MOLECULAR AND CELLULAR BIOLOGY, Aug. 1991, p. 4128-41340270-7306/91/084128-07$02.00/0Copyright C) 1991, American Society for Microbiology

Xeroderma Pigmentosum Complementation Group C Cells RemovePyrimidine Dimers Selectively from the Transcribed

Strand of Active GenesJAAP VENEMA,' ANNEKE VAN HOFFEN,' VERONIKA KARCAGI,'t A. T. NATARAJAN,"2

ALBERT A. VAN ZEELAND,1'2 AND LEON H. F. MULLENDERSL 2*

MGC-Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, Wassenaarseweg 72,2333 AL Leiden,' and J. A. Cohen Institute, Interuniversity Research Institute for Radiopathology

and Radiation Protection, Leiden,2 The Netherlands

Received 13 June 1990/Accepted 20 May 1991

We have measured the removal of UV-induced pyrimidine dimers from DNA fragments of the adenosinedeaminase (ADA) and dihydrofolate reductase (DHFR) genes in primary normal human and xerodermapigmentosum complementation group C (XP-C) cells. Using strand-specific probes, we show that in normalcells, preferential repair of the 5' part of the ADA gene is due to the rapid and efficient repair of the transcribedstrand. Within 8 h after irradiation with UV at 10 J m-2, 70% of the pyrimidine dimers in this strand are

removed. The nontranscribed strand is repaired at a much slower rate, with 30% dimers removed after 8 h.Repair of the transcribed strand in XP-C cells occurs at a rate indistinguishable from that in normal cells, butthe nontranscribed strand is not repaired significantly in these cells. Similar results were obtained for theDHFR gene. In the 3' part of the ADA gene, however, both normal and XP-C cells perform fast and efficientrepair of either strand, which is likely to be caused by the presence of transcription units on both strands. Thefactor defective in XP-C cells is apparently involved in the processing of DNA damage in inactive parts of thegenome, including nontranscribed strands of active genes. These findings have important implications for theunderstanding of the mechanism of UV-induced excision repair and mutagenesis in mammalian cells.

Cells derived from patients with the autosomal recessivedisorder xeroderma pigmentosum (XP) have been shown tobe hypersensitive to the lethal and mutagenic effects of UVas a result of a defect in the recognition or processing ofUV-induced DNA damage (9, 17, 32). Cells from eightindependent complementation groups differ greatly in sensi-tivity to UV (8, 16). Generally, a good correlation existsbetween the level ofUV sensitivity and the extent of overallexcision repair capacity present in XP cells. However, XPcells belonging to complementation group C (XP-C cells)form an exception. Nondividing XP-C cells exhibit a rela-tively high survival despite a low residual repair capacity of15 to 20% compared with normal cells (15). Furthermore,XP-C cells have been shown to be able to restore UV-inhibited RNA and DNA synthesis, a phenomenon notobserved in XP-A and -D cells (22).The relatively good UV resistance of nondividing XP-C

cells may be related to the nonrandom distribution of UV-induced repair synthesis in these cells, initially reported byMansbridge and Hanawalt (21). They showed that someparts of the XP-C genome were repaired proficiently,whereas other parts were not repaired at all. Kantor et al.(14) showed that the transcriptionally active P-actin anddihydrofolate reductase (DHFR) genes were two- to fivefoldenriched in these proficiently repaired regions. Anotherinteresting observation was the association of UV-inducedrepair synthesis in DNA fragments containing the attach-ment sites of chromatin loops at the nuclear matrix (26),presumed to be the anchorage points of active genes. Takentogether, these results suggested that some regions of the

* Corresponding author.t Present address: Department of Biochemistry, National Insti-

tute of Hygiene, 1966 Budapest, Hungary.

genome were repaired efficiently in XP-C cells and that theseregions were critical for cellular survival. Recently, we haveextended these observations by measuring the removal ofUV-induced pyrimidine dimers in specific DNA sequences.It was shown that in XP-C cells the active adenosinedeaminase (ADA) and DHFR genes were repaired to a muchgreater extent than the genome overall. A nontranscribedX-chromosomal locus, termed 754, was not repaired (39).Similar results were obtained by Kantor et al. (14). Theefficient removal of pyrimidine dimers from active geneswould explain the moderate UV sensitivity and the ability torestore UV-inhibited transcription of nonproliferating XP-Ccells.

In contrast to normal human cells, however, XP-C cellsrepaired the different gene fragments analyzed with differentefficiencies (39). It was found that normal human fibroblastsconsistently showed a nearly complete removal of pyrimi-dine dimers from both the ADA and DHFR genes within 24h after UV irradiation at 10 J m-2. In XP-C cells, only the 3'part of the ADA gene was repaired to the same extent as innormal cells. The 5' parts of both the ADA and DHFR geneswere repaired to a lower extent (approximately 60%), al-though still much higher than the 15% observed in thegenome overall. The above-mentioned results were obtainedwith double-stranded DNA probes (39). Using single-stranded probes, Mellon et al. (24) showed that even withinthe active DHFR gene, considerable differences existedbetween repair of the two DNA strands in the gene. Thenontranscribed strand was repaired either not at all (Chinesehamster ovary cells) or very slowly (normal human cells)compared with the very rapid and complete repair of thetranscribed strand. This prompted us to hypothesize that theextent of repair of active genes in XP-C cells was determinedby the transcriptional organization of the gene and that XP-C

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SELECTIVE REPAIR OF THE TRANSCRIBED STRAND IN XP-C CELLS 4129

cells might perform efficient repair of the transcribed strandonly. The nearly complete repair of the 3' part of the ADAgene could be explained by the presence of a convergenttranscription unit overlapping the ADA gene at its 3' end as

was recently found in human fibroblasts (18).This report describes the results of the analysis of dimer

removal in the human ADA and DHFR genes with strand-specific probes. Probes were made from single-strandedDNA fragments and used to detect strand-specific repair innormal human and XP-C fibroblasts. We show that in the 5'part of the ADA gene and in the DHFR gene of normal cells,the transcribed strand was repaired faster than the nontran-scribed strand. In XP-C cells, repair in both gene fragmentswas restricted to the transcribed strand. In the 3' part of theADA gene, we observed efficient repair of both strands innormal and XP-C cells.

MATERIALS AND METHODS

Cell lines and culture conditions. Primary normal human(VH16) and XP-C (XP1TE) fibroblasts were cultured inHam's F10 medium (without hypoxanthine and thymidine)supplemented with 15% fetal calf serum and antibiotics.Cells were prelabeled with [3H]thymidine (0.05 ,uCi/ml, 80Ci/mmol). For irradiation, cells were grown to confluence in94-mm petri dishes for 10 days with regular medium changes.

Analysis of pyrimidine dimer removal. The removal ofpyrimidine dimers from specific DNA sequences was ana-

lyzed essentially as described previously (39). Briefly, cellswere irradiated with UV (254 nm) at 10 J m-2 and eitherlysed immediately (t = 0 h) or incubated for up to 24 h in thepresence of bromodeoxyuridine (to allow for separation ofparental and replicated DNA). After incubation, cells werelysed and high-molecular-weight DNA was purified by phe-nol and chloroform extractions followed by ethanol precip-itation. The DNA was restricted with either BclI, EcoRI, or

KpnI (Pharmacia) and centrifuged to equilibrium in CsCldensity gradients. Gradients were fractionated, and fractionscontaining parental-density DNA were pooled, dialyzedagainst TE (10 mM Tris [pH 8.0], 1 mM EDTA), and ethanolprecipitated. Equal amounts of DNA were either treated ormock treated with the dimer-specific enzyme T4 endonucle-ase V (27) and electrophoresed in 0.6% alkaline agarose gels.T4 endonuclease V specifically incises DNA at the site of apyrimidine dimer, resulting in a decrease of the amount offull-length DNA fragments. Removal of pyrimidine dimers isvisualized as the reappearance of the full-length fragments inthe lane containing enzyme-treated DNA. The DNA was

transferred to Hybond N+ membranes (Amersham) byvacuum Southern blotting (Pharmacia-LKB Vacugene 2016)and hybridized with 32P-labeled gene-specific probes. Afterautoradiography, the films were scanned with a Video den-sitometer (Biorad), and the amount of dimers was calculatedfrom the relative band densities in the lanes containing DNAeither treated or not treated with T4 endonuclease V, usingthe Poisson expression.

Preparation of strand-specific probes. The three PstI frag-ments comprising the human ADA cDNA (BA containingexons 1 to 5 [partly], BO containing exons 5 [partly] to 11,and BE containing exon 12; 2) were subcloned in M13SSEV18 and -19 vectors (3). A genomic 690-bp EcoRI-HindlIl fragment from intron V of the DHFR gene (43) wasalso cloned into these vectors. The orientation of all DNAfragments was confirmed by sequence analysis. The SSEVvectors contain a modified polylinker (compared withM13mpl8 and -19) which is able to form a stem-loop struc-

A1exon2 3 4 567-101112

1 11 111 1I

Bcll EcoRIB 12 3 4 5 6 exon

10 kb KpnlFIG. 1. Genomic maps of the human ADA (A) and DHFR (B)

genes showing the positions of exons and relevant restriction sites.The solid line in the DHFR gene indicates the position of the intronV probe (43). The cDNA probes used to detect the ADA BcII andEcoRI fragments comprised exons 1-5 (partly) and exon 12, respec-tively. The maps are derived from references 42 and 44, respec-tively.

ture in the single-stranded form. This stem-loop structurecontains an EcoRI site and thereby allows for the separationof single-stranded cloned inserts from vector sequences.Isolation and purification of inserts was performed essen-tially as described by Biernat et al. (4). Typically, 50 jig ofsingle-stranded DNA was digested with 200 U of EcoRI for2 h at 37°C. The EcoRI digest was size separated on a 1.5%agarose gel, and the single-stranded insert was excised fromthe gel and purified by electroelution. The fragment was thenlabeled by filling in the 3' recessed EcoRI site (see Fig. 2)with [ct-32P]dATP and Klenow DNA polymerase. The spec-ificity of the probes was checked by including spot blotscontaining both orientations of the fragment used as a probeas well as the original SSEV vectors in the hybridizationreactions. Corresponding double-stranded probes were pre-pared by random primer extension (11) of the purifieddouble-stranded DNA fragments.

RESULTSThe removal of pyrimidine dimers was analyzed in two

adjacent restriction fragments in the human ADA locus (12)and in one restriction fragment from the DHFR gene (14),using procedures developed by Bohr et al. (6). The 19.9-kbBclI fragment is located in the 5' part of the ADA gene andcompletely resides within the coding unit (Fig. 1). The18.5-kb EcoRI fragment lies in the 3' part of the gene andcontains 9 kb of flanking sequences. The 20-kb KpnI frag-ment is entirely located within the transcribed sequences ofthe DHFR gene. To measure repair in the individual DNAstrands in all fragments, we used strand-specific DNAprobes prepared with a recently developed M13 vectorsystem (3). The so-called SSEV18 and -19 vectors arederived from the common M13mpl8 and -19 vectors andcontain a modified polylinker region which is able to form astem-loop structure in the single-stranded form. As can beseen from Fig. 2, a fragment that has been cloned into thepolylinker can be separated from the vector sequences byrestriction with EcoRI and size separation on agarose gels.Purified fragments were labeled by filling in the 3' recessedEcoRI site with [a-32P]dATP and Klenow DNA polymerase(4). In this way, we were able to obtain DNA probes whichwere generally over 100-fold specific for one strand, asmeasured by the intensity of the hybridization signal with thetwo complementary single-stranded DNA fragments.

Figure 3 shows the results of an experiment measuringrepair of pyrimidine dimers in the ADA BclI fragment.Filters were consecutively hybridized with probes specific

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4130 VENEMA ET AL.

TABLE 1. Removal of pyrimidine dimers from respective strandsin the ADA BcII fragment in normal human (VH16)

and XP-C (XP1TE) fibroblasts

Strand probed tRepair Meana % repair (SEM)time(h) VH16 XPlTEBoth 2 11 (4) 4 (4)

4 28 (7) 19 (1)8 54 (5) 42 (6)

24 92 (1) 60 (4)

Transcribed 2 20 (7) 26 (14)4 41 (11) 44 (9)8 70 (10) 79 (6)

24 100 (4) 100 (12)

Nontranscribed 2 0 (2) 0 (1)4 14(5) 6(4)8 30 (2) 0 (9)

24 81 (6) 18 (3)

a Of two independent determinations.

FIG. 2. Schematic diagram of the isolation of single-strandedDNA fragments from SSEV18 and -19 vector clones (adapted fromreference 4).

for both the transcribed strand and the nontranscribedstrand. In this assay, the presence of pyrimidine dimers isvisualized as a decrease in the amount of full-length frag-ments in the lane containing DNA treated with the dimer-specific enzyme T4 endonuclease V. With increasing time,repair of pyrimidine dimers will result in the reappearance ofthe band in the lanes containing T4 endonuclease-treatedDNA. Normal cells performed a rapid and complete repair ofdimers within 24 h after UV irradiation (Fig. 3A). XP-C cells(Fig. 3B) exhibited a slightly slower and incomplete repair.However, when the pyrimidine dimer content was examinedby using a probe specific for the transcribed strand, bothXP-C and normal cells showed a very fast and completeremoval of dimers. The nontranscribed strand revealed adramatic difference between the two cell types: in normalcells repair still occurred, albeit at a lower rate and to asomewhat lower extent than in the transcribed strand, butXP-C cells hardly removed any dimers from this strand.To quantify repair measurements, autoradiograms werescanned and dimer removal was calculated from the relativedensities of the bands in the treated and nontreated lanes,using the Poisson expression. As shown in Table 1, normalcells performed fast and efficient repair of the ADA BcIIfragment: over 50 and 90% of the pyrimidine dimers wereremoved within 8 and 24 h after UV irradiation, respectively.The transcribed strand was repaired even faster, with 70% ofthe dimers removed by 8 h. The nontranscribed strand wasrepaired at a rate similar to that of the genome overall (39).Table 1 also shows that XP-C cells were able to repair thetranscribed strand in a way indistinguishable from wild type.The nontranscribed strand was not significantly repaired.The repair observed with double-stranded probes in XP-Ccells is therefore entirely caused by the complete repair ofthe transcribed strand only.Having established the existence of strand-specific repair

in the BclI fragment, the same question was addressed forthe EcoRI fragment (Fig. 1). The resulting autoradiogramsare shown in Fig. 4, and the results are quantified in Table 2.

In contrast to the situation in the 5' part of the gene, wefound no significant differences when repair of either strandin this fragment was probed. Both XP-C and normal cellsshowed complete repair of the two strands. Within 8 h afterUV irradiation, 70% of the induced pyrimidine dimers wereremoved from both strands, and repair was essentiallycomplete after 24 h.

time (hr) 0 2 4 8 24

T4 endo - + -+ -+± + -+

-p - mo -_. DS

_..- TS

NTSA.

_1 _ _ _ DS

TS

NTSB

FIG. 3. Autoradiograms measuring removal of UV-induced py-rimidine dimers from the ADA BclI fragment. Cells were irradiatedwith UV at 10 J m-2 and incubated for 0, 2, 4, 8, and 24 h. DNA waspurified, restricted with BcIl, and either treated (+) or not treated(-) with T4 endonuclease V. After alkaline gel electrophoresis andSouthern blotting, filters were consecutively hybridized with adouble-stranded cDNA probe (DS) and cDNA probes specific forthe transcribed (TS) or nontranscribed (NTS) strand. (A) Normalhuman (VH16) cells; (B) XP-C (XP1TE) cells.

digestion

with EcoRI

insert

0 TA cA -T

C- C

T -AA - TC-0CC - C0-T AT AA TA T

C - CC-C¢-CA-T0-C0-C- C

C-CC A

T 0

SSEV

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SELECTIVE REPAIR OF THE TRANSCRIBED STRAND IN XP-C CELLS 4131

time (hr) 0 2 4 8 24

T4 endo - + - + + + +

__mm_wm a- DS

time (hr) 0 8 24

T4 endo - + - +

_ DS

t*aW.1wsIV lr TS

-a _mt___m NTSA.

0 *A, lp TS

* NTS

A.

_ lop _ __olm D- OS

DS

__- OWN_ _ NTSB.

FIG. 4. Autoradiograms measuring dimer removal from theADA EcoRI fragment. DNA was isolated and processed as de-scribed in the legend to Fig. 3 except for the EcoRI restriction. Inthis case, TS and NTS are to be read as ADA template strand andnon-ADA template strand, respectively. (A) Normal human (VH16)cells; (B) XP-C (XP1TE) cells.

Since the transcriptional organization in the ADA gene isquite unusual, we wondered whether that might influence therepair pattern observed for the individual strands. There-fore, removal of pyrimidine dimers was also measured in aninternal fragment of the DHFR gene (Fig. 1). Repair in thisfragment was analyzed at 8 and 24 h after UV irradiation.Normal human cells performed efficient repair of the DHFRgene, and repair was even faster in the transcribed strand(Fig. 5A). XP-C cells performed efficient repair of the

TABLE 2. Removal of pyrimidine dimers from respective strandsin the ADA EcoRI fragment in normal human (VH16)

and XP-C (XP1TE) fibroblasts

Repair Mean' % repair (SEM)Strand probed tm htime (h) VH16 XP1TE

Both 2 13 (5) 24 (3)4 40 (4) 41 (5)8 77 (7) 80 (12)

24 98 (1) 100 (12)

Transcribed 2 10 (2) 15 (1)4 40 (3) 29 (10)8 65 (16) 71 (13)

24 95 (10) 100 (7)

Nontranscribed 2 5 (4) 28 (7)4 31 (3) 41 (3)8 63 (17) 85 (14)

24 96 (10) 100 (12)a Of two independent determinations.

& llw_I4SP4 TS

A

NTS

B.FIG. 5. Autoradiograms measuring dimer removal from the

DHFR KpnI fragment. Repair was analyzed at 8 and 24 h afterirradiation with UV at 10 J m-2. Filters were hybridized with a690-bp genomic EcoRI-HindIII fragment from intron V (43) recog-nizing both strands (DS), the transcribed strand (TS), and nontran-scribed strand (NTS), respectively. (A) Normal human (VH16)cells; (B) XP-C (XP1TE) cells.

transcribed strand, whereas virtually no dimer removaloccurred in the nontranscribed strand of the DHFR gene(Fig. SB). Note that the normal human cell line that we usedapparently contains a polymorphism for the KpnI restrictionsite. It was confirmed that the upper band corresponded insize with the DHFR fragment detected in XP-C cells. There-fore, the results were quantified only for the large band. Therate and extent of repair in the DHFR gene were similar tothose seen in the ADA BcII fragment (Table 3). Normalhuman as well as XP-C cells performed efficient repair of the

TABLE 3. Removal of pyrimidine dimers from respective strandsin the DHFR KpnI fragment in normal human (VH16)

and XP-C (XP1TE) fibroblasts

Strand probed Repair Mean' % repair (SEM)time (h) VH16 XP1TE

Both 8 44 (11) 33 (4)24 91 (6) 59 (8)

Transcribed 8 58 (12) 47 (3)24 100 (3) 84 (2)

Nontranscribed 8 33 (6) 8 (3)24 71 (18) 14 (5)

aOf two independent determinations.

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4132 VENEMA ET AL.

transcribed strand. In XP-C cells, the nontranscribed strandof the DHFR gene was not repaired significantly.

DISCUSSION

This study was undertaken to further elucidate the natureof the domains that are repaired in XP-C cells. Pyrimidinedimer removal was analyzed in the ADA and DHFR genes

by using strand-specific probes. The induction of damagewas found to be the same for both strands in all fragmentsanalyzed. In the Bcll fragment of normal human cells,completely located inside the coding unit in the 5' part of theADA gene, it was found that at early times after UVirradiation the transcribed strand is repaired much fasterthan the nontranscribed strand. The nontranscribed strand isrepaired at the slow rate seen for the genome overall (39).Preferential repair of the transcribed strand also occurred inthe 20-kb KpnI fragment of the DHFR gene, although therepair rate was slightly lower than that found in the ADABcll fragment. In the 3' ADA EcoRI fragment, both strandsare repaired at a rate similar to that of the transcribed strandin the Bcll fragment. The absence of strand specificity in thisfragment and the rapid repair are likely to be caused bytranscription of both strands (18). These results indicate thatin primary normal human cells, preferential repair of theADA gene is due to the rapid repair of the transcribedstrand. They are consistent with previous data (24) obtainedfor the human DHFR gene.

In XP-C cells, repair of the transcribed strand occurs at arate indistinguishable from that of normal cells. However,the nontranscribed strand is not repaired significantly. Se-lective repair of the transcribed strand can explain thedifferent repair efficiencies of different gene fragments ana-lyzed in XP-C cells (14, 39). The strand-specific repair foundin various restriction fragments in XP-C cells also influencesthe use of the Poisson expression to calculate repair values.When double-stranded probes are used, it is clear that theassumption of homogeneous repair is no longer true, leadingto an overestimation of repair (see Discussion in reference24). Given the UV dose and fragment sizes used in this andthe previous study (39), values of up to 65% can be obtainedwhere one would expect 50% to be the maximum possiblevalue. The data further support a model in which removal ofpyrimidine dimers from the cellular genome is carried out bya number of distinct pathways (for a discussion, see refer-ences 33 and 34). One mechanism is involved in the repair ofinactive sequences including nontranscribed strands of ac-tive genes, whereas another system deals with the removalof damage from the transcribed parts of the genome. The twopathways may share common factors, but they are at leastpartially independent. The factor defective in XP-C cells isapparently involved in repair of nontranscribed sequencesonly, resulting in the inability to recognize or repair pyrim-idine dimers without the help of a cofactor which is associ-ated with transcription itself. Identification of the XP-Ccomplementing gene could help to elucidate this defect. Inthis regard, it is also noteworthy that recently the yeastRAD16 excision repair mutant was shown to have a repairphenotype similar to that of XP-C cells, i.e., rapid repair ofthe active MATot locus and no repair of the repressed HMLotlocus (37).The transcriptional organization of the ADA EcoRI frag-

ment requires closer examination. The data known so farimply that both DNA strands in this fragment containtranscription units which terminate in the middle of thefragment. The two transcripts overlap by at least 1 kb (18). It

is interesting that a very similar organization was found forthe murine ADA gene (1). The two transcription units at the3' end of the murine ADA gene overlap by about 3 kb.Therefore, in the ADA EcoRI fragment about 50% of eachstrand is transcribed, whereas the other 50% is presumablynot transcribed (Fig. 1). Nevertheless, this is not reflected inthe repair values of XP-C cells, although these cells do notrepair nontranscribed DNA. This could be due to a numberof reasons. First, strictly speaking the site of transcriptiontermination is still ill defined in mammalian cells, and forseveral genes transcription has been reported to proceedbeyond the poly(A) signal for up to 4 kb (30, 31). If this alsoholds for the ADA gene, it might aid in a higher repairefficiency of the EcoRI fragment. A more precise determi-nation of the extent and strand specificity of repair would beachieved by measuring dimer removal in two fragmentsencompassing either the 5' or the 3' half of the EcoRIfragment.

Second, the mechanism of preferential repair itself mayinfluence the repair efficiency of the EcoRI fragment. So far,the mechanism of transcription-coupled repair is unresolved,and possible models have been suggested in which eitherrepair enzymes are directly complexed with the transcriptionapparatus or the stalled RNA polymerase at the site of alesion forms an efficient signal for directing repair toward thetranscribed strand (23, 34). Therefore, it is conceivable thatthe repair process, once initiated by transcription, continuesbeyond the coding sequences. However, the processivity ofthis process seems to be limited since the nontranscribedstrand of the BclI fragment is not repaired in XP-C cells,although this strand lies downstream of the convergenttranscription unit. Additional factors determining the extentof this repair could correspond with several features knownto be related to active chromatin such as DNase I sensitivity(10), methylation (7, 13), and the size of the chromatin loopin which the gene is located. For a number of mammaliangenes, the limits of the DNase I sensitivity domain have beenshown to coincide with the location of matrix attachmentregions to define a topologically sequestered functional unit(20, 29, 35). That domain size may also play a role in DNArepair is suggested by results presented by several groupsinvestigating repair in the Chinese hamster DHFR gene (5),the Chinese hamster (28) and human (19) metallothioneingene family, and the human P-actin gene (14). In all cases, itwas found that the efficiently repaired region was consider-ably larger than the gene of interest. Moreover, it is inter-esting that the average size of chromatin loops (50 to 100 kb)corresponds well with the average size of the proficientlyrepaired regions in XP-C cells (14, 15, 21).The fast and efficient repair of the transcribed strand of

active genes in XP-C cells provides an explanation for theonly moderate UV sensitivity of nondividing cells (16) andthe restoration of UV-induced inhibition of transcription(22). Furthermore, the process of UV-induced mutagenesisis likely to be influenced by these repair characteristics (36).This is illustrated by the observation by Vrieling et al. (40)that the presence or absence of DNA repair markedlyinfluenced the strand specificity of UV-induced mutations inChinese hamster V79 cells and a UV-sensitive derivative,VH1. It was found that point mutations in the hypoxanthinephosphoribosyltransferase (HPRT) gene induced by UV at 2J m-2 in wild-type V79 cells were mainly caused by photo-products originally present in the nontranscribed strand.This suggests that preferential repair of the transcribedstrand prevents the expression of mutations by photoprod-ucts in this strand (38, 41). In the mutant VH1 cells,

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SELECTIVE REPAIR OF THE TRANSCRIBED STRAND IN XP-C CELLS 4133

however, the great majority of the mutations originated fromphotoproducts present in the transcribed strand. VH1 cellsare completely deficient in dimer removal from the HPRTgene (unpublished results). Similarly, it is an interestingquestion as to what effect the strand-specific repair in XP-Ccells has on UV-induced mutagenesis compared with normalcells.

Finally, our results indicate that with respect to pyrimi-dine dimer removal, XP-C cells exhibit a repair patternidentical to that of Chinese hamster cells. Both cell typesperform a low level of pyrimidine dimer removal in thegenome overall but exhibit efficient repair of the transcribedstrand in active genes. The relatively high UV resistance ofstationary XP-C cells demands a further analysis of therepair in these cells directed to (6-4) photoproducts. Chinesehamster cells perform a fast and efficient repair of (6-4)photoproducts from their entire genome, whereas XP-C cellsare as deficient in (6-4) photoproduct removal as in dimerremoval when assayed in the genome overall (25). It ispossible that XP-C cells perform preferential repair of (6-4)photoproducts in active genes. Analysis of the fine structureof (6-4) photoproduct removal should help unravel the role ofboth DNA lesions in UV-induced mutagenesis and cytotox-icity.

ACKNOWLEDGMENTS

We are indebted to U. B. Gobel (Department of Medical Micro-biology, University of Freiburg, Freiburg, Germany) for the kindgift of the SSEV18 and -19 vectors and to C. A. Smith (Departmentof Biological Sciences, University of Stanford, Stanford, Calif.) forthe gift of the DHFR intron V probe. We thank A. R. Filon (thislaboratory) and C. Terleth (Department of Molecular Genetics,University of Leiden) for valuable help in preparation of T4 endo-nuclease V.

This work was supported by the association of the University ofLeiden with the Netherlands Organization for Scientific Research(Medigon contract 900-501-074) and Euratom (contract B16-E-166NL).

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2. Berkvens, T. M., E. J. A. Gerritsen, M. Oldenburg, C. Breukel,J. T. H. Wijnen, H. van Ormondt, J. M. Vossen, A. J. van derEb, and P. Meera Khan. 1987. Severe combined immunodeficiency due to a homozygous 3.2 kb deletion spanning thepromoter and first exon of the adenosine deaminase gene.

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4. Biernat, J., U. B. Gobel, and H. Koster. 1989. New bacterio-phage vectors for the large scale preparation of single strandedinsert DNA. J. Biochem. Biophys. Methods 19:155-168.

5. Bohr, V. A., D. S. Okumoto, L. Ho, and P. C. Hanawalt. 1986.Characterization of a DNA repair domain containing the dihy-drofolate reductase gene in Chinese hamster ovary cells. J. Biol.Chem. 261:16666-16672.

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