Molecular characterization of an acidic region deletion mutant of

© 2000 Oxford University Press

Nucleic Acids Research, 2000, Vol. 28, No. 16 3151–3159

Molecular characterization of an acidic region deletion mutant of Cockayne syndrome group B protein Morten Sunesen, Rebecca R. Selzer1, Robert M. Brosh Jr1, Adayabalam S. Balajee1, Tinna Stevnsner and Vilhelm A. Bohr1,* Department of Molecular and Structural Biology, University of Aarhus, DK-8000 Aarhus C, Denmark and 1Laboratory of Molecular Genetics, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA Received March 15, 2000; Revised and Accepted June 22, 2000

ABSTRACT Cockayne syndrome (CS) is a human genetic disorder characterized by post-natal growth failure, neurological abnormalities and premature aging. CS cells exhibit high sensitivity to UV light, delayed RNA synthesis recovery after UV irradiation and defective transcription-coupled repair (TCR). Two genetic complementation groups of CS have been identified, designated CS-A and CS-B. The CSB gene encodes a helicase domain and a highly acidic region N-terminal to the helicase domain. This study describes the genetic characterization of a CSB mutant allele encoding a full deletion of the acidic region. We have tested its ability to complement the sensitivity of UV61, the hamster homolog of human CS-B cells, to UV and the genotoxic agent N-acetoxy2-acetylaminofluorene (NA-AAF). Deleting 39 consecutive amino acids, of which ∼60% are negatively charged, did not impact on the ability of the protein to complement the sensitive phenotype of UV61 cells to either UV or NA-AAF. Our data indicate that the highly acidic region of CSB is not essential for the TCR and general genome repair pathways of UV- and NA-AAF-induced DNA lesions. INTRODUCTION Cockayne syndrome (CS) is a rare genetic disorder with an autosomal recessive inheritance pattern. Afflicted individuals suffer from growth retardation, cachetic dwarfism, mental retardation and progressive neurological dysfunction and accelerated aging (1). At the cellular level, CS is characterized by high sensitivity to UV light and, following UV exposure, CS cells exhibit reduced survival and delayed recovery of RNA synthesis even though unscheduled DNA synthesis remains normal (2). These observations have been explained by the fact that CS cells are defective in the accelerated rate of repair of the transcribed strand of active genes, transcriptioncoupled repair (TCR), while maintaining proficient global

genome repair (GGR) of UV-induced DNA damage (3–5). The defective TCR in CS cells is not only found after UV irradiation but is also seen after exposure to certain agents that cause oxidative DNA damage (6–8). The CSB gene was originally cloned by its ability to complement the UV-sensitive phenotype displayed by the hamster mutant cell line UV61 (9). More recently, gene-specific repair studies have demonstrated that the human CSB gene corrects the TCR defect in the hamster CS-B cell line UV61 (10). Characterization of the CSB gene revealed that it harbors an acidic amino acid stretch, a glycine-rich region, two putative nuclear localization signal sequences and seven putative helicase domains (11). Based on sequence homology, CSB belongs to the SWI/SNF family of helicases from superfamily 2 (12). All proteins in this family contain seven conserved helicase motifs similar to those found in DNA/RNA helicase families, however, no overt DNA unwinding activity has been shown for any of the proteins from this family (13). The CSB protein is a DNA-stimulated ATPase but fails to unwind DNA as measured by conventional helicase assays (14,15). Members of the SWI/SNF family are involved in transcription regulation, chromatin remodeling and DNA repair, which include actions such as disruption of protein–protein and protein–DNA interactions (reviewed in 16). The CSB gene product could possibly have a similar function. The molecular mechanism of TCR in mammalian cells and the role played by the CSB protein in this repair pathway remain elusive (13,17). Several recent in vitro studies found a direct link between CSB and RNA polymerase II (RNAPII) transcription elongation. The CSB protein interacts with the elongating form of RNAPII and stimulates its transcription elongation activity (15,18,19). Furthermore, a RNAPII–CSB– DNA–RNA quaternary complex has the ability to interact with two subunits of the basal transcription and repair factor TFIIH (20). It is speculated that CSB may facilitate repair of active RNAPII genes by recruiting repair factors to the site of DNA damage (14,20). Recent studies have implicated the CSB protein directly or indirectly in other cellular processes than TCR. CS-B cells are hypersensitive to the genotoxic agent N-acetoxy-2acetylaminofluorene (NA-AAF). In mammalian cells this carcinogen generates dG-C8–AF adducts (21), which are

*To whom correspondence should be addressed. Tel: +1 410 558 8162; Fax: +1 410 558 8157; Email: [email protected] Present address: Adayabalam S. Balajee, Center for Radiological Research, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA

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mainly repaired by GGR. This observation suggests that the hypersensitivity to NA-AAF in CS-B cells is not due to a TCR impairment per se, but rather a defect in either recruiting essential repair/transcription factors or recycling them. Also, it was demonstrated that CS-B cell lines have slower kinetics of proliferating cell nuclear antigen (PCNA) relocation after both UV and oxidative damage (22,23), which suggests that CSB is involved in recruiting various repair factors to the site of DNA damage. Furthermore, CS cells undergo apoptosis at much lower doses of DNA damaging agents than normal cells (24). The blockage of RNAPII at the lesion is thought to be the trigger for apoptosis and the increased apoptosis in CS cells is attributed to defective repair of the transcribed strand (25,26). Recently we demonstrated that a fully functional CSB protein with an intact ATPase domain is required to modulate the UV-induced apoptotic response (27). However, it has not been investigated whether other domains of the CSB protein are equally important for the increased apoptosis seen after DNA damage in CS-B cells. Altogether, information addressing the specific function of various domains in the CSB protein is still very limited. In a recent study, we demonstrated that a point mutation in motif II in the ATPase domain of the CSB protein completely abolishes its ability to function in survival, RNA synthesis recovery, gene-specific repair and apoptosis upon UV irradiation of Chinese hamster ovary cells (27,28). Also, Citterio et al. (29) recently showed that motif I of the ATPase domain may be partly dispensable for RNA synthesis recovery in human CS-B cells, indicating that other domains are also essential for the biological function of CSB. In addition to the helicase and ATPase domains, the CSB protein contains a region in the N-terminal portion, which is strikingly rich in acidic amino acids (60% of a 39 amino acid stretch are acidic). Such an acidic region is a common feature found in a number of nuclear proteins, e.g. proteins involved in transcriptional activation (30,31), regulation of DNA binding (32) and protein–protein interactions (33,34). The fact that CSB belongs to a family of proteins of which some members are involved in chromatin remodeling and specific DNA– protein interactions (reviewed in 16) suggests that the acidic region may mediate an important function of the CSB protein. In addition to a putative role in chromatin remodeling, acidic regions have been characterized as protein domains involved in transcriptional activation (35,36). In fact it has been shown that acidic transcriptional activation domains can interact directly with factors from the basal RNAPII transcriptional machinery (37–39). Furthermore, acidic regions have very recently been implicated in targeting the chromatin-modifying activities of histone acetyltransferase and the SWI/SNF complex to specific promoter sequences where transcription requires the remodeling or modification of nucleosomes (40,41). In nucleotide excision repair (NER), the putative damage recognition factor XPA interacts with the ERCC1 subunit of the ERCC1-XPF repair nuclease (33). Mutational studies delineated the minimal ERCC1-binding region of XPA to a cluster of acidic amino acids (33,42) and if the cluster was removed, the DNA binding activities of XPA were destroyed (42), as well as the ability to complement the UV-sensitive phenotype of an XP-A cell line (43). Thus far the functional significance of the region strikingly rich in acidic amino acids in the CSB protein remains unknown, but it may play an

important role in the repair and/or transcription functions of CSB. To better understand the functional role of various domains of the CSB protein, the construction of site-specific CSB mutants was undertaken. Recent CSB mutant constructions have demonstrated an absolute requirement for an intact ATPase domain in biochemical and genetic complementation studies of the UV-sensitive phenotype of CS-B (28,29). The present study describes the generation and characterization of a full acidic region deletion mutant of the CSB protein in the repair-deficient hamster cell line UV61. MATERIALS AND METHODS Cell lines and culture conditions The AA8 and UV61 cell lines used in this study and their repair characteristics were previously described (44). AA8 is a repairproficient wild-type hamster cell line. The UV61 cell line, derived from AA8, belongs to rodent complementation group 6 and is homologous to human CS-B cells. UV61 cells transfected with the mammalian expression vector pcDNA3.1 (abbreviated to pc3.1; Invitrogen, San Diego, CA) or pc3.1 containing the wild-type human CSB gene are designated UV61/pc3.1 and UV61/pc3.1-CSBwt, respectively. UV61 cells transfected with pc3.1-CSB containing a 39 amino acid deletion of the acidic region are designated UV61/pc3.1CSB∆Ac. All the cells were routinely grown in Ham’s F-10 and DMEM (1:1) media containing 400 µg/ml geneticin (Gibco BRL, Paisley, UK). Construction of pc3.1-CSB mutant and wild-type constructs pcBLsSE, a bacterial phagemid containing the f1 origin of replication and the entire human CSB gene, was used for sitedirected mutagenesis by published procedures (45). A description of the deletion mutation introduced in the CSB gene is shown in Figure 1. The oligonucleotide 5'-ACCATCTCCAGACAGCCAAGGTCTCCTTGC-3' was used to precisely delete 39 amino acids (356–394) in the CSB protein. The CSB gene, containing the wild-type or mutant sequence, was cloned into the mammalian expression vector pc3.1 to yield pc3.1-CSB by previously described methods (28). The site-directed mutation in the CSB gene was verified by sequencing of RT–PCR products. The entire CSB coding sequence of pc3.1-CSBwt was sequenced to verify that the coding sequence was intact. Selection of stable transfectant cell lines CsCl-purified pc3.1-CSB plasmids (100 µg) were linearized with PvuI (200 U) at 37°C for 16 h. The DNA sample was treated with proteinase K (200 µg/ml) in 1 M lithium acetate and 0.1% SDS at 50°C for 2 h, phenol extracted and ethanol precipitated. UV61 cells were transfected with pc3.1 and pc3.1-CSB plasmids using liposomes (N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammonium-methyl sulfate; Boeringer Mannheim, Indianapolis, IN) according to the manufacturer’s procedure. The transfected cells were trypsinized and transferred to a 10 cm2 dish and geneticin was added to a final concentration of 400 µg/ml for selection of antibiotic-resistant cells. After 10 days selection, the surviving

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cells were trypsinized and seeded for isolation of clones (50 cells/10 cm2 dish). Individual colonies were isolated and re-seeded for UV and NA-AAF experiments.

orthodiagnostic cytofluorograph system equipped with a 488 nm excitation source was used to measure the DNA content of the cells.

UV and NA-AAF clonogenic survival assays

Detection of apoptosis by DNA fragmentation

UV61 transfectant cell lines were trypsinized and 300 cells were seeded per 10 cm dish and allowed to grow for 16 h. For UV treatment, the cells were irradiated with the indicated doses of UVC light (254 nm). The cells were grown for 7 days, washed with phosphate-buffered saline (PBS), fixed with methanol and stained with methylene blue. The stained cells were washed and blue colonies were counted to determine the clonogenic survival of cells. Treatment of cells with NA-AAF was essentially performed according to previously published protocols (21). In brief, cells were incubated with the indicated concentrations of NA-AAF for 30 min at 37°C in complete medium. After incubation the cells were washed twice with PBS and fresh medium was added. The cells were then treated as described above to determine colony-forming units.

For detection of DNA fragmentation after UV exposure, ∼2 × 106 cells were seeded in a 10 cm2 dish and allowed to grow for 2 days. Cells were then irradiated with 6 J/m2 UVC (254 nm) and both the attached and floating cells were harvested 36 h post-treatment. The cell pellet was resuspended in 1 ml of lysis buffer (50 mM Tris–HCl, pH 8.0, 20 mM EDTA, 150 mM NaCl, 50 µg/ml proteinase K and 0.5% SDS) and incubated overnight at 37°C. Genomic DNA was isolated by the salting out procedure (48). RNA was digested by treatment with DNase-free RNase (10 µg/ml) for 3 h at 37°C. DNA was pelleted and resuspended in TE buffer. An aliquot of 10 µg DNA from each cell line was electrophoresed on a 1% agarose gel and analyzed for the presence of nucleosomal laddering indicative of apoptosis (49).

Gene-specific repair

PCNA complex formation: protein extraction and western blotting

Treatment of cells in culture with UV light and subsequent isolation of genomic DNA were essentially performed according to previously published techniques (46). DNA was resuspended in TE buffer and the concentration was measured spectrophotometrically at 260 nm. The number of cyclobutane pyrimidine dimers (CPDs) per 14 kb CHO dihydrofolate reductase gene (DHFR) fragment was estimated using T4 endonuclease V as described previously (47). T4 endonuclease V-treated and untreated DNA samples (10 µg) were electrophoresed under alkaline conditions on a 0.5% agarose gel. DNA was transferred to a Hybond N+ membrane (Amersham Pharmacia Biotech, Piscataway, NJ) by Posiblot (Stratagene, La Jolla, CA) using standard protocols. The membrane was treated in prehybridization buffer (0.342 M Na2HPO4 + 0.088 M NaH2PO4, pH 7.2, 7% SDS and 2 mM EDTA) for a minimum of 2 h. Double-stranded DNA probes were prepared with a random primed labeling kit (Amersham Pharmacia Biotech) using a 3.4 kb DHFR template. Membranes were hybridized with the probe at 68°C overnight and non-hybridizing probe was removed in stringent washes. The blots were visualized by PhosphorImager analysis and quantitated using ImageQuant software. Fluorescence staining detection of apoptotic cells after UV irradiation A mixture of propidium iodide (PI) (0.5 µg/ml) and fluorescein diacetate (FDA) (5 µg/ml) was used to distinguish apoptotic and necrotic cells from viable cells. UV61 cell lines were nonirradiated or irradiated with either 3 or 6 J/m2 UVC (254 nm) and stained with FDA/PI 36 h post-irradiation. The cells were examined under a microscope and representative images of apoptotic and viable cells were captured for each cell line and time point. Cell cycle analysis Cells were scraped off and washed with PBS and fixed in 70% ethanol. Fixed cells were centrifuged and washed once with PBS before RNase treatment (100 µg/ml, 30 min at 37°C). Subsequently, the cells were stained with PI (1 µg/ml, 30 min at room temperature) prior to DNA content analysis. An

Cells were grown to ∼80% confluence in 10 cm2 dishes, washed with PBS and grown in a low serum-containing medium (0.2% serum) for at least 3 days. Cells were washed and either non-irradiated or irradiated with 20 J/m2 UVC (254 nm). The cells were then incubated for 30 min in low serum-containing medium and proteins were extracted according to previously published protocols (50). The obtained protein samples were sonicated for 30 s and protein concentration was determined using the Bradford Protein Assay kit (Bio-Rad, Hercules, CA). Insoluble proteins (25 µg) from each cell line were separated on a NuPage 4–12% Bis–Tris gel (Novex, San Diego, CA) and transferred onto PVDF membrane following standard procedures (Novex). The membrane was incubated with TBS-T (20 mM Tris–HCl, pH 7.4, 137 mM NaCl and 0.2% Tween 20) containing 5% non-fat dried milk (NFDM) for a minimum of 60 min at room temperature. The membrane was subsequently incubated with mouse monoclonal anti-PCNA antibody (PC10; Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:1000 dilution in TBS-T + 5% NFDM for 1 h, followed by three washes in TBS-T. Horseradish peroxidase-conjugated anti-mouse IgG antibody (Jackson Immunochemicals, West Grove, PA) was used as secondary antibody (1:2000) and incubated with the membrane for 1 h in TBS-T + 5% NFDM. After repeated washes in TBST, the PCNA signal was detected using the Western Blotting Luminol Reagent (Santa Cruz Biotechnology). RESULTS Establishment of UV61 transfectant cell lines We have previously characterized a 10 acidic amino acid deletion and a four acidic amino acid substitution in the N-terminal acidic region of the CSB protein and found no effect of these mutations (28). Because the remaining acidic residues in the highly acidic region may have been sufficient to retain function in TCR, we constructed a site-specific mutation in the CSB gene, CSB∆Ac, which encodes a mutant with all 39 amino acids of the acidic region deleted (∼60% acidic amino acids)


Figure 1. A schematic presentation of the CSB protein containing the conserved ATPase/helicase motifs and the acidic region. A site-directed mutation was introduced in the acidic region precisely deleting 39 consecutive amino acids, of which 22 (56%) are acidic residues. UV61/pc3.1-CSBwt and UV61/ pc3.1-CSB∆Ac denote isogenic UV61 transfectant cells harboring the wildtype CSB gene or the acidic deletion mutant, respectively. The previously characterized mutations either substituted acidic residues at positions 381, 382, 384 and 385 with alanines or encoded a deletion of 10 consecutive acidic residues (378–387) of the CSB protein (see also 28).

(Fig. 1). To obtain isogenic stable transfectants, we followed the same cloning and transfection strategy as previously published (28; see also Materials and Methods). Analysis of CSB expression Whole cell lysates and cytoplasmic and nuclear extracts of hamster CS-B transfectant cell lines were tested by immunoblot to detect CSB protein using a rabbit polyclonal anti-CSB antibody raised against the C-terminal 158 amino acids of CSB, overproduced as a protein A fusion product in Escherichia coli graciously provided by Dr J. Hoeijmakers (Erasmus University, Rotterdam) (14). Although the anti-CSB antibody effectively reacted with native CSB protein expressed in human cell lines GM0637D and HeLa by immunoblot analysis (unpublished results), we were unable to detect CSB protein in the UV-resistant hamster cell line UV61/ pc3.1-CSBwt. We were able to detect CSB protein expression in the human CSB stable transfectant cell lines CS1AN/ pc3.1-CSBwt and CS1AN/pc3.1-CSB∆Ac at an equal level (Selzer et al., unpublished data). Thus the CSB protein containing the 39 amino acid deletion of the acidic region is stably expressed in the human CS-B cell line. The immunoblot analysis of hamster CS-B transfectant cell lines also proved negative using an affinity purified rabbit polyclonal antibody against a recombinant fragment of the CSB protein (amino acids 528–1222) kindly provided by Drs C. Selby and A. Sancar (University of North Carolina, Chapel Hill, NC) (15). The lack of detection of CSB protein in the hamster transfectant cell lines may be accounted for by several explanations. The level of CSB protein may be quite low in the hamster CSB transfectant cell lines, but at a sufficient level to complement the TCR deficiency by the wild-type CSB protein. Alternatively, the CSB protein may be poorly recognized by the CSB antibody when the human protein is present in a preparation (whole cell lysate or cytoplasmic or nuclear fraction) in a background of hamster proteins. To quantitate the relative levels of CSB expression in UV61/ pc3.1-CSBwt and UV61/pc3.1-CSB∆Ac, we performed relative quantitative RT–PCR experiments (QuantumRNA module; Ambion, Austin, TX). The QuantumRNA module provides a method for comparing relative transcript abundances

standardized by co-amplification of a highly conserved fragment of 18S rRNA as an invariant internal standard. The CSB transcript and 18S rRNA were analyzed during the exponential phase of amplification to determine an accurate level of relative expression. Initial experiments were performed to determine the optimal conditions for the linear range of amplification of the CSB fragment. Once this was established, the amplification efficiency of the 18S rRNA standard was optimized to the efficiency of amplification of the CSB transcript by adjusting the ratio 18S:Competimers. Competimers are modified 18S rRNA primers blocked at their 3'-ends to prevent extension by DNA polymerase (QuantumRNA module; Ambion). PCR cycling of the CSB fragment and 18S RNA standard was performed in a single tube. For CSB cDNA amplification, the primers 5'-GGTGTTAGGTGGCTGTGGGAATT-3' (3F) and 5'-GTATCTCGTAAGACACACATGCACAC-3' (3R) were used, which produce a 671 bp product from the coding sequence of CSB mRNA. The RNA used for cDNA synthesis was isolated from UV61 transfectant cell lines by the procedure described above. cDNA synthesis was performed by incubating 2.5 µg total RNA, 250 ng random decamer primer (Ambion) and 500 U Superscript II at 42°C for 50 min. For PCR amplification of cDNA products, 5 U AmpliTaq Gold (Perkin Elmer, Norwalk, CT) was used according to the manufacturer’s procedures. In addition to the standard reaction components, PCR mixtures contained 20 pmol CSB primers 3F and 3R and 20 pmol 18S RNA primer:Competimer (9:1) in a total volume of 50 µl. Reaction products were amplified by 31 cycles (94°C, 1 min; 67°C, 1.5 min; 72°C, 3 min). The reaction products (10 ml) were loaded on a 1.2% agarose–1× Tris–borate/EDTA gel. DNA species were visualized with Sybr-Green stain using a Fluoroimager and quantitated using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The ratio of CSB transcript to 18S RNA standard (671 nt CSB fragment/488 nt 18S RNA internal standard) for UV61/pc3.1-CSBwt and UV61/pc3.1-CSB∆Ac were determined to be 0.8 and 0.9, respectively (data not shown). The results demonstrate that the wild-type and mutant CSB transcripts are expressed with approximately the same efficiency in the UV61 transfectant cell lines. UV survival Using the classical clonogenic survival assay, we determined the effect of UV irradiation on the survival rate of the UV61 transfectant cell lines compared with the parental cell line AA8. As previously demonstrated, transfection of UV61 with the CSB wild-type allele restored UV resistance to a level comparable with that in the parental wild-type cell line AA8 (10,28). Similarly, CSB∆Ac conferred UV-resistance to a level equal to UV61/pc3.1-CSBwt and AA8 (Fig. 2), suggesting that the acidic region of CSB is not critical for cell survival after UV-induced DNA damage. Gene-specific repair CS-B cells fail to show preferential repair of transcriptionally active genes after UV irradiation (3,5). To directly assess if the acidic region of CSB plays any significant role in TCR, we sought to determine whether transfection of the CSB∆Ac mutant allele into UV61 was able to change the repair capability of the cells. The accumulated results of at least three independent quantitative Southern blots measuring the amount


Figure 2. Clonogenic UV survival assay. AA8 and isogenic clonal populations of UV61 transfectant cell lines were irradiated as described in Materials and Methods. Survival data is expressed as the fraction of UV-irradiated cells for ing colonies compared to the non-irradiated cells and represents the average ± SD of at least three independent experiments.

of CPDs in the DHFR gene at 8 and 24 h after UVC irradiation is presented in Table 1. Sixty-three percent of the CPDs in the DHFR gene were repaired after 24 h in UV61/pc3.1-CSB∆Ac, compared to 71% in AA8 and 46% in UV61/pc3.1-CSBwt. As expected, the UV61/pc3.1 cell line is almost completely defective in gene-specific repair of DHFR. Our results demonstrate essentially the same repair capacity for AA8 and UV61/pc3.1CSB∆Ac, suggesting that the acidic region is dispensable for the function of CSB in TCR. Table 1. Induction and removal of CPDs in the DHFR gene Cell line

Initial CPDsa

Repair (%) 8h

24 h

AA8

1.55 ± 0.05

35.5 ± 4.95

71.5 ± 10.6

UV61/pc3.1-CSBwt

1.53 ± 0.06

20.2 ± 10.7

46.3 ± 15.28

UV61/pc3.1-CSB∆Ac

1.61 ± 0.13

32.8 ± 6.41

63.0 ± 13.2

UV61/pc3.1

1.39 ± 0.06

5.7 ± 5.41

–4.6 ± 6.8

Mean ± SD are calculated from three independent experiments. aInitial CPD is presented as the average number of CPDs per DHFR fragment (14 kb).

Apoptosis in UV61 transfectant cell lines We next examined the apoptotic response of the UV61 transfectant cell lines after UV irradiation. The introduction of CPDs into DNA after UV irradiation causes a block to the elongating RNAPII (51). Presumably, the blocked polymerase signals an apoptotic event (25). It has been shown that CS cells are more prone to undergo UV-induced apoptosis, suggesting that the blockage of RNAPII at unrepaired CPDs on the transcribed strand is responsible for the onset of apoptosis (24,27).

We have previously shown that the functional integrity of the ATPase domain of CSB is essential for its anti-apoptotic function while two minor mutations in the acidic region are not (27). To gain further insight into the significance of the acidic region of CSB in UV-induced apoptosis, we employed both microscopic and cell sorting analyses. PI and FDA staining was used as a qualitative tool to visualize UV-induced apoptosis and cell sorting as a quantitative method. FDA only stains the cytoplasm and nucleus of viable cells, while necrotic and apoptotic cells are stained with PI. We tested apoptotic cell death in the UV61 transfectant cell lines after UV irradiation. While apoptosis was efficiently induced in UV61/pc3.1, the UV61/pc3.1-CSB∆Ac cell line turned out to be as resistant to UV-induced apoptosis as UV61/pc3.1-CSBwt, approximately 5–10-fold more resistant to UV-induced programmed cell death than the vector control (Fig. 3A and B). In addition to fluorescent staining and sub-G1 quantification, the nucleosomal degradation pattern, a characteristic feature of apoptotic cells (52), was also analyzed. The UV61 transfectant cell lines were irradiated with 6 J/m2 and DNA was isolated 36 h later. In agreement with the microscopic and fluorescence results, UV-induced laddering was only observed in the UV61 cell line transfected with the vector alone (Fig. 4). UV61/ pc3.1-CSBwt and UV61pc3.1-CSB∆Ac both demonstrated resistance to UV-induced apoptosis, as judged by the nucleosomal degradation pattern (Fig. 4). Taken together, these results suggest that the acidic region of CSB is not important in rescuing the cells from the apoptotic pathway of cell death after UV irradiation. PCNA relocation PCNA is an indispensable factor in both replication and NER. The common function of PCNA in these processes is to assist in initiation of DNA synthesis. Upon UV irradiation, PCNA relocates to the chromatin where it mediates a number of protein interactions (53). The relocation of PCNA after UV irradiation seems to reflect the DNA synthesis step in NER. In a recent study, it was demonstrated that PCNA relocation after UV irradiation was at an intermediate level in UV61 cells compared to NER-proficient and NER-deficient cell lines (22). The defect was attributed to abrogated TCR but may involve alternative protein–protein interactions of the CSB protein. To investigate whether the acidic deletion mutant of the CSB protein affects the UV-induced relocation of PCNA, we irradiated the UV61 transfectant cell lines with 20 J/m2 and harvested soluble and insoluble protein fractions after 30 min (see Materials and Methods). In agreement with previously published results, the PCNA relocation observed in UV61/ pc3.1 30 min post-incubation was 61% of that seen in UV61 cells transfected with the wild-type CSB gene (Fig. 5A and B). The CSB mutant allele CSB∆Ac was able to complement the PCNA relocation deficiency of UV61 to a level comparable to the wild-type level. This suggests that the acidic region of CSB is not involved in the protein–protein interactions leading to relocation of PCNA from the soluble to the insoluble protein fraction. NA-AAF survival studies The UV-mimetic DNA-damaging agent NA-AAF induces bulky lesions in DNA that are processed by NER. NA-AAF


A

B

Figure 3. Analysis of UV-induced apoptosis in UV61 transfectant cell lines. (A) Cells in exponential growth were irradiated as described in Materials and Methods and stained with FDA and PI to detect apoptotic cell death. The viable FDA stained cells appeared green, while the necrotic and apoptotic cells appeared red in color. Representative images for the individual cell lines were captured 36 h after UV irradiation. (B) Cells entering apoptosis were quantitated using fluorescenceactivated cell sorting analysis. Apoptosis data is expressed as the fraction of UV-irradiated cells with sub-G1 DNA content compared to the irradiated UV61/pc3.1 cells and represents the average ± SD of four independent experiments.

reacts preferentially with guanine and generates the deacetylated form (dG-C8–AF) and the acetylated form (dG-C8–AAF). It has been shown that the major lesion induced by NA-AAF in mammalian cells is dG-C8–AF, which is removed by CS cells with the same kinetics as in normal cells (21). However, human CS cell lines are sensitive to the cytotoxic effects of NA-AAF and lack the ability to resume RNA synthesis after treatment with this agent (54), indicating that processes other than TCR are defective in human CS cells. We tested hamster transfectant cell lines for sensitivity to NA-AAF treatment and asked if the acidic region of CSB had any functional role in this TCR-independent pathway. The UV61 transfectant cell lines were evaluated for survival as a function of NA-AAF dose (Fig. 6). In parallel with the human CS-B cell lines (21), we found that UV61/pc.3.1, the hamster homolog of CS-B, is highly sensitive to NA-AAF treatment and that wild-type CSB is able to complement UV61 in resistance to NA-AAF. This is the first demonstration of complementation of NA-AAF sensitivity in UV61 cells by the human CSB gene. Moreover, transfection of the acidic mutant CSB allele CSB∆Ac was also able to complement the NA-AAF sensitivity to a level similar to that of the wild-type CSB allele. We conclude that UV61, the hamster homolog of the human CS-B cell line, exhibits the same sensitivity pattern to the genotoxic agent NA-AAF and that the acidic region of the CSB protein is not important for the TCR-independent pathway of processing NA-AAF-induced DNA damage. DISCUSSION Using genetic and biochemical approaches, we have evaluated the repair function of the acidic region of the CSB gene in both the TCR and GGR pathways. CSB∆Ac encodes a CSB mutant which contains a deletion of 39 consecutive amino acids of

which ∼60% are negatively charged. Surprisingly, we found that the CSB∆Ac mutant allele of the CSB gene was able to complement UV61 cells in various genetic and biochemical assays to the same level as that observed when the UV61 cell line was complemented with the wild-type CSB gene. The acidic region deletion mutant of CSB functions similarly to wild-type CSB in cell survival, gene-specific repair of CPDs, apoptosis and PCNA relocation after UV irradiation. The acidic region deletion mutant of CSB was also able to complement the sensitivity of UV61 to the genotoxic agent NA-AAF. These results indicate that the acidic region of the CSB protein is not required for CSB function in these general pathways. Structure–function analysis of the CSB protein is still at a very early stage. Citterio et al. (29) reported that replacement of an invariant lysine with an arginine (K538R) in motif I of the ATPase domain abolished the ATPase activity of the protein. When this CSB mutant protein was microinjected into the cytoplasm of human CS-B cells, the authors reported a 38% recovery of RNA synthesis after UV treatment when compared to transcription in non-irradiated wild-type cells. This suggested that additional domains of the CSB protein are involved in resumption of RNA synthesis after UV treatment. In a previous paper we characterized the functional significance of a mutation in motif II of the ATPase domain in CSB. We substituted a conserved glutamic acid residue in the Mg2+binding pocket with an uncharged glutamine residue (28) and found that a point mutation in motif II of the ATPase domain completely abolished the ability of the CSB protein to complement the UV-sensitive phenotype of UV61 in survival, RNA synthesis recovery and gene-specific repair (28). Structure– function studies of this kind indicate that CSB participates in rather complex scenarios where different domains may play individual roles.


Figure 4. Agarose gel electrophoretic analysis of UV-induced DNA fragmentation in UV61 transfectant cell lines. Cells in exponential growth phase were either irradiated with UV at 6 J/m2 or non-irradiated. Genomic DNA was harvested at 36 h post-UV irradiation. Genomic DNA (10 µg) from non-irradiated (–) and irradiated (+) cells was loaded onto a 1% agarose gel to determine the nucleosome-like degradation pattern characteristic of apoptotic cell death. The sizes of four HindIII restriction fragments of λ are shown on the left.

We previously examined two minor CSB mutations of the acidic region. CSB mutants harboring either a 10 acidic amino acid deletion or a four acidic amino acid substitution were characterized but no overt effect was found (28). However, the possibility existed that the remaining 11 acidic residues of the acidic region were able to fulfill the requirements of CSB. Similarly, a mutant in the acidic region of the RAD6 protein, deleting 19 out of 23 residues, retained considerable activity when compared to a complete deletion of the acidic region (55). We examined this possibility in this report by deleting the entire acidic region of CSB, excluding any residual function mediated by acidic amino acids remaining in this region of the CSB protein. A number of studies have reported that acidic regions play key roles in transactivation of transcription (56,57). Transcription deficiencies have also been observed in cells from CS-B patients (58–60) and, in a reconstituted system, purified CSB enhanced the rate of transcription (19). Also, SWI/SNF is a chromatin remodeling complex that activates/facilitates the expression of certain genes (16), and it was recently shown that one mechanism of targeting the SWI/SNF complex to the promoter region of specific genes is via an interaction with an acidic activator (40,41). It would therefore be tempting to speculate that CSB may have combined a SWI/SNF-like function with an endogenous acidic region responsible for gene targeting.

Figure 5. UV-induced PCNA relocation in UV61 transfectant cell lines. Cells were arrested at G1 by serum starvation and UV irradiated (+) or non-irradiated (–). The soluble and insoluble protein fractions were harvested 30 min postUV irradiation. (A) The detergent-insoluble protein fractions (25 µg) were analyzed by 4–12% SDS–PAGE, blotted onto PVDF membrane and reacted with anti-PCNA and anti-lamin B antibodies. Lamin B serves as an internal control for equal loading. (B) Histogram of the relative fluorescence intensity of PCNA relocation expressed as the ratio between PCNA in the insoluble protein fraction after UV irradiation compared to PCNA in the non-irradiated control fraction. Data is expressed relative to PCNA relocation in UV61/pc3.1CSBwt. Loading differences were corrected for using the lamin B signal.

Another role undertaken by an acidic protein region is the mediation of protein–protein interactions. It has been suggested that CSB could function as a factor recruiting other proteins (14,20). This function could be mediated through the acidic region. Makino et al. very recently demonstrated that the TATA-binding protein-interacting protein 120 was able to stimulate RNAPII transcription by recruiting RNAPII to the promoter site and that an acidic region was required for this function (61). Also, one of the putative damage recognition factors in NER, XPA, harbors an acidic region that is essential for interaction with the 5' NER endonuclease ERCC1-XPF (33,42). It has been suggested that XPA serves as a scaffold for the DNA damage-dependent assembly of the human excision nuclease complex (62). Deleting the acidic amino acid cluster in XPA disrupted the interaction with ERCC1 and destroyed its ability to function properly in repair (43). CSB has also been suggested to be involved in recruiting proteins to the lesion in TCR (18,21). In analogy to XPA, the acidic region of CSB


Figure 6. NA-AAF sensitivity of isogenic clonal populations of UV61 transfectant cell lines. Cells were treated as described in Materials and Methods. Survival data are expressed as the fraction of NA-AAF-treated cells forming colonies compared to the non-treated cells and represents the average of two independent experiments.

could be fundamental for its function in TCR. On the basis of these convincing reports, we set out to examine the importance of the acidic region of the CSB protein in the context of TCR activities. In contrast to the importance of the ATPase domains of CSB in TCR, the acidic region seems to be dispensable for the TCR-dependent and -independent functions of CSB we have tested. The possibility exists that the acidic region is a structural component of the CSB protein and when the region is deleted, the conformation may be conserved as in the wild-type. An alternative explanation is that these acidic amino acids are involved in non-essential sites of protein–protein interactions and that absence of these interactions are not detected in the assays we employed in this report. Perhaps the acidic region plays a role in repair of oxidative DNA damage, a question we have not addressed here. Clearly, more direct structure–function approaches will be valuable in order to advance the understanding of the complex molecular functions of the CSB protein and eventually understanding molecular pathogenesis of CS. ACKNOWLEDGEMENTS We thank J. Crest for help with the cell cycle and fluorescence experiments. We appreciate the technical help of A. May and U. Henriksen. M.S. and T.S. were supported by the Danish Center for Molecular Gerontology and T.S. was also supported by the Danish Medical Research Council, grant no. 9503202. REFERENCES 1. Nance,M.A. and Berry,S.A. (1992) Am. J. Med. Genet., 42, 68–84. 2. Mayne,L.V. and Lehmann,A.R. (1982) Cancer Res., 42, 1473–1478.

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