Cell lines from xeroderma pigmentosum

Bioscience Reports 3, 667-674 (1983) Printed in Great Britain

667

Cell lines from xeroderma pigmentosum complementation

group A lack a single-stranded-DNA-binding activity Urs KUHNLEIN, Siu Sing TSANG, Opal LOKKEN, Silvian TONG, and Daniel TWA Environmental Carcinogenesis Unit, British Columbia Cancer Research Centre, 601 West 10th Avenue, Vancouver, B.C., Canada VSZ IL3; and Department of Medical Genetics, University of British Columbia, Vancouver, B.C., Canada V6T IW5 (Received 28 June 1983)

Human fibroblasts and HeLa ceils contain two major DNA-binding activities for superhelical DNA, which can be separated by phosphocellulose chromatography. The DNA-binding a c t i v i t y which e l u t e s f i r s t from the column coelutes with and is probably identical to a single-stranded-DNA-binding activity. The second a c t i v i t y has been characterized previously. It binds p r e f e r e n t i a l l y to super-helical DNA containing DNA d a m a g e , but does not bind to single-stranded DNA. Five cell lines derived from patients with the repairdeficiency syndrome xeroderma pigmentosum (XP) were analyzed for the presence of these binding activities. Four of the cell lines were from the A-complementation group and one was from the D-complementation group of XP. The binding activity with preference for d a m a g e d DNA was preSent in all cell lines. The single-stranded-DNA-binding activity was present in the XP-D ceil line but was absent or reduced in all of the four XP-A ceil lines tested. Mammalian cells have an excision repair system which can remove a wide spectrum of DNA damage, including damage caused by u.v. light (1). This repair system is defective in xeroderma pigmentosum patients (1,2). As a consequence, they are very sensitive to sunlight and have an approx. 10 000=fold higher frequency of skin cancer. The defect in DNA repair appears to be in an early step of the pathway: either the recognition of DNA damage by cellular proteins or the incision of DNA near DNA damage (3,z~). Complementation studies indicate that these early steps are quite complex, involving at least seven different complementation groups (5-7). Attempts to identify a structural protein or an enzyme activity which is lacking in XP cells have so far been unsuccessful. The only exceptions are cell lines which belong to the D-complementation group of XP. C e l l s from this complementation group lack an apurinic e n d o n u c l e a s e which cleaves DNA at the 5' side of apurinic sites (8-10). The role of this enzyme in DNA repair is still obscure. 01983

The Biochemical Society

668

KUHNLEIN ET AL.

In this c o m m u n i c a t i o n , we r e p o r t a c o m p a r a t i v e analysis of DNA-binding activities in normal cell lines and XP cell lines. XP cell lines f r o m the A - c o m p l e m e n t a t i o n group Iack a single-strandedDNA-binding activity. This binding activity is the dominant singlestranded-DNA-binding activity in repair-proficient cells. It contains two maior binding p r o t e i n s with mol.wts, of 48 000 and 56 000 daltons. The 4g 000-dalton protein may be identical to a protein isolated from the Novikoff hepatoma ( l l ) .

M a t e r i a l s and M e t h o d s Cell lines

Cell line 207 was a gift from Dr. S. Wood of the Department of Medical Genetics, University of British Columbia. It was derived from a skin biopsis from a 32-year-old mate Caucasian. The HeLa cell line was from Flow Laboratories Inc., and the XP ceil lines were from the Human Genetics Mutant Cell [Repository of the National Institute of H e a l t h , C a m d e n , New 3 e r s e y . Cell line XP2NE belongs to the D-complementation group and originates from a 23-year-old Caucasian female (12). Cell lines XP1CA and XPSCA were derived from an 8-year-old female and a Y-year-old male, respectively. The latter two patients were from apparently unrelated Egyptian families and differed in the severity of symptoms (13). XP12BE (5V40) and XP20S (SV~0) are SV40-transformed cells from the A-complementation group. They a r e d e r i v e d from a 7-year-old Caucasian female and a 7-year-old 3apanese female, respectively (It;,15). The cell repository numbers of XP c e l l lines in the order listed are GM0~35, GM2990, GM299L;, GMtttt29A, and GM4312A. DNA-binding assay 3H-Labelled PM2 DNA was prepared as described previously (16). U.v. irradiation of DNA was carried out at a concentration of 0.5 mM DNA nucleotide in 10 mM Tris/HCl (pH 7.5) at 0 ~ with a 6-watt GE GIST8 germicidal lamp at a dose of 1200 3/mZ. For the preparation of l i n e a r DNA, PM2 DNA was t r e a t e d with MSPI e n z y m e as recommended by the supplier (New England Biolabs). MSPt cleaves PM2 DNA at a single site ( i 7 ) . The cleaved DNA was extracted with phenol/chloroform ( I : t ) and dialyzed extensively. Single-stranded DNA was prepared from linear DNA by alkali denaturation. The standard DNA-binding assay mixture (300 ~d) contained 10 mM Tris/HC1 (pH 7.5), 2 mM EDTA, 139 fmol of 3H-labelled PM2 DNA molecules, 175 mM NaCl, and an appropriate aliquot of DNA-binding protein. The mixture was incubated for 10 min on ice. The assay mixture was then diluted with 1.7 ml of ice-cold 10 mM Tris/HCl (pH 7.5) and 100 mM NaC% and filtered immediately through a GF/C glass fiber filter (Whatman) at a flow rate of 10-30 ml/min. The filter was washed with an additional two vol. of t.7 ml of buffer, dried under a h e a t tamp, and t h e r a d i o a c t i v i t y determined by liquidscintillation counting. A unit of DNA-binding activity is defined as the amount o5 protein which retains 1 fmol of DNA molecules.

DNA BINDING IN XERODERMA PIGMENTOSUM CELLS

669

Preparation and chromatography of cell extracts

Fibroblasts were grown in plastic tissue-culture flasks (77# cm2) with minimal essential Eagle's medium (Gibco) containing 10% fet al calf serum, 100 pg/ml penicillin, 30 lJg/ml streptomycin sulfate, 100 IJg/ml kanamycin, and 2.5 IJg/ml fungizone. Cells were harvested near confluency in batches of 2# flasks, yielding a total of 5-6 x 107 ceils. At the time of harvest the number of passages was between 8 and 13. C e l l s w e r e h a r v e s t e d by t r y p s i n i z a t i o n , washed three times with phosphate-buffered saline, and stored as a pellet in liquid nitrogen. Extracts were prepared by suspending the pellets in # ml of 50 mM Tris/HCl (pH 7.5), 1 mM EDTA, and I mM DTT. The cells were disrupted by sonication and the sonicate was centrifuged for 50 min at 50 000 r.p.m, in a Beckman 50 Ti rotor. T he supernatant was made 10% in glycerol and 0.# M in NaCl and filtered through a DEAE-cellulose column (2 ml). The fractions containing DNA-binding activity were pooled and were dialyzed overnight against 500 ml of l0 mM potassium phosphate (pH 7.#), 10% glycerol, 1 mM EDTA, and 1 mM DTT (buffer A). The dialysate was centrifuged at 15 000 r.p.m, for 15 min in a Beckman Ti rotor to remove a preci pi t at e which formed during dialysis. The dialyzed e x t r a c t s which contained between 8.5 and 12.9 mg of protein were applied to a 3.5-ml phosphocellulose column. The column was washed sucessively with 3 ml of buffer A, 3-5 ml of buffer A containing 50 mM instead of 10 mM potassium phosphate, and finally eluted with a 30-ml linear gradient from 50 mM to 500 mM potassium phosphate (pH 7.#) containing 10% glycerol, 1 mM EDTA, and I mM DTT. Fractions of 0.5 ml were collected and made 200 IJg/ml in 8-1actoglobulin. Aliquots of 20 tJl of the column fractions were assayed immediately for DNA-binding activity. H e L a c e l l s and S V # 0 - t r a n s f o r m e d XP-A cells were grown and analyzed in the same manner, e x cept that (i) the extraction buffer contained 0.4 M NaCI, (ii) the volume o5 the phosphocellulose column was 8 ml, (iii) the column was eluted with an 80-ml linear gradient from 50 mM to 600 mM potassium phosphate, and (iv) fractions of 2 ml were collected and made #0% in glycerol and stored at -15~ without 8-1actoglobulin.

Results

and D i s c u s s i o n

Th e p h o s p h o c e l l u l o s e - c h r o m a t o g r a p h y p r o f i l e s of DNA-binding a c t i v i t y from ext r a c t s from normal human fibroblasts and XP cell lines from the D- and A-complementation groups are shown in Fig. 1. With e x t r a c t s from normal fibroblasts, the DNA-binding activity eluted in two m a j o r peaks , at 150-300 mM potassium phosphate (PI) and 300-500 mM potassium phosphate (PII). A similar profile was obtained with e x t r a c t s from the XP-D cell lines. The • cell e x t r a c t , however, lacked or had reduced amounts of the act i vi t y which eluted between 150 and 300 mM potassium phosphate. The recoveries of DNA-binding activity from phosphocellulose of the e x t r a c t s analyzed in Fig. l, as well as of a second e x t r a c t of the A c e l l l i n e XPSCA and an e x t r a c t of the unrelated A cell line XP1CA, are summarized in Table 1. The t o t a l recovery of a c t i v i t y and the r e l a t i v e amount of a c t i v i t y in PII was s i m i l a r in all f i v e

670

KUHNLEIN

ET

AL.

100 Normal cells

80

0.4

60

j/'

40

02

e-

E

20

o

L)

s

100

z 9

. ,,/

XP5CA ( A - g r o u p ) .,"

80

5

6o

02

40

~ I,Ll

~

20

N 100 L9 z z < z s

XP2NE C D - g r o u p ) 80

0,4 6O

40

02

20

20

40

6O

FRACTION NUMBER

Fig. I. Phosphocellulose chromatography of extracts from cell lines 207 (normal), XP5CA (A-group), and XP2NE (D-group). Chromatography and assay procedure are described in Materials and Methods. O U.v.-irradiated PM2 DNA; A, untreated PM2 DNA. extracts. PI was p r e s e n t in a p p r o x i m a t e l y equal a m o u n t s in n o r m a l and X P - D cell lines, but was r e d u c e d s e v e r a l - f o l d in all XP-A cell extracts. Q u a l i t a t i v e l y similar d i f f e r e n c e s in the elution of D N A binding a c t i v i t y f r o m phosphocellulose w e r e o b s e r v e d b e t w e e n H e L a

DNA BINDING IN XERODERMA PIGMENTOSUM CELLS

Table I.

671

Recovery of DNA-binding activity from phosphocellulose chromatography for untransformed fibroblasts

Passage

Binding activity i~ DEAE eluate (unite/mg protein)

PII

PI/PII

207 (normal)

i0

2060

45

220(100%)

5!9(100%)

0.42

XP2NE (D-group)

I0

2120

27

159(72%)

378(73%)

0.42

XP5CA (A-group)

I0

1740

32

28(13%)

411(79%)

0.07

13

1900

35

32(14%)

546(105%)

0.06

8

2140

35

52(23%)

648~125%)

Cell line

% Recovery of binding activity from P-cell column

Binding activity per mg protein applied to the P-cell column (units)* P!

Ratio of activities

0,08 XPICA (A-stoup) *The activity of individual fractions was determined~ summed between 150 and 300 mM potassium phosphate (PI) and between 300 and 500 mM potassium phosphate (PII), and divided by the amount of protein applied to the column.

cells and SV#0-transformed XP-A cells (Fig. 2; Table 2). Again, the level of Pt was reduced in XP-A (SV#0) cell lines. In contrast to untransformed XP-A cells, however, the SV~0-transformed XP-A ceils had v e r y high l e v e l s of PII. Such high levels of PII might be incompatible with the presence of PI and may be the reason why many laboratories have been unsuccessful in reintroducing DNA-repair genes into SV#0-transformed XP-A cell lines. PII has been characterized previously ( l g ) . It binds to u.v.-lightor acetoxyacetylaminofluorene-induced binding sites with a dissociation constant of # x I0-11 M, has a strict requirement for a superhelical DNA substrate, and does not bind to single-stranded DNA. PI s i m i l a r l y r e q u i r e s s u p e r h e l i c i t y when assayed with doublestranded DNA. However, in contrast to PII, it also binds to singlestranded DNA. Fig. 3 shows the same phosphocellulose chromatograms as in Fig. 2 assayed for single-stranded-DNA-binding activity. The major peak of activity obtained with HeLa cell extracts coelutes and is presumably identical to PI. This activity is reduced 3- to 6-fold in the two SV#0-transformed XP-A cell lines (Table 2). PI can be f u r t h e r p u r i f i e d by s i n g l e - s t r a n d e d - D N A cellulose chromatography. Sedimentation analysis and SDS/gel electrophoresis indicate that it contains two single-stranded-DNA-binding proteins of

Table 2. Recovery of DNA-binding activity from phosphocellulose chromatography for transformed fibroblasts* U.v.-irradiated-DNAbinding activity (units)

Single-stranded-DNAbinding activity (units)

PI

PII

PI

PII

1390(100%)

1880(100%)

8620(100%)

1730(100%)

XPI2BE (SV40)

750(54%)

3500(186%)

2940(34%)

1460(84%)

XP2OS (SV40)

610(44%)

4600(245%)

1280(15%)

1880(109%)

HeLa

*The extraction buffer contained 0.4 M NaCI (see Materials and Methods).

KUHNLEIN

672

ET

AL.

0.6 300 0.4 200

0.2

100

o

,~ 400 0.6

~300 C

0.4

200

0 . 2 0z

'~ 100

o

v

Fig. 2. Phosphoeellulose chromatography of e x t r a c t s f r o m HeLa c e l l s ~ XP12BE (SV40) cells~ and X P 2 0 S (SV40) cells. The two XP cells are transformed with SV40 and belong to the A-complementation group The extraction buffer used was 0.4 M in NaCl (see Materials and M e t h o d s ) . O ~ U.v.-treated DNA; 9 untreated DNA. The scale in the lowest panel is reduced by a factor of two. 9

~. o O 80o

XP 2 0 8

I" 0.

(8V40)

O

1-

(

- #g 000 and M r = 56 000.

cells.

0.

0.4

400

0.2

200

2O

3O

FRACTION

M

0.6

600

< z

10

xi;-

/

40

50

NUMBER

Both of these proteins are absent in

A single-stranded-DNA-binding protein of M r = from single-stranded-DNA cellulose at the same has been p r e v i o u s l y i s o l a t e d f r o m N o v i k o f f d e s t a b i l i z e s the helix of double-stranded DNA potymerase B. This protein may be identical

#g 000, which elutes ionic strength as PI, hepatoma ( l l ) . It and stimulates DNA to one of the two

DNA-bindin 8 proteins which are missing in XP-A cells 9

DNA BINDING IN X E R O D E R M A

PIGMENTOSUM CELLS

673

HeLa cells

9

8

7 _6

% x

3

c

30

:3

40

>" ).-

50 XP 12 BE ( S V 4 0 )

_6 z_ Z

30

40

Z

50 XP 2 0 S (SV 4 0 )

6

!

3'O FRACTION

40

50

NUMBER

Fig. 3. Binding to single-stranded DNA. The same column f r a c t i o n s as in F i g . 2 were a s s a y e d f o r single-stranded-DNA-binding activity. The p a n e l s are a l i g n e d so t h a t t h e s a l t g r a d i e n t s c o i n c i d e .

Acknowledgements This work was supported by the Medical R e s e a r c h Council and the National C a n c e r I n s t i t u t e of Canada. Siu Sing Tsang was a R e s e a r c h Student and Dr. Urs Kuhnlein was a R e s e a r c h Scholar of the National C a n c e r I n s t i t u t e of Canada. The authors wish to thank Mrs. G. Wood for c a r r y i n g out the t i s s u e - c u l t u r e work. References

i. Francis AA, Snyder RO, Dunn WC & Regan JD (1981) Mutation Res. 831 159-169. 2. Friedberg EL, Ehmann UK & Williams JI (1979) Adv. Radiat. Biol. 8~ 85-174. 3. Zelle B & Lohman PHM (1979) Mutation Res. 62, 363-368.

674

KUHNLEIN ET

AL.

4. Fornace AJ~ Kohn KW Jr & Kann HE Jr (1976) Proc. Natl. Acad. Sci. U.S.A. 737 39-43. 5. Kraemer K H i de Weerd-Kastelein EA, Robbins JH, Keijzer W 9 Barrett SF i Petinga RA & Bootsma D (1975) Mutation Res. 337 327-340. 6. Arase S 7 Kozuka T 9 Tanaka K 9 Ikenaga M & Takebe H (1979) Mutation Res. 59r 143-146. 7. Keijzer W, Jaspers NGJ 9 Abrahams PJ9 Taylor AMR 9 Arlett CF9 Zelle B 9 Takebe H r Kihmont PDS & Bootsma D (1979) Mutation Res. 629 183-190. 8. Kuhnlein U~ Penhoet EE & Linn S (1976) Proc. Natl. Acad. Sci. U.S.A. 739 1169-1173. 9. Kuhnlein U r Lee B 9 Penhoet EE & Linn S (1978) Nucl. Acids Res. 59 951-960. i0. Mosbaugh DW & Linn S (1980) J. Biol. Chem. 2559 11743-11752. Ii. Koerner TJ~ & Meyer RR (1983) J. Mol. Biol. 258, 3126-3133. 12. Lehman ARr Kirk-Bell $9 Arlett CF, Harcourt SAr de WeerdKastelein EA r Keijzer W & Hall-Smith P (1977) Cancer Res. 379 904-910. 13. Hashem N, Bootsma D r Keijzer W, Green A, Coriell L r Thomas G & Cleaver JE (1980) Cancer Res. 409 13-18. 14. Lin PFr Slate DL r Lawyer FC & Ruddle FH (1980) Science 2099 285-287. 15. Takebe H r Nii S r Ishii M & Utsumi H (1974) Mutation Res. 259 383-390. 16. Kuhnlein Ur Tsang SS & Edwards J (1979) Mutation Res. 64, 167-182. 17. Wang JC (1974) J. Mol. Biol. 87, 797-816. 18. Tsang SS & Kuhnlein U (1982) Biochim. Biophys. Acta 697, 202-212.