THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, NO. 29, Issue of October 15, pp. 19786-19789, 1991 Printed in U.S.A.

Identification and Characterization of xpac Protein, the Gene Product of the Human XPAC (Xeroderma Pigmentosum Group A Complementing) Gene*

(Received for publication, June 3, 1991)

Naoyuki MiuraS, Iwai Miyamoto, Hiroshi Asahina, Ichiro Satokata, Kiyoji Tanaka, and Yoshio Okada From the Institute for Molecular and Cellular Biology, Osaka University, 1-3 Yamada-Oh, Suita, Osaka 565, Japan

We have cloned human xeroderma pigmentosum group A complementing (XPAC) cDNA that encodes a “zinc finger” protein with a predicted size of 31 kDa. To detect the xpac protein in cells, we raised antibody against a recombinant human xpac protein. Using this antibody, we identified the xpac protein in the nucleus of cells. In normal human cells, 40- and 38-kDa pro- teins were detected by sodium dodecyl sulfate-poly- acrylamide gel electrophoresis. A reduced amount of the smaller protein was detected in XP 39OSSV cells, which show low UV sensitivity, and no xpac proteins were detected in XP 2OSSV cells, which show high UV sensitivity. These levels of xpac proteins in xeroderma pigmentosum cells were determinants of heterogeneity of the DNA repair defect in group A xeroderma pig- mentosum. Synthesis of the xpac protein did not in- crease after UV irradiation.

DNA repair is considered to play a key role in maintaining DNA integrity on attack by various carcinogens and mutagens including sunlight. The nucleotide excision repair pathway is a major pathway for DNA repair in mammals. However, nothing is known about the proteins participating in DNA excision repair in humans. Since xeroderma pigmentosum (XP)’ is a rare hereditary disease of a defect in DNA excision repair (1, 2), studies on the defective genes in XP should be helpful in understanding the DNA excision repair mechanism. There are seven complementing groups of XP, group A-G (3), indicating that at least seven different gene products are involved in the DNA excision repair pathway.

Weeda et al. (4) recently cloned the excision repair cross- complementing rodent repair deficiency 3/xeroderma pigmen- tosum group B complementing gene, which encodes a protein of 782 amino acids and is suggested to be a DNA helicase.

Previously, we cloned the gene defective in group A XP cells and named it the XPAC (xeroderma pigmentosum group A complementing) gene (5). Nucleotide sequence analysis of human XPAC cDNA showed that the encoded protein is 273

* This work was supported by a Grant-in-Aid from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed The Institute for Molecular and Cellular Biology, Osaka University, 1-3 Yamada-Oka, Suita, Osaka 565, Japan. Tel.: 06-877-5238; Fax: 06-875-2468.

The abbreviations used are: XP, xeroderma pigmentosum; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; UDS, unscheduled DNA synthesis; XPAC, xeroderma pigmentosum group A complementing.

amino acids long and has one zinc finger motif (6). In molec- ular genetic analyses, we found that XP 2OSSV cells from a XP A patient with severe clinical symptoms contained little of the smaller mRNA and that there was a G-C transversion in the splicing acceptor site in intron 3 of the XPAC gene (7). Moreover, we found that in XP 39OSSV cells from a XP A patient with mild symptoms, the steady-state level of XPAC mRNA was similar to that of normal cells but that a nonsense mutation was present in the 228th codon in both alleles,2 predicting a truncated xpac protein lacking the 46 carboxyl- terminal amino acids. In the present study, we identified and characterized the xpac proteins in normal cells and XP A cells.

MATERIALS AND METHODS

Human XPAC cDNA was cut at the Fnu4HI and Aha111 sites at Production of a Recombinant xpac Protein in Escherichia coli-

nucleotide 190 and 860 (6), respectively. The fragment was filled in and ligated into the BamHI site of PET-3c (8) using a BamHI linker. The ligated plasmid, PET-XP2, was transformed into BL21(DE3), and the cells were treated with 1 mM isopropylthiogalactoside for 2 h to induce the xpac protein. The recombinant xpac protein was purified as described previously (9) except that 6 M guanidine HCI was used instead of 8 M urea. Rabbits were immunized with 0.3 mg of the purified recombinant xpac protein to obtain antiserum. The anti- xpac antibody was then purified on protein A-Sepharose (Pharmacia LKB Biotechnology Inc.).

Metabolic Labeling and Immunoprecipitation-WI38VA13 cells, HeLa cells, XP 2OSSV cells, and XP 39OSSV cells were cultured as described previously (7). They were then labeled for 2 h with [”SI methionine (0.4 mCi/ml) or [32P]H3P04 (0.5 mCi/ml) in methionine- free or phosphate-free minimum essential medium, washed with phosphate-buffered saline, and solubilized in radioimmune precipi- tation buffer (50 mM Tris-HC1, pH 7.5, 0.15 M NaCI, 1% Nonidet P- 40, 0.5% deoxycholate, 0.1% SDS) containing protease inhibitors (2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 2 pg/ml of aprotinin, leupeptin, and antipain). Solubilized samples, precleared with 40 p1 of protein A-Sepharose, were incubated with anti-xpac antibody for 1 h and then with 20 p1 of protein A-Sepharose for 1 h. The protein A-Sepharose was then washed three times with radioim- mune precipitation buffer. Immunoprecipitates were subjected to 12.5% SDS-PAGE under reducing conditions. After electrophoresis, the gel was impregnated with Enhance (Du Pont-New England Nu- clear) and dried for fluorography.

In Vitro Transcription and in Vitro Translation of xpac Protein- Human XPAC cDNA was cut at the Mae1 andAhaIII sites (nucleotide -24 and 860) (6 ) , filled in, ligated with an EcoRI linker, and inserted into the EcoRI site of pGEM7Zf(+) (Promega Biotec) to construct the pGM-H19-WS plasmid. In vitro transcripts were obtained as described previously (10) and translated in uitro with a rabbit retic- ulocyte lysate (Amersham N90) in the presence of [35S]methionine.

Wheat Germ Agglutinin-Agarose Chromatography-Labeled mate- rials were loaded onto a column of wheat germ agglutinin-agarose

I. Satokata, K. Tanaka, N. Miura, M. Narita, T. Mimaki, Y. Satoh, S. Kondo, and Y. Okada, submitted for publication.

19786

Characterization of xpac Protein 19787 (Honen Corp., Tokyo) equilibrated with 50 mM Tris-HC1, pH 7.5, containing 0.15 M NaCI, 0.1% Nonidet P-40 and 1 mM dithiothreitol. The column was washed with 5 column volumes of the same buffer and developed with 0.3 M N-acetylglucosamine in the same buffer.

Immunofluorescent Staining of xpac Protein"WI38VA13 cells, XP 2OSSV cells, and XP 39OSSV cells were cultured on coverslips, fixed with 3.5% formalin in phosphate-buffered saline, and permeabilized with 0.2% Triton X in phosphate-buffered saline. Anti-xpac serum or preimmune serum was used as the first antibody and fluorescien isothiocyanate-conjugated goat anti-rabbit IgG (Zymed, CA) as the second.

Microinjection of a HeLa Cell Extract into XP Cells-Preparation of a HeLa cell extract, microinjection of this extract, and assay of unscheduled DNA synthesis (UDS) were performed as described previously (11).

RESULTS AND DISCUSSION

To identify and characterize the xpac protein, the product of the human XPAC gene, we produced a recombinant xpac protein in E. coli. The expected protein was a fusion protein with 14 extra amino acids (Met-Ala-Ser-Met-Thr-Gly-Gly- Gln-Gln-Met-Gly-Arg-Ile-Arg) and the 209 carboxyl-terminal amino acids of the xpac protein. SDS-PAGE analysis of the purified protein showed that it migrated to a position corre- sponding to a molecular mass of 34 kDa (Fig. l a ) . Rabbits were immunized with this purified recombinant xpac protein to obtain anti-xpac antiserum.

We used immunoprecipitation analysis with anti-xpac serum to identify the xpac protein in the cells. Normal human cells, WI38VA13 cells, HeLa cells, XP group A cells, XP 2OSSV cells, and XP 39OSSV cells were labeled with [35S] methionine for 2 h, solubilized, and used for immunoprecipi- tation. On SDS-PAGE analysis (Fig. lb) , no specific proteins were precipitated with preimmune serum (lanes 1-3). But with anti-xpac serum, two proteins with molecular masses of 40 and 38 kDa were specifically precipitated from prepara- tions of WI38VA13 cells ( l a n e 5 , arrow) and HeLa cells (data not shown). Two bands of protein of 35 and 83 kDa were detected in the preparation from XP 39OSSV cells ( l a n e 6, arrowhead). In contrast, no specific proteins were precipitated from the preparation from XP 2OSSV cells ( l a n e 4) . Untrans- fected XP 2OSSV cells and pcD2h19- (SV40 promoter-driven human xpac expression plasmid) (6) transfected XP 2OSSV cells were also processed for immunoprecipitation with anti- xpac serum in the same way. As shown in Fig. IC, two bands of 40 and 38 kDa were detected in pcD2hl9-transfected XP POSSV cells (lane 1 ) but not in the parental XP 2OSSV cells ( l a n e 2) . From these results, we conclude that the proteins migrating at positions corresponding to molecular masses of 40 and 38 kDa were xpac proteins. The findings that no xpac proteins were detected in XP 2OSSV cells and that xpac proteins in XP 39OSSV cells were 5 kDa smaller than those in WI38VA13 cells are in good agreement with the results of our previous (7) and recent' molecular genetic analyses. How-

TABLE I Effect of anti-xpac serum on the ability of the HeLa cell extract to

complement XP A cells

Recipient cells Injected substances Grain number' cell (UDS)

FS3 (normal) Buffer 44 f. 8 (100%) GM5509 (XP A) Buffer 2 rf: 1 (4%)

HeLa cell extract 44 f. 8 (100%) HeLa cell extract 45 rf: 11 (102%)

+ preimmune serum

+ anti-xpac serum

HeLa cell extract 3 f. 1(6%)

v) v) 0 - * *

kDa g r R R s Z kDa 200-

-"I-

kDa 92.5-

110- 69- 84-

46-

47-

30- 33- - 24-

16- 14.3-

-92.5

-69

-46 " - c 4

-I

c

4

.30

,14.3

1 2 3 4 5 6

FIG. 1. a, Coomassie staining of purified recombinant xpac protein separated by SDS-PAGE. b and c, fluorogram of immunoprecipitates from various cell lines separated by SDS-PAGE. For b, WI38VA13 cells (lanes 2 and 5), XP 2OSSV cells (lanes 3 and 4) , and XP 39OSSV cells (lanes 1 and 6) were metabolically labeled, solubilized, and immunoprecipitated with preimmune serum (lanes 1-3) and anti- xpac serum (lanes 4-6). Note that the amount of trichloroacetic acid- precipitable material from XP 39OSSV cells was twice those from WI38VA13 and XP 2OSSV cells. For c, untransfected XP 2OSSV cells (lane 2 ) and pcD2h19 plasmid-transfected XP 2OSSV cells (lane 1 ) were processed as for b and immunoprecipitated with anti-xpac serum.

1 2 3 4 5 " .

kDa

92.5-

69-

46-

+ +

30-

FIG. 2. Comparison of proteins translated in uiuo and in vitro. Lune 1, immunoprecipitates from in vivo-labeled WI38VA13 cells; lane 2, in vitro translation products of in vitro transcripts of pGM-HlS-WS; lane 3, in vitro translation products of in vitro tran- scripts of pGEM'IZf(+) (control); lane 4, immunoprecipitates of lane 2 with anti-xpac serum; lane 5, immunoprecipitates of lane 3 with anti-xpac serum.

ever, densitometric measurement showed that the amount of xpac protein in XP 39OSSV cells was about one-fifth that in WI38VA13 cells (Fig. Ib, lanes 5 and 6 ) . As the steady-state levels of XPAC mRNA in XP 39OSSV cells and WI38VA13 cells were similar, this fact suggests that carboxyl-terminal truncation might decrease the stability of the xpac protein in the cells.

For further characterization of the xpac protein, we synthe- sized it by in vitro translation of in vitro transcripts from the cloned XPAC cDNA. As shown in Fig. 2, the in vitro trans- lated proteins had identical electrophoretic mobilities ( l a n e 2) to those of in vivo-labeled proteins ( l a n e 1 ), and the two

19788 Characterization of xpac Protein

bands were recognized by anti-xpac serum ( l a n e 4) . Moreover, in a pulse-chase experiment in WI38VA13 cells in which the cells were pulse-labeled for 10 min and chased in Dulbecco's modified Eagle's medium for various times, the ratio of the intensities of the 40- and 38-kDa bands remained constant from 0 min to 4 h of chase (data not shown). When WI38VA13 cells and XP 2OSSV cells were metabolically labeled with ["P]H3P04, solubilized, and immunoprecipitated with anti- xpac serum, no 32P incorporation into the two bands of xpac protein was detected (data not. shown).

Sometimes nulcear proteins have 0-linked N-acetylglucos- amine (GlcNAc) monosaccharide residues (12). To test whether the xpac proteins also have GlcNAc residues, we translated them in vitro (as described above) and loaded them on a wheat germ agglutinin-agarose column. The 40- and 38- kDa proteins were detected in the flow through fraction but not in the eluate with 0.3 M GlcNAc (data not shown). Thus, we obtained no evidence that the xpac protein was posttrans- lationally modified or processed.

FIG. 3. Indirect immunofluorescent staining of xpac protein in various cells. XP 2OSSV cells (A and D), WI38VA13 cells ( B and E ) , and XP 39OSSV cells (C and F) were cultured on coverslips and fixed. Preimmune serum (A-C) or anti-xpac serum (D-F) was used as the first antibody and fluorescein isothiocyanate-conjugated anti-rabbit IgG as the second.

46-

30

14.3.

1 2 3 4 5 6 7 8 9 1 0

FIG. 4. Effect of UV irradiation on the synthesis of xpac protein. WI38VA13 cells were irradiated with various doses of UV light, and 2 h (lanes 1-5) and 4 h (lanes 6-10) later, they were labeled for 30 min with [3sS]methionine and solubilized. Equal counts of trichloroacetic acid-precipitable material of solubilized samples at each time were used for immunoprecipitation. Lanes 1 and 6,O J/m2 (control); 2 and 7, 0.5 J/m2; 3 and 8, 1.0 J/m2; 4 and 9, 2.0 J/m2; 5 and 10, 4.0 J/m2.

If the translated protein starts from the second methionine at amino acid position 37, the difference in sizes of the proteins starting at the first and second methionines should be about 4 kDa. In a preliminary experiment in which a mutant cDNA construct that contained the second methio- nine as the initial translation site was transfected into XP POSSV cells, we detected two bands with molecular masses of 36 and 34 kDa. The possibility that double translation initiation results in two proteins is excluded. So because two bands of xpac protein of exactly the same sizes as in vivo products were obtained on in vitro translation of in vitro transcripts from a single cDNA, we tentatively consider that the 38-kDa protein is a degradation product of the 40 kDa protein. The possibility that the 40-kDa protein is a posttrans- lationally modified form of the 38-kDa protein cannot, how- ever, yet be excluded.

To determine the intracellular localization of the xpac protein, we stained cells by the indirect immunofluorescent technique. As shown in Fig. 3, in WI38VA13 cells, the xpac protein was mainly localized in the nucleus (panel E ) . In XP 39OSSV cells also, the xpac protein was weakly but clearly stained in the nucleus ( punel F) . In XP 2OSSV cells, however, no staining of the xpac protein was detected in either the cytoplasm or the nucleus (panel D). These findings that no xpac proteins were detected in XP 2OSSV cells and reduced amounts (about one-fifth) of truncated xpac protein were detected in the nucleus in XP 39OSSV cells may explain why patient XP 20s has severe symptoms, while patient XP 390s has mild ones.

The level of UvrA protein, which is a zinc finger protein involved in DNA excision repair in E. coli, increased 10-fold on SOS induction (13). Therefore, we examined whether synthesis of the xpac protein increased after UV irradiation. For this, WI38VA13 cells were irradiated at up to 4 J/m2, and 2,4, and 8 h later, control and UV-irradiated cells were labeled with [35S]methionine for 30 min, solubilized, and processed for immunoprecipitation, SDS-PAGE, and fluorography. The de novo synthesis of xpac protein was not changed, 2,4, and 8 h after UV irradiation (Fig. 4 and data not shown).

Finally, we investigated whether anti-xpac serum inhibited the DNA repair activity in a HeLa cell extract. In GM 5509 cells (XP group A cells), no UDS was detected after UV irradiation. When a HeLa cell extract (5 mg/ml) was injected into GM 5509 cells, UDS was restored to the normal level. Preincubation of a HeLa cell extract with preimmune serum did not affect its complementing activity, but its preincuba- tion with anti-xpac serum (0.5 mg/ml) completely abolished its complementing activity to XP group A cells. These results indicate that the xpac protein is the only protein defective in XP group A cells. Consistent with these findings, a XP-A factor that corrects XP A cells was recently purified from HeLa cells and calf thymus by conventional chromatographic procedures and estimated to have a molecular mass of 40-45 kDa by SDS-PAGE.3*4 In the present study, the xpac protein was found to have an apparent molecular mass of 40 kDa (Fig. lb). Thus, the xpac protein seems to be identical to the XP-A factor.

In this study we raised antibody against a recombinant protein and characterized the xpac protein in normal cells and XP A cells. The xpac protein is a nuclear protein with a zinc finger motif and so is probably a DNA-binding protein. However, the functional role of the xpac protein in the DNA repair pathway in human cells is not known. In a preliminary experiment, we found that microinjection of a recombinant

J. H. J. Hoeijmakers, personal communication. ' M. Yamaizumi, personal communication.

Characterization of xpac Protein 19789

xpac protein into XP A cells restored UDS completely. As a der Eb, A. J., and Hoeijmakers, J. H. J . (1990) Cell 6 2 , 777- soluble cell-free system for UV-dependent DNA excision re- 791 pair has been developed (14), further using this cell- 5' Tanaka, K., Satokata, I., Ogita, z', Uchida, T., and Okada, y.

free system, recombinant proteins* and should be 6. Tan&, K., Miura, N., Satokata, I., Miyamoto, I., Yoshida, M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,5512-5516

helpful in determining the role of the xpac protein in the repair pathway.

C., Satoh, Y., Kondo, S., Yasui, A., Okayama, H., and Okada, Y. (1990) Nature 348, 73-76

7. Satokata, I., Tanaka, K., Miura, N., Miyamoto, I., Satoh, Y., Acknowledgments-We thank Dr. Mahito Nakanishi for helpful Kondo, S., and Okada, Y. (1990) Proc. Natl. Acad. Sci. U. S. A.

8. Rosenberg, A. H., Lade, B. N., Chui, D., Lin, S., Dunn, J. J., and Studier, F. W. (1987) Gene ( A n s t . ) 66,125-135

9. Samhrook, J., Fritsh, E. F., and Maniatis, T. (1989) Molecular

1. Cleaver, J. E. (1968) Nature 218,652-656 Cloning: A Laboratory Manual, pp. 17.37-17.41, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

2. Cleaver, J. E., and Kraemer, K. H. (1989) in The Metabolic Basis 10. Struhl, K. (1987) in Current Protocol in Molecular Biology, pp. of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., 10.17.1-10.17.5, John Wiley & Sons, Inc. New York and Valle, D., eds) 6th Ed., pp. 2949-2971, McGraw-Hill Inc., 11. Yamaizumi, M., Sugano, T., Asahina, H., Okada, Y., and Uchida, New York T. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,1476-1479

comments and discussion and Keiko Hirata for technical assistance. 87,9908-9912

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