171

Mutation Research, 235 (1990) 171-180 DNA Repair Elsevier MUTDNA 06378

Selective repair of specific chromatin domains in UV-irradiated cells from xeroderma p i g m e n t o s u m c o m p l e m e n t a t i o n group C George J. Kantor a,b, Linda S. Barsalou a and Philip C. Hanawalt b Department of Biological Sciences, Wright State University, Dayton, OH 45435 and b Department of Biological Sciences, Stanford University, Stanfora~ CA 94305 (U.S.A.) (Received 14 July 1989) (Revision received 3 November 1989) (Accepted 7 November 1989)

Keywords: fl-Actin; Dihydrofolate reductase; Xeroderma pigmentosum complementation group C; XP-C; Preferential repair; DNA excision repair

Summary The limited DNA-excision repair in UV-irradiated nondividing fibroblasts from xeroderma pigmentosum complementation group C (XP-C) occurs in localized chromatin regions generating large DNA segments (at least 30-70 kb) free of pyrimidine dimers. A genomic fraction enriched for this DNA was isolated on the basis of the larger size of the repaired fragments after UV-endonuclease treatment and screened for specific genes. It contains more copies per/tg DNA of two transcriptionally active genes, fl-actin and dihydrofolate reductase, compared to the remaining DNA but an equal number of copies per /~g DNA of an inactive locus termed 754. We confirmed that the active genes were preferentially repaired by measuring the removal of pyrimidine dimers from specific genomic restriction fragments comprising these sequences. These results mean that a unique set of relatively large chromatin domains are repaired in nondividing XP-C cells, even though most of the DNA remains unrepaired. The repaired domains may be those containing the active genes. This specific repair may account for the relatively high UV-resistance of the nondividing cells. In normal cells, a very rapid repair of a restriction fragment containing the/3-actin gene and slow repair of the 754-containing fragment was detected indicating that a similar domain-oriented repair process also exists in these cells. These results are consistent with the previously discovered rapid repair of active genes compared to bulk DNA. Separate damage-recognition systems may exist in human cells for chromatin domains that contain transcribed regions and those that contain no transcribed regions. The latter system may be deficient in XP-C.

Correspondence: Dr. G.J. Kantor, Department of Biological Sciences, Wright State University, Dayton, OH 45435 (U.S.A.).

Abbreviations: DHFR, dihydrofolate reductase; ESS, UV-endonuclease-sensitive sites; HU, hydroxyurea; LS, liquid scintillation; pr, preferentially repaired; UV-endo, UV-specific endonuclease; XP-C, xeroderma pigmentosum complementation group C.

The processes of DNA-excision repair in mammalian cells serve to alleviate some of the lethal, mutagenic and carcinogenic consequences of DNA damage (Hanawalt and Sarasin, 1986; Barbacid, 1986; Kraemer et al., 1984; McCormick and Maher, 1983). While 'the biological consequences of repair are appreciated, the mechanisms are

0921-8777/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

172

understood only in general terms. The recent discoveries of variable repair activities in different genomic regions (Bohr et al., 1987; Smith, 1987) serve to emphasize our lack of knowledge of how damage is recognized and repaired in the nucleoprotein chromatin complex which contains regions of diverse molecular structures and activities. The biological consequences associated with preferential repair of selected genomic regions, such as active genes (Bohr et al., 1986a; Mellon et al., 1987), underscore the necessity of defining these processes. Here we present further evidence for preferential repair of selected chromatin regions in human cells. The regions are specific and relatively large and their repair is associated with greater resistance to UV. The evidence derives from studies with xeroderma pigmentosum (XP) cells from complementation group C (XP-C). Cultured fibroblasts from XP-C patients exhibit only about 10% of normal DNA repair activity (Kantor and Elking, 1988). The cells are very sensitive to UV when colony-forming ability is measured (Andrews et al., 1978) but are relatively resistant compared to other XP strains when maintained as nondividing cells (Kantor and Hull, 1984). The limited repair detected in XP-C cells occurs in an apparent random fashion throughout the genome in proliferating cells (Cleaver, 1986) but in localized genomic regions in nondividing cells (Cleaver, 1986; Mansbridge and Hanawalt, 1983; Kantor and Player, 1986). The localized repair creates large pyrimidine dimer-free segments of DNA that are at least 30-70 kb long (Kantor and Player, 1986). Kantor and Elking (1988) proposed that the UV-resistance of the nondividing cells was the result of repair of specific DNA regions that were important for survival. To explore this proposal, we isolated a DNA fraction enriched in the preferentially repaired DNA (prDNA) from nondividing XP-C cells and examined it for the relative presence of specific genes. The isolation of pr-DNA was based on its large size after digestion of genomic DNA from UV-irradiated cells with a nuclease that nicks D N A specifically at the sites of pyrimidine dimers. We present evidence to show that specific genes have an enhanced representation in the pr-DNA fraction. Based on this and other evidence for preferential repair, we conclude that the small amount of

excision repair in nondividing XP-C cells represents repair of a unique set of chromatin domains. These domains contain transcriptionally active genes. At least some of these domains are relatively large. Evidence obtained with normal human ceils suggests that a similar domain-oriented repair mechanism exists in these cells as well. Materials and methods

Cells and cell culture. Normal human fibroblasts (GM38) were obtained from the Institute for Medical Research (Camden, N J) and XP-C cells (XP4RO, CRL 1260 and XP10BE, CRL 1204) were obtained from the American Type Culture Collection (Rockville, MD). Nondividing populations of cells (Kantor, 1986) were used throughout. Isolation of pr-DNA. DNA previously labeled uniformly with 14C was extracted from XP-C cells 24 h after exposure to UV (20 j/m2). Radiolabeling of cells (Kantor and Elking, 1988) and UVirradiation and D N A extraction procedures (Kantor and Setlow, 1981) were as described. The DNA was fractionated in alkaline sucrose gradients (34 ml, 5-20% sucrose, SW-28 rotor, 27 000 rpm, 20°C, 9 h) after complete digestion with Micrococcus luteus or bacteriophage T4 UV-endonuclease (UV-endo; Carrier and Setlow, 1970; Seawell et al., 1981). Fractions containing the largest molecules representing 15% of the total DNA (see Fig. 1) and about 13 ml of a gradient were pooled, neutralized with HC1, and dialyzed against 0.1 x SSC (80 volumes, one change) and 0.05 x SSC (40 volumes). The volume of the dialysate was reduced by evaporation (Speed Vac Concentrator, Savant Instruments, Inc., Hicksville, NY) resulting in a DNA solution in 1-2 x SSC. About 4 fig of the pr-DNA fraction are recovered from 107 cells. The specific radioactivities of bulk and pr samples were determined from fluorometric (Stout and Becker, 1982) and liquid-scintillation (LS) measurements. Slot-blotting procedures. Just prior to use, DNA solutions were placed in boiling water for 5 min, cooled on ice and made 10 x SSC. Samples were placed onto nitrocellulose using a slot blot apparatus (Minifold II Slot-Blotter, Schleicher and

173 10 - 20 J/m 2 O5

8

~ (l)

6

•"='--

4

6°°POo-oq~_

Repair Synthesis ~,-;.. ,

2

Bulk D N A

•

0'6'o

q

"O i'l"

0

. 0

10

.

i 20

30

Fraction Number ',

80

!

!

!

40

!

I

I

20 1~0 5 Size (kb)

!

1

Fig. 1. Definition of pr-DNA. A culture of nondividing XP4RO cells uniformly labeled in DNA with 14C (O) was UV-irradiated (20 J / m 2) and incubated for 24 h with 3H-dThd ( 0 ) and H U to label repair synthesis. After extraction, the D N A was digested with UV-endo and fractionated on an alkaline sucrose gradient. Sedimentation is from right to left. The hatched area indicates the fractions containing the largest D N A molecules, about 15% of the total DNA and about 50% of the repair synthesis (3H activity). D N A extracted from several parallel cultures treated as described above, with omission of 3H-dThd, was sedimented in a parallel gradient. Fractions with the above defined 15% were pooled as pr-DNA. Fractions from the 14C peak area with 50% of the 14C activity were pooled as bulk DNA. DNA sizes were calculated as described previously (Regan et al., 1971).

Schuell, Inc., Keene, NH) (Anderson and Young, 1985). Filters were baked in a vacuum at 80°C for 2 h, wet uniformly with 2 x SSC, incubated in a prehybridization solution for 4 h and a hybridization solution containing radioactive probe for about 40 h at 42°C, and then exposed to post-hybridization washes using standard procedures (Maniatis et al., 1982). The final washes were in 0.1 x SSC at 65°C for 2 h.

Analysis of restriction fragment repair. Standard procedures (Bohr et al., 1985) to detect repair of pyrimidine dimer-containing specific DNA-restriction fragments, as modified by Mellon et al. (1986), were used. Probes. The fl-actin probes were a 385-bp third intron fragment (IVS-3) in pHflA-IVS3-MS and a 14-kb EcoR1 human genomic fragment containing the /~-actin gene cloned in p14Tf117 (Ng et al., 1985), obtained from P. Gunning (Stanford University). A 253-bp fragment from the first intron

of the human DHFR gene was obtained by digestion of pBH31R1.8 with restriction enzymes Kpnl and Sstl. This plasmid, obtained from G. Attardi (California Institute of Technology), contains a 1.8-kb fragment of the DHFR gene (Yang et al., 1984). The "754 fragment" is a 2.2-kb human X-chromosome HindIII fragment (Hofker et al., 1985). It was obtained from P. Pierson (State University of Leiden) cloned into a pAT-153 vector. All probes were purified after digestion of the plasmids with appropriate restriction enzymes by gel electrophoresis and excision of the appropriate bands from low melting temperature agarose gels. 32p-Labeled probes were obtained using the random primer method (Feinberg and Vogelstein, 1983) with the gel-purified fragments. Results

Definition of pr-DNA. Some DNA regions in nondividing XP-C cells are repaired following exposure to UV while other regions remain unrepaired (Kantor and Elking, 1988; Mansbridge and Hanawalt, 1983). The results of an experiment to detect the preferentially repaired regions, modeled after the original work of Mansbridge and Hanawalt (1983), are presented in Fig. 1 and used to define a pr-DNA fraction. In brief, cells uniformly prelabeled with [a4C]thymidine were placed in a nondividing state by 10-d incubation in culture medium containing 0.5% fetal calf serum. They were then UV-irradiated and incubated with [3H]thymidine to label repair synthesis. After 24 h, the DNA was extracted, digested with UV-endo to nick at remaining dimer sites and sedimented in an alkaline sucrose gradient to size-fractionate the molecules, some of which contain repair patches. Since no detectable DNA replicative synthesis occurs in this period (Kantor and Elking, 1988), the lack of congruity of the 14C and 3H distributions indicates a non-random or localized distribution of repair patches (Mansbridge and Hanawalt, 1983). For the experiments reported here, pr-DNA is defined as the 15 % fraction containing the largest DNA molecules. They are about 30-70 kb long and have associated with them about 50% of the repair synthesis. This DNA fraction was isolated in a parallel experiment, conducted with the [3H]thymidine omitted, and then examined in subse-

174

A. 14 C

32

P

pr

bulk 2730

80

~

B. 2690

240

pr

bulk DNA (ng)

32p

DNA (ng)

32p

33

10

46

21

57

16

87

36

117

20

158

62

193

28

Fig. 2. Enhanced representation of B-actin sequences in preferentially repaired DNA. Samples of pr- and bulk-DNA were slot-blotted onto nitrocellulose membranes and hybridized to a 32p-labeled unique intron fragment (IVS-3) of tlie human /~-actin gene. (A) XP4RO DNA. 200-ng samples of either 14C-labeled bulk or pr-DNA were applied to two adjacent slots. Autoradiograms before hybridization (top row, marked 14C) and after (lower row, marked 32p; a filter blocked the 14C activity) are shown. Next to each slot is the radioactivity (cpm) detected at the end of the experiment by LS methods. (B) Results of a second experiment, in which increasing amounts of XP4RO DNA were applied. Only the autoradiogram taken after hybridization, depicting 32p activity, is shown. The numbers reflect the amounts of DNA loaded (calculated from the 14C activity) and the 32p activity (cpm) hybridized to each sample. quent experiments for the presence of specific genes. A fraction from the a4C peak region was isolated to represent bulk D N A and used concomitantly with p r - D N A . The two fractions were found to have equal 14C specific activities in all experiments.

Enhanced representation of specific genes in prDNA. Equal amounts of D N A from each fraction were applied to the same nitrocellulose membrane and hybridized to a specific gene probe. Autoradiographic exposures of the membranes before hybridization allowed visualization of the adhering a4C-DNA and exposures after hybridization allowed visualization of the hybridized 32p_ probe D N A when a filter blocking ]4C activity was used. After hybridization and autoradiography, the 14C and 32p radioactivities were measured by LS counting of the appropriate sections of the membrane. Results using a probe unique to the single copy transcriptionally active ]3-actin gene are presented in Fig. 2. Two sets of results are presented, each representing an independent experiment that included the entire experimental protocol from cell culture through D N A extractions and D N A hybridization reactions. Both the autoradiographic and LS detection evidence are presented to illustrate the kinds of data obtained and the concurrence of the two detection procedures. The 14C data of part A clearly indicate that nearly equal amounts of the two D N A fractions

were retained on the m e m b r a n e after application (autoradiographic evidence) and hybridization (LS data). However, about three times more 32p activity (i.e., /~-actin probe) was associated with the p r - D N A than with the bulk fraction. In a second experiment, increasing amounts of the two D N A fractions were examined. The D N A masses retained on the m e m b r a n e were calculated from the 14C activity. The autoradiograph depicts the 32p activity only. All of the hybridization results show a greater representation of the /3-actin gene in p r - D N A , with an enhancement of about 2.7 times calculated for the two samples of each fraction containing the most D N A . The enhancement detected in several repeat experiments varied from about 2.5 to 7 times. Similar results were obtained for p r - D N A extracted from a second XP-C strain XP10BE (data not shown). Bulk and p r - D N A were also analyzed for their content of the D H F R gene and an inactive sequence termed the 754 locus. A b o u t twice as much of the D H F R probe associated with p r - D N A (Fig. 3A) c o m p a r e d to b u l k - D N A while an equal a m o u n t of the 754 sequence associated with each D N A fraction (Fig. 3B). In the 754 Expt., 6 equal samples of bulk D N A and 2 unequal samples of ~ f - D N A were analyzed. For the bulk D N A , the P//14C ratios were all within 9% of the mean value of 0.033. For the p r - D N A , ratios of 0.039 and 0.027 were obtained. We conclude that no enrichment was observed for this non-transcribed

175

A. DHFR intron probe

bulk 14C ....... 1410 2870 32p

1520 3140

40 86

B. 754 probe

bulk 14C "~ 2440 2560 2550 2740 2620 2580 32p ~

pr

8876

90 170 pr

Z

2280 1210

90 33

77

82

Fig. 3. Relative amount of the active DHFR gene and inactive 754 locus in pr- and bulk-DNA. XP4RO DNA samples were applied to nitrocellulose and hybridized (A) to a 32P-labeled unique fragment from intron I of the DHFR gene or (B) to a 32p-labeled 2.2-kb HindlII fragment (754) from the human X-chromosome. All membranes were screened for radioactivity as described in Fig. 2. The 14C autoradiographs represent 14C activity on membranes prior to hybridization and the 32p autoradiographs represent 32p activity after hybridization in the same respective slots. Numbers indicate the respective 14C and 32p activities (cpms) detected after hybridization. The amounts of DNA applied were in A, 75 and 150 lag respectively for both bulk- and pr-DNA and in B, 160 ng in each slot for bulk DNA and 160 ng in the top slot and 80 ng in the second slot for pr-DNA. Solution volumes were varied in B so that for bulk DNA, volumes of 40, 80, 120, 200, 400 and 800 /~1, in descending order, were loaded into the slot-blot apparatus while for pr-DNA, volumes of 400 /xl were used for both samples.

sequence. T h e s a m p l e s of b u l k D N A in this exp e r i m e n t were d i l u t e d over a wide range to assess the effect of l o a d i n g different volumes. N o effect was observed. T h e b u l k a n d p r - D N A s were also h y b r i d i z e d to p r o b e m a d e f r o m a 14-kb g e n o m i c f r a g m e n t that c o n t a i n s b o t h the fl-actin gene a n d a highly repetitive alu sequence (fl-17 p r o b e ; J. Leavitt, personal c o m m u n i c a t i o n ) . S a m p l e s c o n t a i n i n g 0.1, 0.2 a n d 0.5 /zg of each e x h i b i t e d the s a m e r a t i o of h y b r i d i z a t i o n p e r / ~ g o f D N A . These results indicate that the i n c r e a s e d h y b r i d i z a t i o n to p r - D N A o b s e r v e d for the two active gene sequences (flactin; D H F R ) are r e l a t e d to the n u m b e r of gene copies in the two fractions r a t h e r t h a n to some a r t i f a c t u a l e n h a n c e m e n t of the h y b r i d i z a t i o n efficiency of p r - D N A , c a u s e d in s o m e u n k n o w n w a y b y the m e t h o d used to f r a c t i o n a t e o r analyze the D N A . A s a n a d d i t i o n a l control, the entire D N A f r a c t i o n a t i o n p r o c e d u r e was c a r r i e d ~out on cells t h a t were lysed i m m e d i a t e l y after i r r a d i a t i o n . T h e D N A f o u n d in the regions of the g r a d i e n t a n d the f r a c t i o n a t e d D N A p r o f i l e usually o c c u p i e d b y prD N A e x h i b i t e d the s a m e h y b r i d i z a t i o n p e r / ~ g of D N A as d i d the b u l k D N A , b o t h to fl-actin a n d D H F R - s p e c i f i c p r o b e s . This indicates that the initial p y r i m i d i n e d i m e r c o n t e n t of these gene regions is the s a m e as in the b u l k o f the D N A , a n d that the greater size of p r - D N A does n o t e n h a n c e its h y b r i d i z a t i o n . T h e efficiency of h y b r i d i z a t i o n of b u l k D N A f r o m cells lysed i m m e d i a t e l y after U V - i r r a d i a t i o n to the fl-actin p r o b e was also det e r m i n e d a n d f o u n d to be i d e n t i c a l to that of the D N A t h a t served as the starting m a t e r i a l for the f r a c t i o n a t i o n , b o t h p r i o r to a n d j u s t after treatm e n t with U V - e n d o . W e c o n c l u d e f r o m all of these c o n t r o l s t h a t the e n h a n c e d h y b r i d i z a t i o n of fl-actin a n d D H F R p r o b e s to p r - D N A does ind e e d reflect a n i n c r e a s e d n u m b e r of gene copies a n d a m o v e m e n t of these sequences into the prD N A fraction as a result of a p o s t - U V i n c u b a t i o n period.

Repair of specific DNA restriction enzyme fragments. T h e a b o v e results suggest that the g e n o m i c regions r e p a i r e d in X P - C cells c o n t a i n specific genes that are p r e f e r e n t i a l l y repaired. To c o n f i r m this, we used a p r e v i o u s l y d e f i n e d proced u r e (Bohr et al., 1985) to m e a s u r e r e m o v a l of

176 endonuclease-sensitive sites (ESS) f r o m g e n o m i c restriction f r a g m e n t s c o n t a i n i n g the /3-actin, D H F R a n d 754 sequences. F o r the small /3-actin gene, we e x a m i n e d a 13.8-kb EcoR1 f r a g m e n t that e n c o m p a s s e s it, b o t h in X P - C a n d r e p a i r - p r o f i cient n o r m a l fibroblasts. T y p i c a l results are shown in Fig. 4. U V - i r r a d i a t e d cells were i n c u b a t e d for various periods, after which the D N A was extracted a n d digested with E c o R I . O n e half of each D N A s a m p l e was further digested with U V - e n d o . Both halves were f r a c t i o n a t e d in alkaline agarose gels. The relative n u m b e r of 13.8-kb fragments c o n t a i n i n g the /3-actin gene in each aliquot was detected after transferring the D N A to a n y l o n m e m b r a n e a n d h y b r i d i z i n g it to the 32p-labeled /3-actin intron sequence IVS-3. Q u a n t i t a t i o n was a c c o m p l i s h e d b y d e n s i t o m e t r i c s c a n n i n g of autor a d i o g r a m s of the h y b r i d i z e d m e m b r a n e s a n d application of the Poisson e q u a t i o n (Bohr et al., 1985). D i m e r s are responsible for fewer full size D N A restriction f r a g m e n t s in the U V - e n d o treated sample. Their repair with time results in an increase in these fragments. F o r n o r m a l cells (part

A), most of the d i m e r s evident i m m e d i a t e l y after U V - i r r a d i a t i o n (10 J / m 2) d i s a p p e a r e d in 8 h. F o r X P - C cells ( p a r t B), their d i s a p p e a r a n c e was cons i d e r a b l y slower b u t still evident b y 24 h. Quantitative analysis of this a n d o t h e r a u t o r a d i o g r a m s are p r e s e n t e d in T a b l e 1. This analysis shows that X P - C cells r e m o v e a b o u t 25% of the ESS in the 13.8-kb /3-actin-containing f r a g m e n t in 8 h and a b o u t 50% in 24 h. N o r m a l cells remove ESS from this f r a g m e n t m o r e r a p i d l y , with nearly 75% excised in 8 h. These rates are c o n s i d e r a b l y faster t h a n those o b s e r v e d for repair of the g e n o m e overall in the respective cells (for G M - 3 8 a n d other n o r m a l cells, see K a n t o r a n d Hull, 1984; K a n t o r a n d Setlow, 1981; T a b l e 1, p a r t D; for X P 4 R O cells, see K a n t o r a n d Elking, 1988). W e o b s e r v e d no r e p a i r of a 754-containing restriction f r a g m e n t in 24 h in X P - C cells a n d very limited r e p a i r in the s a m e p e r i o d ( a b o u t 30% of the ESS r e m o v e d ) in n o r m a l cells ( p a r t B). A limited but significant a m o u n t of r e p a i r of a restriction fragm e n t c o n t a i n i n g D H F R gene sequences was detected in X P - C cells. A b o u t 25% of the ESS were

TABLE 1 REMOVAL OF ENDONUCLEASE-SENSITIVE SITES FROM GENE-SPEC1FIC RESTRICTION FRAGMENTS ESS removed (%) a

DNArestriction fragment

Cell strain

2h

4h

8h

24h

(A)/3-actin (13.8-kb Eco RI fragment; 10 J/m 2)

GM-38

33

54 67

76 77

76 79

10

15 34

54 65

21 26

15 21

39 23

5

0

7

XP4RO (B) 754 (14-kb Eco RI fragment; 10 J/m 2)

GM-38 XP4RO

(C) DHFR (24-kb Hind III fragment; 5 J/m 2)

XP4RO

(D) Genome overall (10 J/m 2)

GM-38 b XP4RO ¢

11

21 33 20

35

48h

56 59

75 5

The average number of ESS in a DNA restriction fragment was calculated using Poisson statistics and the fraction of restriction fragments free of ESS, obtained by densitometer analysis of the autoradiogram in Fig. 4 and other autoradiographs. The initial number of ESS detected was for (A), 1.1/13.8-kb fragment (expected, 1.1); (B) 1.2/14-kb fragment (expected, 1.1); (C) 0.9/23-kb fragment (expected, 1.0). Data in different rows are from separate experiments. b Determined as described previously, relative to other normal cells (Kantor and Hull, 1984). c Kantor and Elking (1988). a

177

A. GM38 Time, h uv-endo

0 --

2 "4".

--

4 "4".

--

8 ,4,'

--

"4".

--

24 -i-

23.6 kb

-

9.4

-

6.6

-

4.4

-

23.6 kb

-

9.4

-

6.6

-

4.4

B. XP4RO Time, h uv-endo

0

4 ÷

--

8 ÷

--

24 "4".

--

-I-

Fig. 4. Detection of repair of a fl-actin-containing D N A restriction fragment in UV-irradiated normal and XP-C cells. EcoRI-restricted DNA samples from UV-irradiated cells that had been incubated for the times indicated were treated with ( + ) or without ( - ) UV-endonuclease, fractionated on alkaline agarose gels and transferred to nylon membranes. Hybridization to the 32p-labeled fl-actin IVS-3 fragment was detected by autoradiography. The sizes (kb) and positions of HindIII-digested DNA markers are shown. (A) GM38 cells, 10 j / m 2 ; (B) XP4RO cells, 10 J / m 2.

removed in 24 h and 50% in 48 h from a 23-kb D H F R fragment (part C). Discussion

Unique repair domains, pr-DNA blotted on nitrocellulose membranes binds more probe D N A specific for the fl-actin or D H F R gene than does b u l l DNA. No experimental anomalies that could account for the observed results were detected. Two different probes, 754 and fl-17, hybridize equally well to both pr-DNA and bulk-DNA. The 754 sequence, which is not efficiently repaired in XP-C cells (van Zeeland et al., 1988; Table 1) should not have an enhanced representation in

pr-DNA. The fl-17 probe should not discriminate between the two D N A fractions because it conrains in addition to the entire fl-actin gene an alu sequence and thus is a general probe for human genomic DNA. Results of several hybridization reactions involving D N A samples from intermediate steps of the overall D N A isolation procedure with the fl-actin intron probe were the same. No unknown factors in any of the D N A fractions that influence the hybridization reactions were detected. The results thus indicate that there are more fl-actin and D H F R gene copies per/~g D N A in pr-DNA than bulk DNA. The enhanced representation of the specific genes in the p r - D N A could reflect either an initial lower level of D N A damage in these regions or active repair in only a small fraction of the cells in low serum medium. Evidence obtained for the fl-actin and DHFR-containing restriction fragments indicates that the first alternative is not the case. Those fragments contain the initial expected number of pyrimidine dimers (Bohr et al., 1986a; Table 1) if dimers are introduced randomly throughout the mammalian genome. In addition, probes unique to the fl-actin and D H F R genes hybridize equally well to a " p r D N A " fraction isolated from cells lysed immediately after UV-exposure and a bulk fraction, an expected result for this " n o repair" condition if dimers are randomly distributed. The second alternative is ruled out by autoradiographic evidence published previously (Kantor and Elking, 1988) showing that all cells in these irradiated populations perform D N A repair synthesis. Thus all of these results indicate that the greater number of fl-actin and D H F R gene copies found in pr-DNA represent preferential repair of these genes. Evidently the limited nonrandom repair found in nondividing XP-C cells represents repair of the same unique chromatin regions throughout the cell population. Recent evidence of others (Bohr et al., 1987; Smith, 1987) shows that some specific genes are preferentially repaired compared to the genome overall in mammalian cells. Bohr et al. (1986b) have discussed the relation of this repair to chromatin domains in rodent cells, based on a map of repair activity associated with different D N A fragments from the D H F R genomic region. They suggested that a chromatin domain somewhat

178

larger than the 30-kb D H F R gene, estimated at 50-80 kb, may define the D H F R preferentially repaired region. Here we have shown by a different approach that the relatively small human /3actin gene, which occupies approx. 3.5 kb on chromosome 7 (Ng et al., 1985), has an enhanced representation in a set of preferentially repaired molecules that are 30-70 kb long. This range is a minimum for the repaired regions since they are found in a population of D N A molecules that have been sheared by the extraction procedure. We suggest from this that the fl-actin gene is preferentially repaired in XP-C cells as part of a relatively large genomic domain and that the D H F R gene, found on chromosome 5 (Maurer et al., 1984), is also preferentially repaired but as part of a different large domain. The preferentially repaired fl-actin and D H F R genes are both transcriptionally active. In contrast, the unrepaired 754 locus is inactive. Others had shown previously that the repaired D N A in XP-C cells is associated with the nuclear matrix (Mullenders et al., 1984) and sensitive to endogenous nucleases (Player and Kantor, 1987), two lines of evidence suggesting that the repaired DNA contains actively transcribed regions. The unique set of domains that are repaired in nondividing XP-C cells may thus be those that contain the transcriptionally active genes.

Restriction fragment analysis. Evidence obtained by examining UV-damaged DNA-restriction fragments containing the fl-actin and D H F R genes and the 754 locus in XP-C cells corroborates the slot-blot evidence for specific repair domains. Repair activity is detected in /3-actin-containing and DHFR-containing fragments but not in a 754-containing fragment. Based on other experiments that provided quantitative data for both total genomic repair and domain-oriented repair, Kantor and Elking (1988) had suggested that the overall genomic repair detected in XP-C cells probably occurs exclusively in only a few domains. The present results are further evidence that repair is limited to selected regions. The results presented in Table 1 are from experiments that employed probes for both strands of the DNA-restriction fragments. These kinds of probes cannot be used to detect the strand-specific

repair detected by others for the D H F R gene (Mellon et al., 1987). The results in Table 1 are calculated assuming conditions that permit the use of Poisson statistics. We recognize that those conditions may not hold for our calculations, since the repair activity associated with each strand may be different. We present these results in this fashion to facilitate comparisons to the earlier results of others obtained using similar procedures. In addition, these results indicate the occurrence or absence of repair of specific genes and confirm the slot-blot evidence for preferential repair in XP-C cells. Bohr et al. (1986a) previously reported that a restriction fragment containing D H F R is not efficiently repaired in XP-C cells (about 10% of dimers removed in 24 h). We detect a somewhat greater repair level for the D H F R fragment (30%). Observations made at extended post UV-periods confirm the occurrence of repair activity (50% in 48 h). We conclude from our data of two independent types of experimental measures, gene representation in pr-DNA and repair of restriction fragments, that a moderate level of repair occurs in the D H F R gene, a conclusion at variance with the earlier one (Bohr et al., 1986a). Also we find relatively efficient repair in the active/3-actin gene and others (van Zeeland et al., 1988) report similar results for the adenosine deaminase (ADA) gene. The transcriptionally active genes studied thus far in XP-C cells are preferentially repaired. While there is no clear reason for the variance with the earlier D H F R report, we suggest that different cell culture methods might account for the observed difference. Cleaver (1986) showed recently that proliferating XP-C cells have the same level of repair activity as nondividing cells, but the activity is in apparent random locations throughout the genome, at least as detected by the kind of experiment described in Fig. 1 of this report. We used nondividing cells while Bohr et al. (1986a) used actively growing cells. The extent of enhancement observed for a specific gene in pr-DNA should relate to the rate of gene-specific restriction fragment repair. Using these data, we estimated an expected enhancement ratio of about 4 for the /3-actin gene. We have observed enhancement ratios for this gene varying from 2.4 to 7.0 for several repeat DNA extrac-

179 tions, results that are in reasonable agreement with our estimation.

Preferential repair in normal cells. A repair process targeted to specific chromatin domains is expected in normal human cells because the XP-C repair process is probably a subset of the overall DNA-excision repair scheme. The results of Mellon et al. (1986) showing that the D H F R gene is repaired more rapidly in human cells than is the bulk of the DNA are evidence for such a process. The very rapid repair found here for the fl-actin restriction fragment and the very slow repair found for the 754-containing sequence is direct evidence for the existence of chromatin fractions that are repaired at different rates and thus a targeted process in normal fibroblast cells. The pr-DNA fraction isolated from XP-C cells may thus also represent the preferentially repaired fraction in similar cultures of normal cells. As with XP-C cells, the genes preferentially repaired are transcriptionally active (fl-actin, D H F R , ADA) (Mellon et al., 1986; van Zeeland et al., 1988) while the poorly repaired 754 sequence is transcriptionally inactive (van Zeeland et al., 1988), an observation consistent with the proposal that transcriptional activity promotes DNA-excision repair (Bohr et al., 1987; Mellon et al., 1986). Previous studies of gene-specific repair have employed genomic restriction fragments that are for the most part contained within the transcription unit of the gene (for example, see Bohr et al., 1986b; van Zeeland et al., 1988). Results acquired with these fragments are a direct indication of gene-specific repair activity. The fl-actin-containing fragment studied here is otherwise, being about 4 times larger than the fl-actin transcription unit (3.5 kb of the 14-kb EcoR1 fragment). The m R N A initiation site is located about 3.2 kb from the 5' end of the 14-kb fragment (Ng et al., 1985). The very rapid repair of this 14-kb fragment is indicative not only of the repair of the fl-actin gene, but of both upstream and downstream sequences. Since the transcriptional activity of these other sequences is unknown, their dependence on this activity for preferential repair cannot be judged. Further study of both transcriptional and repair activities of this fragment is warranted. Conclusions. Results of quantitative slot-blot hybridization screens and of experiments quanti-

tating repair of specific restriction fragments are consistent and indicate that relatively large genomic regions containing specific genes are preferentially repaired in nondividing XP-C cultures. The specific regions include transcriptionally active genes and do not include at least one inactive gene. Thus the specific regions may be the chromatin domains containing active genes. While the repair of specific gene-containing domains occurs, the repair rate of restriction fragments specific for these genes is slower than that for the same genes in normal cells. Thus, the repair defect in XP-C cells is one that affects not just the inactive sequences but the entire genome. However, the result of the defect is retention of repair activity targeted almost exclusively to the unique set of domains. Associated with the specific domain repair in nondividing XP-C cells is a relative resistance to lethal UV effects (Kantor and Elking, 1988). Thus the regions repaired are probably responsible for genetic information that is important to survival.

Acknowledgements We thank J. Leavitt, C.A. Smith, A.K. Ganesan, I. Mellon, P. Gee, L. Ho and A. Islas for many helpful discussions, E. Wauthier for preparation of T4 endonuclease V, I. Mellon for D H F R plasmids, P. Gunning for fl-actin plasmids and P. Pierson for the 754 plasmid. We appreciate the considerable effort of C.A. Smith in the preparation of the manuscript. This work was supported by a National Research Service award from the National Cancer Institute (CA08306) and a professional development award from Wright State University to G.J.K., and by a PHS Outstanding Investigator Research Grant No. CA44349 to P.C.H.

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