleic Acids Res. 23:2715-2723[Abstract/Free Full Text].

FRIEDBERG, E. C., G. C. WALKER and W. SIEDE, 1995 DNA Repair and Mutagenesis. ASM Press, Washington, DC.

GANCEDO, J. M., 1998  Yeast carbon catabolite repression. Microbiol. Mol. Biol. Rev. 62:334-361[Abstract/Free Full Text].

HAPPEL, A. M., M. S. SWANSON, and F. WINSTON, 1991  The SNF2, SNF5 and SNF6 genes are required for Ty transcription in Saccharomyces cerevisiae. Genetics 128:69-77[Abstract].

HIRSCHHORN, J. N., S. A. BROWN, C. D. CLARK, and F. WINSTON, 1992  Evidence that SNF2/SWI2 and SNF5 activate transcription in yeast by altering chromatin structure. Genes Dev. 6:2288-2298[Abstract/Free Full Text].

HOLSTEGE, F. C., E. G. JENNINGS, J. J. WYRICK, T. I. LEE, and C. J. HENGARTNER et al., 1998  Dissecting the regulatory circuitry of a eukaryotic genome. Cell 95:717-728[Medline].

IMBALZANO, A. N., H. KWON, M. R. GREEN, and R. E. KINGSTON, 1994  Facilitated binding of TATA-binding protein to nucleosomal DNA. Nature 370:481-485. [see comments][Medline].

KINGSTON, R. E. and G. J. NARLIKAR, 1999  ATP-dependent remodeling and acetylation as regulators of chromatin fluidity. Genes Dev. 13:2339-2352[Free Full Text].

KINGSTON, R. E., C. A. BUNKER, and A. N. IMBALZANO, 1996  Repression and activation by multiprotein complexes that alter chromatin structure. Genes Dev. 10:905-920[Abstract/Free Full Text].

KREBS, J. E., M. H. KUO, C. D. ALLIS, and C. L. PETERSON, 1999  Cell cycle-regulated histone acetylation required for expression of the yeast HO gene. Genes Dev. 13:1412-1421[Abstract/Free Full Text].

LEADON, S. A. and D. A. LAWRENCE, 1992  Strand-selective repair of DNA damage in the yeast GAL7 gene requires RNA polymerase II. J. Biol. Chem. 267:23175-23182[Abstract/Free Full Text].

LE PAGE, F., E. E. KWOH, A. AVRUTSKAYA, A. GENTIL, and S. A. LEADON et al., 2000  Transcription-coupled repair of 8-oxoGuanine: requirement for XPG, TFIIH, and CSB and implications for Cockayne syndrome. Cell 101:159-171[Medline].

LOMMEL, L., S. GREGORY, K. BECKER, and K. SWEDER, 2000  Transcription-coupled DNA repair in yeast transcription factor IIE (TFIIE) mutants. Nucleic Acids Res. 28:835-842[Abstract/Free Full Text].

LORCH, Y., M. ZHANG, and R. D. KORNBERG, 1999  Histone octamer transfer by a chromatin-remodeling complex. Cell 96:389-392[Medline].

MELLON, I., G. SPIVAK, and P. C. HANAWALT, 1987  Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene. Cell 51:241-249[Medline].

MOREIRA, J. M. and S. HOLMBERG, 1999  Transcriptional repression of the yeast CHA1 gene requires the chromatin-remodeling complex RSC. EMBO J. 18:2836-2844[Medline].

MURPHY, D. J., S. HARDY, and D. A. ENGEL, 1999  Human SWI-SNF component BRG1 represses transcription of the c-fos gene. Mol. Cell. Biol. 19:2724-2733[Abstract/Free Full Text].

NANCE, M. A. and S. A. BERRY, 1992  Cockayne syndrome: review of 140 cases. Am. J. Med. Genet. 42:68-84[Medline].

NASMYTH, K. A. and S. I. REED, 1980  Isolation of genes by complementation in yeast: molecular cloning of a cell-cycle gene. Proc. Natl. Acad. Sci. USA 77:2119-2123[Abstract/Free Full Text].

NEIGEBORN, L. and M. CARLSON, 1984  Genes affecting the regulation of SUC2 gene expression by glucose repression in Saccharomyces cerevisiae. Genetics 108:845-858[Abstract/Free Full Text].

NEIGEBORN, L., K. RUBIN, and M. CARLSON, 1986  Suppressors of SNF2 mutations restore invertase derepression and cause temperature-sensitive lethality in yeast. Genetics 112:741-753[Abstract/Free Full Text].

PETERSON, C. L. and I. HERSKOWITZ, 1992  Characterization of the yeast SWI1, SWI2, and SWI3 genes, which encode a global activator of transcription. Cell 68:573-583[Medline].

PETERSON, C. L. and J. W. TAMKUN, 1995  The SWI-SNF complex: a chromatin remodeling machine? Trends Biochem. Sci. 20:143-146[Medline].

PRAKASH, S. and L. PRAKASH, 2000  Nucleotide excision repair in yeast. Mutat. Res. 451:13-24[Medline].

ROTH, F. P., J. D. HUGHES, P. W. ESTEP, and G. M. CHURCH, 1998  Finding DNA regulatory motifs within unaligned noncoding sequences clustered by whole-genome mRNA quantitation. Nat. Biotechnol. 16:939-945. [see comments][Medline].

SAMBROOK, J., E. F. FRITSCH and T. MANIATIS, 1989 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

SMERDON, M. J., J. BEDOYAN, and F. THOMA, 1990  DNA repair in a small yeast plasmid folded into chromatin. Nucleic Acids Res. 18:2045-2051[Abstract/Free Full Text].

SWEDER, K. S. and P. C. HANAWALT, 1992  Preferential repair of cyclobutane pyrimidine dimers in the transcribed strand of a gene in yeast chromosomes and plasmids is dependent on transcription. Proc. Natl. Acad. Sci. USA 89:10696-10700[Abstract/Free Full Text].

SWEDER, K. S. and P. C. HANAWALT, 1994  The COOH terminus of suppressor of stem loop (SSL2/Rad25) in yeast is essential for overall genomic excision repair and transcription-coupled repair. J. Biol. Chem. 269:1852-1857[Abstract/Free Full Text].

SWEDER, K. S., R. A. CHUN, T. MORI, and P. C. HANAWALT, 1996  DNA repair deficiencies associated with mutations in genes encoding subunits of transcription initiation factor TFIIH in yeast. Nucleic Acids Res. 24:1540-1546[Abstract/Free Full Text].

UESONO, Y., A. TOH-E, and Y. KIKUCHI, 1997  Ssd1p of Saccharomyces cerevisiae associates with RNA. J. Biol. Chem. 272:16103-16109[Abstract/Free Full Text].

VAN GOOL, A. J., R. VERHAGE, S. M. SWAGEMAKERS, P. VAN DE PUTTE, and J. BROUWER et al., 1994  RAD26, the functional S. cerevisiae homolog of the Cockayne syndrome B gene ERCC6. EMBO J. 13:5361-5369[Medline].

VAN HOFFEN, A., A. T. NATARAJAN, L. V. MAYNE, A. A. VAN ZEELAND, and L. H. F. MULLENDERS et al., 1993  Deficient repair of the transcribed strand of active genes in Cockayne's syndrome cells. Nucleic Acids Res. 21:5890-5895[Abstract/Free Full Text].

VENEMA, J., L. H. F. MULLENDERS, A. T. NATARAJAN, A. A. VAN ZEELAND, and L. V. MAYNE, 1990  The genetic defect in Cockayne syndrome is associated with a defect in repair of UV-induced DNA damage in transcriptionally active DNA. Proc. Natl. Acad. Sci. USA 87:4707-4711[Abstract/Free Full Text].

VERHAGE, R. A., A. J. VAN GOOL, N. DE GROOT, J. H. HOEIJMAKERS, and P. VAN DE PUTTE et al., 1996  Double mutants of Saccharomyces cerevisiae with alterations in global genome and transcription-coupled repair. Mol. Cell. Biol. 16:496-502[Abstract].

WARNER, J. R., 1991  Labeling of RNA and phosphoproteins in Saccharomyces cerevisiae. Methods Enzymol. 194:423-428[Medline].

WERNER-WASHBURNE, M., J. BECKER, J. KOSIC-SMITHERS, and E. A. CRAIG, 1989  Yeast Hsp70 RNA levels vary in response to the physiological status of the cell. J. Bacteriol. 171:2680-2688[Abstract/Free Full Text].

WINSTON, F. and M. CARLSON, 1992  Yeast SNF/SWI transcriptional activators and the SPT/SIN chromatin connection. Trends Genet. 8:387-391[Medline].

This article has been cited by other articles:

				Z. Feng, W. Hu, E. Komissarova, A. Pao, M.-C. Hung, G. M. Adair, and M.-s. Tang Transcription-coupled DNA Repair Is Genomic Context-dependent J. Biol. Chem., April 5, 2002; 277(15): 12777 - 12783. [Abstract] [Full Text] [PDF]