175

Mutation Research, DNA Repair, 254 (1991) 175-184 © 1991 Elsevier Science Publishers B.V. 0921-8777/91/$03.50 ADONIS 092187779100059C

MUTDNA 06422

Establishment of a monoclonal antibody recognizing cyclobutane-type thymine dimers in DNA: a comparative study with 64M-1 antibody specific for (6-4)photoproducts Terumi Mizuno 1, Tsukasa Matsunaga 1, Makoto Ihara 2 and O s a m u Nikaido 1 I Division of Radiation Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920 and 2 Department of Biology, Nara Medical University, Kashihara, Nara 634 (Japan) (Received 11 May 1990) (Revision Received 31 July 1990) (Accepted 2 August 1990)

Keywords: Monoclonal antibody; Thymine dimer; (6-4)Photoproduct; Photoreactivation; DNA repair

Summary We obtained a monoclonal antibody (TDM-1) binding to 313-nm UV-irradiated DNA in the presence of acetophenone. The binding of TDM-1 to 254-nm UV-irradiated DNA was not reduced with the subsequent irradiation of 313-nm UV. Furthermore, the treatment of UV-irradiated DNA with photolyase from E. coli and visible light exposure reduced both the antibody binding and the amount of thymine dimers in the DNA. A competitive inhibition assay revealed that the binding of TDM-1 to UV-irradiated DNA was inhibited with photolyase, but not with 64M-1 antibody specific for (6-4)photoproducts. These results suggest that TDM-1 antibody recognizes cyclobutane-type thymine dimers in DNA. Using TDM-1 and 64M-1 antibodies, we differentially measured each type of damage in DNA extracted from UV-irradiated mammalian cells. Repair experiments confirm that thymine dimers are excised from UV-irradiated cellular DNA more slowly than (6-4)photoproducts, and that the excision rates of thymine dimers and (6-4)photoproducts are lower in mouse NIH3T3 cells than in human cells.

UV light has carcinogenic, mutagenic and killing effects on mammalian cells (Setlow, 1978; Suzuki et al., 1981; Maher et al., 1982). It has been thought for a long time that the cyclobutane-type pyrimidine dimer is the dominant DNA lesion involved in the biological effects of UV. Recently, (6-4)photoproduct has been analyzed compara-

Correspondence: Dr. O. Nikaido, Division of Radiation Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920 (Japan).

tively with pyrimidine dimer as another type of DNA damage relevant to the biological implications (Cleaver et al., 1988; Mitchell, 1988; Mitchell and Nairn, 1989). It is essential to distinguish and quantify each photoproduct in order to understand the causal relationship between each type of DNA damage and its subsequent biological effects. At present, only the immunological method is capable of detecting both pyrimidine dimers and (6-4)photoproducts at physiological UV doses. Although antisera raised against UV-irradiated DNA

176

have been extensively utilized (Levine et al., 1966; Tan, 1968; Lucas, 1972; Cornelis et al., 1977; Wakizaka and Okuhara, 1979; Mitchell and Clarkson, 1981; Eggset et al., 1983; Wani et al., 1984), the multiple specificity of polyclonal antibodies appears to be not suitable for the selective detection of a specific lesion. In this respect, monoclonal antibodies recognizing each type of DNA damage are the best choice for this kind of study, because of its monoclonal nature. Furthermore, if the epitope recognized by the monoclonal antibody is characterized in detail, the immunological method would be more reliable. In this report, we established a monoclonal antibody (TDM-1) directed against UV-induced DNA damage, and carefully characterized the epitope recognized by the antibody, compared with the 64M-1 antibody specific for (6-4)photoproducts, as already established (Mori et al., 1988; Matsunaga et al., 1990), and with E. coli photolyase specific for pyrimidine dimers (Sancar and Sancar, 1988). We demonstrated that TDM-1 antibody specifically recognized cyclobutane-type thymine dimers in DNA. Furthermore, by using the 2 well-characterized monoclonal antibodies, TDM-1 and 64M-1, we differentially detected thymine dimers and (6-4)photoproducts in cellular DNA, and also analyzed differential repair of both kinds of DNA damage in mammalian cells. Materials and methods

Chemicals Calf thymus DNA (Type 1) was purchased from Sigma (St. Louis, MO). Single-stranded DNA (ssDNA) was prepared by heating double-stranded DNA (dsDNA, dissolved in 10 mM phosphatebuffered saline (PBS), pH 7.4) for 10 min in boiled water followed by rapid cooling in an ice bath. Oligo(dT)8, oligo(dTdC)4 and oligo(dC)8 were prepared with a System-1 Plus DNA Synthesizer (Beckman, Palo Alto, CA). Bovine serum albumin (Fraction V) was purchased from Armour Pharmaceutical Co. (Kankakee, IL). Methylated bovine serum albumin (mBSA) was synthesized by the method of Sueoka and Cheng (1962). Tritiated thymidine-labeled DNA (3H-DNA) was prepared according to the method reported previously (Mori et al., 1988).

Cells and media A mouse myeloma cell line (P3-NSI/1 -Ag4-1, hypoxanthine guanine phosphoribosyl transferasedeficient (HPRT , Kohler et al., 1976) as a fusion partner was purchased from Flow Laboratories, Inc. (McLean, VA) and cultured in Dulbecco's modified Eagle's medium (DMEM, Nissui Seiyaku, Tokyo) supplemented with 10% fetal bovine serum (M.A. Bioproducts, Walkersville, MD). Normal human embryonic fibroblasts, HE-4, were cultured in the same medium, and mouse NIH3T3 cells were cultured in DMEM supplemented with 10% calf serum (HyClone, Logan, UT). UV irradiation of DNA and various oligonucleotides DNA and oligonucleotides were irradiated with UV using 3 germicidal lamps (GL-10, predominantly 254 nm, Toshiba, Osaka) at room temperature. The dose rate was monitored with a Topcon UV radiometer (Tokyo Kogaku Kikai, Tokyo). Monochromatic UV irradiation (313 nm) to DNA was performed by the Okazaki Large Spectrograph (Watanabe et al., 1982) at the National Institute for Basic Biology, Okazaki. Preparation of immunogen Immunogen was prepared according to the method of Plescia (1968). An aliquot of 1 mg/ml of calf thymus DNA solution in 10 mM PBS (pH 7.4) was mixed with an equal volume of 20 mM acetophenone (Wako, Osaka) and irradiated with 313-nm UV (300 kJ/m2). After irradiation, acetophenone was removed by dialysis against PBS. Then the irradiated DNA solution was mixed with an equal volume of mBSA (500 /~g/ml in sodium chloride-sodium citrate buffer at pH 7.0 containing 0.15 M NaCI and 0.015 M Na-citrate (SSC)). This mixture was emulsified with an equal volume of complete Freund's adjuvant (Difco Laboratories, Detroit, MI). Immunization of mice BALB/c mice (6 weeks old, Sankyo Labo Service, Toyama) were injected intraperitoneally with 0.5 ml (125 /~g of DNA) of UV-dsDNAmBSA emulsified with complete Freund's adjuvant. The same immunizations were carried out 3 and 6 weeks later. One week after the last

177

immunization, the mice were given a booster injection of 313-nm UV-irradiated dsDNA conjugated with mBSA into the tail vain.

at 2000 rpm for 5 min and the supernatant was used as the antibody source throughout this study.

Detection of antibody binding Cell fusion and cloning Three days after the booster injection, the spleens were excised from the mice and dissociated by passage through 100-mesh steel gauze. Cell fusion was performed by the method of Moil et al. (1988) with a slight modification. 1 x 107 exponentially growing mouse myeloma cells were washed with serum-free D M E M and mixed with 1 x 108 immunized spleen cells followed by centrifugation. 1 ml of prewarmed 45% polyethylene glycol 4000 (Sigma) was added to the cell pellet over a period of 1 rain and the cells were incubated for 90 s at 37°C. Then 45 ml of serumfree D M E M was added and the mixture was further incubated for 5 rain at 37°C. The cells were centrifuged and the pellet was suspended in 50 ml of growth medium. 1 ml of the cell suspension was dispensed into each well of a 24-well culture plate (Nunc, Denmark) which had been inoculated with macrophages (BALB/c, 1-2 × 10 6 cells/well) 24 h before. 24 h later, 1 ml of fresh growth medium containing 2 × 10 - 4 M hypoxanthine, 8 × 10 - 7 M amethopterin and 3 . 2 x 10 -s M thymidine ( x 2 H A T medium, Littlefield, 1964) was added to each well. The cells were fed with fresh HAT medium every 3 days for 2 weeks and subsequently with the growth medium. Each well was tested for antibody activity by means of the enzyme-linked immunosorbent assay (ELISA) mentioned below, and the cells in the promising wells were cloned twice by limiting dilution in a 96-well culture plate (Becton Dickinson Labware, Lincoln Park, N J).

Preparation of TDM-1 and 64M-1 antibody The medium of clone TDM-1 or 64M-1 cultured for 4 days was collected and centrifuged at 1500 rpm for 15 min. The supernatant was mixed with an equal volume of ice-cold saturated ammonium sulfate solution (pH 7.0), left at 4°C for 1 h and centrifuged at 4°C for 30 min (10 000 rpm). The pellet was dissolved in a small volume of water and dialyzed against 10 volumes of PBS over night at 4°C. The dialysate was centrifuged

Direct binding of monoclonal antibody to antigen was measured by the ELISA. Details of the method have been described elsewhere (Matsunaga et al., 1990). In brief, polyvinylchloride flat-bottom microtiter plates (Dynatech, Alexandria, VA) precoated with protamine sulfate (Sigma) were incubated with UV-irradiated calf thymus ssDNA or dsDNA (50 n g / 5 0 / d / w e l l ) in PBS at 37°C for 20 h. After drying, the plates were washed 5 times with PBS containing 0.05% Tween 20 (PBS-T). The plates were incubated with 1% newborn calf serum in PBS at 37°C for 90 min to avoid nonspecific binding of the antibody and washed again. 100 /~1 of hybridoma supernatant or of TDM-1 antibody solution was added to the wells (in triplicate) and the plates were incubated at 37°C for 90 min. After the incubation, the plates were washed as described above and then incubated with 100 #1 of goat anti-mouse IgM conjugated with peroxidase (Cooper Biomedical Inc., Malvern, PA) at 37°C for 90 min. The plates were washed 3 times with PBS-T and subsequently washed twice with citrate-phosphate buffer (pH 5.0). Then, 100 ~tl of substrate solution containing 0.04% o-phenylene diamine and 0.007% H202 in citrate-phosphate buffer was added to each well. After 30 rain incubation at 37°C, 2 M H2SO 4 was added to stop the reaction, and absorbance at 490 nm was measured using an Immuno Reader N J-2000 (Japan Spectroscopic Co., Tokyo). The mean values of 3 wells were calculated, and the background value without antibody was subtracted. For a competitive inhibition assay, 50 ng/50/~1 of UV-irradiated dsDNA was added and dried to coat the well. TDM-1 solution giving 50% of the maximum binding to the immobilized D N A was mixed with various concentrations of the competitors. Percent inhibition was calculated as follows: Percent inhibition = [ 1 . 0 - ( A 2 -

Aa)/(A

1-

A3) ] x 100

where A 1 is the absorbance at 490 nm without c o m p e t i t o r , A 2 is the absorbance with competitor and A 3 is the background absorbance (without antibody).

178

Determination of cyclobutane-type thymine dimers high-performance liquid chromatography (HPLC) The method used for the determination of thymine dimers in D N A has been described previously (Mori et al., 1988). The amount of thymine dimers in D N A was expressed as the percent radioactivity of thymine dimers in total thymine. The induction rate of thymine dimers in D N A irradiated with 254-nm UV was 0 . 0 0 2 4 % / J / m 2.

Preparation of photolyase from E. coli When p K M 3 constructed with tac-phr fusion was introduced into E. coli JM107, photolyase constituted more than 10% of the total cellular proteins by induction with isopropyl-/3-D-thiogalactopyranoside (IPTG). To purify photolyase, bacterial cells grown in L broth containing I P T G were prepared. The procedures of purification were based on the methods described by Sancar et al. (1984). After sonication of the cells, the crude proteins were fractionated by a m m o n i u m sulfate precipitation. The enzyme fraction was applied on phenyl-sepharose CL4B, hydroxylapatite, DEAEagarose, DNA-cellulose and phenyl-sepharose CL4B columns in the order. Purified photolyase was detected as a single band in SDS-PAGE by Coomassie blue staining. The final yield was 11.2 mg from 27.5 g E. coli.

Treatment of UV-irradiated DNA with photolyase and uisible light exposure The procedure used for photolyase treatment of D N A and visible light exposure in this study is a slight modification of the method described by Sancar et al. (1984). In brief, 20 /tg of 3H-DNA irradiated with 1 k J / m 2 of UV was used as a substrate. The substrate and various concentrations of photolyase were mixed in 1000 ~1 reaction buffer containing 50 m M T r i s - H C l , pH 7.2, 10 m M NaC1, 1 m M E D T A and 10 mM dithiothreitol. The mixture in a 35-mm plastic dish covered with a 0-52 filter (to cut off below 340 nm, Corning Glass Works, Corning, NY) was exposed to photoreactivating light (3 fluorescent lamps, FL10EX, National, Osaka) at a distance of 4.5 cm. Samples were removed at intervals and treated with proteinase K (200 /~g/ml in PBS, Merck, Darmstadt, F.R.G.) at 37°C for 1 h in the dark. After the treatment, D N A was purified according

to the method of Marmur (1961), and using ELISA and H P L C techniques the binding of TDM-1 antibody was related to the amount of thymine dimers in DNA.

UV irradiation of cells and DNA extraction HE-4 and N I H 3 T 3 cells were grown to semiconfluence in 10-cm culture dishes (Becton Dickinson Labware) in humidified air with 5% CO 2 at 37°C. The medium was removed and the cells were washed twice with cold Dulbecco's phosphate buffered saline (D-PBS) prior to UV irradiation with 2 germicidal lamps at 0.2 j / m 2 / s . After various incubation times for repair, the cells were washed with D-PBS and lysed in 1 ml of cell lysis buffer (10 mM Tris-HC1 (pH 8.0)/1 m M N a z E D T A (pH 8.0)/100 m M N a C I / I % SDS). The cell lysate was collected using a rubber policeman and incubated with 200 ~ g / m l of proteinase K at 37°C overnight. D N A was extracted with the p h e n o l / C I A (chloroform : isoamyl alcohol, 24 : 1) procedure. After ethanol precipitation, D N A was dried and dissolved in TE buffer (10 m M Tris-HC1 (pH 7.5)/1 mM N a 2 E D T A ) , and subsequently treated with pancreatic RNase ( 5 0 / z g / m l ) at 37°C for 1 h. Then, D N A was reextracted as described above. Precipitated D N A was dissolved in PBS and its concentration was quantitated by measuring absorbance at 260 nm.

Measurement of thymine dimers and (6-4)photoproducts' using an ELISA For repair experiments, the extracted D N A was sonicated with a sonicator (TOMY, Tokyo) so that the fragments were about 0.5 kb, denatured and added (150 ng for the 10 J / m 2 repair assay or 100 ng for the 20 and 40 repair assays) to each well of the ELISA plate. Percent of initial antibody binding sites at each repair time was calculated as follows: Relative absorbance

a t 4 9 0 n m = [( B 2 - B 3 ) / ( B 1 - B 3)] × 100

where B 1 is the absorbance at 0 h of repair time, B 2 is the absorbance at various repair times and B 3 is the absorbance of unirradiated samples (0 J / m 2, 0 h). To determine (6-4)photoproducts in D N A using 64M-1 antibody, a biotin-streptavidin system was

179

adopted to a direct ELISA. 125 ng of sample DNA, which was extracted from UV-irradiated mammalian cells and denatured, was added to the well of the ELISA plate. The method was the same as described above, except that goat antimouse IgG(H + L) conjugated with biotin, (Fab')2 fragment (affinity purified grade, Zymed, San Francisco, CA), and then streptavidin conjugated with peroxidase (Zymed) were used instead of goat anti-mouse IgM conjugated with peroxidase. Results

Screening of hybridomas A total of 195 out of 480 wells were shown to contain hybridomas. Supernatants from these wells were tested for binding to UV-ssDNA and UVdsDNA and 7 gave positive results. After further cloning, one clone showed the highest binding activity against UV (10 kJ/m2)-irradiated ssDNA and dsDNA. This clone, designated TDM-1, was used for further study. The TDM-1 antibody was found to be the IgM 0¢) class by isotype determination. Binding of TDM-1 antibody to various UV-irradiated DNAs We examined the dose dependence of the antibody binding to UV-irradiated DNA using ELISA (Fig. 1). The binding of the antibody to dsDNA increased linearly as a function of UV dose. The candidate UV lesion recognized by the antibody was at first assumed to be a cyclobutane-type thymine dimer in DNA, because of the use of DNA irradiated with 313-nm UV in the presence of acetophenone as an immunogen, which predominantly contained abundant thymine dimers (about 17% in total thymine, data not shown) as a UV photoproduct (Lamola, 1969). Therefore, the binding of the antibody to DNA irradiated with 313-nm UV in the presence of acetophenone was examined (Fig. 2a). The binding of TDM-1 to DNA increased in a 313-nm UV dose-dependent manner corresponding to the induction rate of thymine dimers determined by HPLC, indicating that thymine dimer could be the UV lesion recognized by TDM-1 antibody. The inability of 64M-1 to bind to the DNA showed that (6-4)photoproducts were not induced in the DNA.

I

i

i

400

600

!

,

0.8

E c-

0.6

o O~

~ 0.4 t-

e~

0.2

0 0

200

254-nm UV d o s e

800

1000

(J/m 2)

Fig. 1. Binding of TDM-1 antibody to D N A irradiated with various doses of 254-nm UV. 10 ng of UV-irradiated calf thymus d s D N A was added to each well of the ELISA plate, and assayed as shown i~ Materials and methods.

To clarify whether TDM-1 antibody can recognize (6-4)photoproducts in DNA, 254-nm UVirradiated DNA (20 k J / m 2) was exposed to various doses of 313-nm UV, which is known to photolyze the (6-4)photoproduct (Patrick, 1970; Ikenaga et al., 1970) and isomerize to Dewar photoproduct (Taylor and Cohrs, 1987; Taylor et al., 1990). The binding of 64M-1 to the DNA decreased with increasing doses of 313-nm UV as shown in Fig. 2b, whereas the binding of TDM-1 to the DNA remained unchanged with 313-nm UV. These results suggest that TDM-1 does not recognize (6-4)photoproducts in DNA.

Base sequence specificity of TDM-1 binding Base sequence specificity of TDM-1 binding to pyrimidine dimers was analyzed using various concentrations of oligo(dT)8, oligo(dTdC)4 and oligo(dC) 8 as competitors in a competitive inhibition assay. When the synthetic oligonucleotides were irradiated with 10 k J / m 2 of UV, only oligo(dT) 8 inhibited TDM-1 binding to immobilized UV-dsDNA in a concentration-dependent manner (Fig. 3). We could not observe any competition by oligo(dTdC)4 or oligo(dC)8 even at the highest concentration.

180 100 0.6

•

i

a

I

•

TDM-I///'i

/,o

o.s

E c ¢) o~

,/

0.4

0.3

10

60

t,'O

o/o/

0.2

o. I I-

or-

II

O 40

v

,_E

•

•

64M-1

•

--0-0~t0

"~

•.

TDM-1

100

20

o ~y_

-- l

~u

E

6

=

o , , , m - , - I ,, 10

50

64M-1

b ~

0 0

I 25

I 50

I 75

~ ..... W ,

3 I Competitor"

0.3

0.1

(Hg)

Fig. 3. Competitive inhibition of TDM-1 antibody binding to d s D N A (50 ng) irradiated with 313-nm UV (160 k J / m 2) in the presence of acetophenone by various concentrations of oligonucleotides irradiated with 10 k J / m 2 of 254-nm UV.

•i

2 ~"

UV-oligo(dTdC)4 UV-oligo(dC)8

A

/0

- 12

~'I~T~.I~~ :

80

UV-ol i g o ( d T ) 8

1

2

I I I00

0

313-nm UV dose (J/m 2) Fig. 2. Binding of T D M - I antibody to D N A irradiated with 313-nm UV in the presence of acetophenone (a) and to D N A irradiated with 254-nm UV (20 k J / m 2) and subsequently with 313-nm UV up to 100 k J / m 2 (b). 10 ng of calf thymus d s D N A was added to each well of the ELISA plate. The a m o u n t of thymine dimers in D N A was determined by H P L C (open symbols). The binding of 64M-1 antibody to the D N A s (50 ng) was also measured.

that the antibody does not recognize any UV lesions other than thymine dimer. Fig. 4 suggests that the damage recognized by TDM-1 antibody, which seems to be thymine di,

looo 1 ~ E

,

,

_

,

QI 3

i

800

=

Inhibition of TDM-1 binding to UV-irradiated DNA E. coli photolyase Since cyclobutane-type pyrimidine dimer but not (6-4)photoproduct is the substrate for E. coli photolyase (Brash et al., 1985), we studied the binding of the antibody to D N A which was irradiated with UV and subsequently treated with photolyase at various molar ratios under visible light exposure (Fig. 4). The percent of thymine dimers in total thymine, assayed by HPLC, decreased with increasing time of visible light exposure and with increasing concentrations of photolyase. The binding of the antibody to D N A decreased in a similar manner (Fig. 4). It is noteworthy that TDM-1 antibody does not bind to the D N A in which all thymine dimers are monomerized (PR : dimer = 1 : 1, 30-rain exposure), suggesting

600

2

i

g 400

~

1 ~-

"~ 200

0 0

I 10 PR time

' 20 (rain)

~w)-- 0 A

30

Fig. 4. Effect of photoreactivation on TDM-1 antibody binding to UV-irradiated DNA. 3 H - D N A irradiated with 1 k J / m 2 of 254-nm UV was treated with various concentrations of photolyase and exposed to visible light for various times. The binding of TDM-1 was expressed as UV dose equivalent (closed symbols). The a m o u n t of thymine dimers in D N A was determined by HPLC (open symbols). The molar ratios of photolyase against thymine dimer are 0 : 1 (squares); 0.5:1 (triangles); 1 : 1 (circles).

181 0.8

TABLE 1 COMPETITIVE INHIBITION (%) OF TDM-1 ANTIBODY BINDING TO UV-IRRADIATED DNA (1 kJ/m 2) BY VARIOUS PROTEINS Competitor Photolyase 64M-1 a BSA

E

1:10

19.4 3.4 0.1

44.3 4.3 - 0.1

O

0.6

"O

Molar ratio (thymine dimer : competitor) 1:1

i

i

. Thymine

i

!

.(6-;)p.o'tooroduc

dimer

HE-4

/T

OHE-4

NIH3T3

/

0.4

0.2

I t I I

a The amount of 64M-1 showing maximum binding to immobilized UV-irradiated DNA (20 kJ/m 2) was designated 1.

0

10

20

30

254-nm

40

I 10

UV dose

I 20

I 30

I 40

(J/m 2 )

Fig. 5. Induction of thymine dimers and (6-4)photoproducts in DNA of mammalian cells irradiated with various doses of 254-nm UV. The sample DNAs were extracted from UVirradiated cells, and then denatured and assayed by ELISA using TDM-1 (a) or 64M-1 (b) antibody.

mers, is also the substrate for photolyase. W e p r e s u m e d that T D M - 1 b i n d i n g to U V - i r r a d i a t e d D N A m a y be competitively i n h i b i t e d with photolyase. We e x a m i n e d the b i n d i n g of T D M - 1 to U V - i r r a d i a t e d D N A in the presence of photolyase in the dark (Table 1). Photolyase i n h i b i t e d T D M - 1 b i n d i n g to U V - i r r a d i a t e d D N A in a concentrat i o n - d e p e n d e n t m a n n e r . The result reveals that T D M - 1 a n t i b o d y a n d photolyase competitively b i n d to the same k i n d of D N A damage. A similar experiment using 64M-1 a n t i b o d y was performed. Since the 64M-1 a n t i b o d y was f o u n d to be of the IgG2b (x) subclass, we can

selectively measure the binding of each antibody in the TDM-1 and 64M-1 mixture using anti-IgM or anti-IgG, respectively, as the second antibody conjugated with peroxidase in ELISA. Table 1 shows that the 64M-1 antibody recognizing (64)photoproducts, as well as BSA, never influences TDM-1 binding to UV-irradiated DNA.

120

I00

80

~ 4o

~~

• •

20

20

J/m 2 40 J/m 2

(6-4) p hotoproduc t ~7 40 J/m 2

6

12

I 18

--~" 24 0 Repair

time

I 6

I 12

I 18

I 24

(hr)

Fig. 6. Removal kinetics of antibody-binding sites in mammalian ceils irradiated with 254-nm UV. HE-4 (a) and NIH3T3 (b) cells were irradiated with various doses of 254-nm UV and incubated for various periods. The DNAs were extracted, denatured and assayed by ELISA using TDM-1 (e, A, II) or 64M-1 (v) antibody. Removal of 64M-1 antibody-binding sites was analyzed only in cells irradiated with 40 J/m 2 of 254-nm UV.

182 Induction and removal of thymine dimers and (64)photoproducts in UV-irradiated mammalian cells We tried to detect thymine dimers in UVirradiated cellular DNA by ELISA using TDM-1 antibody. HE-4 and NIH3T3 cells were exposed to various doses of UV, and the extracted DNA was used for determination of thymine dimers (Fig. 5a). Induction of (6-4)photoproducts was also measured by 64M-1 antibod~¢ using the same samples (Fig. 5b). Absorbance at 490 nm in ELISA, showing the binding of TDM-1 and 64M-1 antibodies, increased linearly with UV dose. We analyzed the removal kinetics of the antibody-binding sites in HE-4 and NIH3T3 cells irradiated with various UV doses (Fig. 6). Increasing the UV doses resulted in an increasing percent of TDM-1 antibody-binding sites remaining at 24 h. Fig. 6 also shows that human HE-4 cells excised both TDM-1 and 64M-1 antibody-binding sites more efficiently than did mouse NIH3T3 cells. Furthermore, loss of TDM-1 antibody-binding sites in the UV-irradiated cells examined in this experiment was processed more slowly than that of 64M-1 antibody-binding sites, confirming the results reported by Mitchell et al. (1985). Discussion

The TDM-1 antibody, which was established in this study, bound to 254-nm UV-irradiated DNA (Fig. 1) and 313-nm UV-irradiated DNA in the presence of acetophenone (Fig. 2). In addition, the binding of TDM-1 antibody to 254-nm UV-irradiated DNA decreased with the pretreatment of the DNA with photoreactivation (monomerization of cyclobutane-type pyrimidine dimers, Fig. 4), but not with subsequent irradiation of 313-nm UV (photoisomerization of (6-4)phot0product to Dewar photoproduct (Fig. 2). The binding of TDM-1 antibody is entirely parallel to the amount of thymine dimers in DNA determined by HPLC, but not to 64M-1 binding to the DNA (Fig. 2). We conclude that TDM-1 antibody recognizes cyclobutane-type pyrimidine dimers, but not (64)photoproducts. Competitive inhibition assay with photolyase and with 64M-1 [antibody (Table 1) confirms this conclusion. Furthermore, TDM-1 antibody did not bind to OsO4-treated DNA, indi-

cating that it does not recognize thymine glycols (data not shown). The base sequence specificity of TDM-1 binding to pyrimidine dimers was analyzed in Fig. 3. Only UV-oligo(dT)8 exhibited an inhibitory effect in a concentration-dependent manner. Umlas et al. (1985) reported that the relative induction rates of pyrimidine dimers in TT, T C / C T and CC sequences were 6.7:3.2: 1, respectively, in defined-sequence DNA irradiated with 5 k J / m 2 of UV. Mitchell and Clarkson (1984) reported that TT dimers in poly(dT) were induced about 10 times more than CC dimers in poly(dC) when irradiated with 10 k J / m 2 of UV. Garces and Davila (1982) also reported that the induction of TT dimers and T C / C T dimers in 3H-DNA irradiated with 10 k J / m 2 of UV reached a maximum level, at which the percent of the 2 dimers in total thymine is 3.84% and 0.81%, respectively. Although the actual yields of various cyclobutane pyrimidine dimers in each of the UV-irradiated oligonucleotides used in the present study have not been determined, we easily find that TDM-1 antibody specifically recognizes cyclobutane-type dimer formed between thymine and thymine. Table 1 also shows the interaction between TDM-1 antibody and other UV damage binding proteins. Photolyase inhibited TDM-1 binding to UV-DNA in the dark, indicating that both proteins competitively bound to the same DNA damage, which is the thymine dimer. We also found that pretreatment of UV-irradiated DNA with TDM-1 antibody inhibited the monomerization of thymine dimers following the photoreactivating procedure (data not shown). On the other hand, 64M-1 antibody, which has been characterized in detail (Matsunaga et al., 1990), does not influence the binding of TDM-1 to UV-DNA (Table 1), suggesting that the UV lesions recognized by the 2 antibodies are completely different. Using the 2 monoclonal antibodies, TDM-1 and 64M-1, we could detect thymine dimers as well as (6-4)photoproducts induced in mammalian cells. The repair experiments shown in Fig. 6 reveal the following points: (1) increasing the UV dose results in an increasing percent of antibodybinding sites remaining at 24 h; (2) human HE-4 cells excise thymine dimers and (6-4)photoprod-

183

ucts more efficiently than do mouse NIH3T3 cells; (3) thymine dimers are removed from both types of mammalian cells more slowly than (6-4)photoproducts. Other monoclonal antibodies specific for thymine dimers have already been established (Strickland and Boyle, 1981; Roza et al., 1988). The repair kinetics of thymine dimers in Fig. 6 is similar to the data in normal human fibroblasts obtained by Roza et al. (1988), whereas it is extremely different from that in xeroderma pigmentosum variant fibroblasts and biopsy material from a normal population determined by aUVssDNA-1 antibody, which was established by Strickland and Boyle (1981) (Roth et al., 1988). On the other hand, the data reported by Mitchell et al. (1985), who used antisera raised against thymine dimers and (6-4)photoproducts, are consistent with our results. A comparison of repair kinetics of UV lesions in mammalian cells suggests the necessity of strict characterization of the epitopes recognized by the antibodies used in their experiments. In conclusion, we succeeded in establishing and characterizing a monoclonal antibody directed against cyclobutane-type thymine dimers in DNA, in addition to the 64M-1 antibody specific for (6-4)photoproducts, as already established. By using the 2 well-characterized monoclonal antibodies with high specificity, we demonstrated the induction and differential repair of thymine dimers and (6-4)photoproducts in mammalian cells.

Acknowledgements The irradiation with 313-nm UV was carried out under the NIBB Cooperative Research Program for the Okazaki Large Spectrograph (87-506). We are grateful to Dr. M. Watanabe and Mr. M. Kubota of the National Institute for Basic Biology, for helping us in the 313-nm UV irradiation. We are also grateful to Prof. B.W. Fox of Paterson Institute for Cancer Research for his critical reading of the manuscript and suggestions. This work was supported in part by Grants-in-Aid for Cancer Research (Nos. 01010036 and 01010040), and for Scientific Research (Nos. 63621510 and 02454547), from the Ministry of Education, Science and Culture of Japan, and by a

grant from the Hokkoku Foundation for Cancer Research (1989).

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