Mutation Research, DNA Repair, 273 (1992) 49-56
49
© 1992 Elsevier Science Publishers B.V. All rights reserved 0921-8777/92/$05.00
MUTDNA 06470
Biochemical heterogeneity in xeroderma pigrnentosum complementation group E Scott K e e n e y , H a r r i s o n W e i n a n d S t u a r t L i n n Division of Biochemistry and MolecularBiology, Barker Hall, University of California, Berkeley, CA 94720 (U.S.A.) (Received 26 April 1991) (Revision received 14 June 1991) (Accepted 18 June 1991)
Keywords: DNA repair; DNA-binding proteins; Proteins, DNA-binding; UV irradiation; XP complementation group E; DNA, UV-irradiated
Summary Cells from two patients with xeroderma pigmentosum complementation group E (XP-E) have been shown to lack an activity which binds specifically to UV-irradiated DNA (Chu and Chang, 1988). We investigated the occurrence of this binding activity in cell strains from nine additional, unrelated XP-E patients and found that all but one of these strains contained normal levels of the binding protein. Furthermore, the binding activity from these XP-E strains was indistinguishable from that of normal controls in thermal stability, behavior on ion-exchange chromatography, and electrophoretic mobility of protein-DNA complexes, indicating that there were no gross structural alterations in the protein. The association of XP-E with a deficiency in DNA-damage binding protein in cells from 3 of 12 XP-E patients (compared to 0 of 20 non-XP-E controls) is statistically significant (p < 0.05), but there is no obvious correlation between the biochemical defect and the clinical or cellular characteristics of individual patients. Implications of these findings for the role of the binding protein in XP-E are discussed.
Xeroderma pigmentosum (XP) is a rare genetic disease characterized by a clinical and cellular hypersensitivity to UV irradiation (see Cleaver and Kraemer, 1989, for a recent review). Patients show dermatologic abnormalities, including thickening and hyperpigmentation of sunlight-exposed skin, and develop sunlight-induced malignancies
Correspondence: Dr. Stuart Linn, Division of Biochemistry and Molecular Biology, Barker Hall, University of California, Berkeley, CA 94720 (U.S.A.), Tel. (510) 642-7583, Fax (510) 643-5035.
at an earlier age than usual. Cells from XP patients show hypersensitivity to killing by UV irradiation, and this cellular phenotype correlates with a defect in excision repair of UV-induced DNA lesions. Individuals with XP are divided into 7 complementation groups (A through G) and an excision-proficient variant group (XP-V). Considerable effort has focused on elucidating the molecular basis of the DNA-repair defects of the various XP groups. Recently, genes which are defective in XP-A (Tanaka et al., 1989) and XP-B (Weeda et al., 1990) have been identified by molecular genetic techniques. Other approaches
50 include the use of permeabilized cells (Keeney and Linn, 1990), microinjection (Hoeijmakers et al., 1990), or an in vitro plasmid repair system (Wood et al., 1988; Hoeijmakers et al., 1990) to assay for protein factors which correct the defect of XP ceils. Alternatively, XP ceils have been screened for defects in enzymatic activities which are thought to be required for excision repair. Using the latter approach, Chu and colleagues reported that cells from two XP-E patients lack a protein which specifically binds to UV-damaged DNA (Chu and Chang, 1988). This activity is apparently identical to a protein previously identified in human placenta (Feldberg and Grossman, 1976). Several lines of evidence implicate this protein in DNA repair. First, it is absent from some DNA repair-defective XP-E cells. Second, treatment of cells in culture with the DNA-damaging agents mitomycin C or UV induce elevated levels of the DNA-damage binding activity (Hirschfeld et al., 1990). Third, tissue-culture cells which have acquired resistance to the antitumor drug cisplatin also have elevated levels of the DNAdamage binding protein, which binds to cisplatininduced DNA lesions (Chu and Chang, 1990; Chao et al., 1991). Because the original observation that XP-E cells lack the DNA.damag¢ binding protein was made for cells from consanguineous patients, designated XP2RO and XP3RO, we extended the survey to include a number of unrelated patients recently documented in Japan (Fujiwara et al., 1985; Kawada et al., 1986; Kondo et ai., 1988, 1989). We report here that a cellular deficiency in DNA-damage binding activity is not a general feature of all XP-E patients and that cells from the majority of XP-E patients express apparently normal binding protein at levels comparable to those found in repair-proficient strains. We discuss the implications of these findings for the role of the binding protein in XP-E. Materials and methods
Materials Phage PM2 ['~H] DNA was prepared as described (Kuhnlein et al., 1976), Synthetic polynucleotides were from Midland Certified Reagent
Co. For gel shift assays, the Bgll-Ncol fragment (40 bp) from the SV40 early promoter was cut from the plasmid pUC-HSO, end-labeled with the Klenow fragment of DNA polymerase I and [a-32P]dTI'P (Amersham, 3000 Ci/mmole), and purified by nondenaturing polyacry!amide gel electrophoresis. Plasmid pUC-HSO, a derivative of pUC19 containing the 200 bp HindllI-SphI fragment from the SV40 replication origin, was generously provided by Dr. M. Botchan, U.C. Berkeley. Ceil strains and culture Normal human fibroblasts (strain F65, obtained from the Naval Biomedical Research Laboratory, Oakland, CA) were cultured in Dulbecco's modified essential medium (Gibco) with 10% fetal bovine serum. XP-E fibroblast strain GM2415 (XP2RO) and lymphoblast cell line GM2450D (XP3RO) were obtained from the Human Genetic Mutant Repository, Camden, NJ. XP-E strains XP43TO, XP70TO, XP80TO, XPS1TO, XP82TO, XP89TO, XP93TO and XP95TO (Kawada et ai., 1986; Kondo et al., 1988, 1989; S. Kondo, personal communication) were graciously provided by Dr. Seiji Kondo, Tokyo Medical and Dental University. Strain XP24KO (Fujiwara et al., 1985) was generously supplied by Dr, Yoshisada Fujiwara, Kobe University School of Medicine. All XP fibroblast strains were cultured in Eagle's minimal essential medium (Whittaker) supplemented with 20% fetal bovine serum and a 2-fold concentration of essential and nonessential amino acids and vitamins. All fibroblast strains were grown in the presence of penicillin, streptomycin, and 5 mM L-glutamine at 37"C in a 5% 0 3 2 atmosphere. Lymphoblast cultures were grown in RPMI 1640 medium with 15% fetal bovine serum. Manipulations of the above cultures and media were carried out under yellow light to avoid exposure of cells to white fluorescent light. HeLa cells were grown in suspension culture in Joldik's modified Eagle's medium (Gibco) with 5% calf serum and penicillin and streptomycin. HeLa cells were collected by centrifugation and fibroblasts were harvested by scraping from the surface of culture dishes into ice-cold phosphate-buffered saline. Cell extracts were pre-
51
pared by dounce homogenization as described (Nishida et al., 1988) except that the extract was dialyzed against 10 mM PDG buffer (10 mM KPO4, pH 7.5, 1 mM D T r , 10% (v/v) glycerol), containing the protease inhibitors PMSF (1 raM), TPCK (10 /tg/ml), leupeptin (0.5 /~g/ml), and aprotinin (0.5 p,g/ml).
Assays Binding of protein to UV-irradiated DNA was assayed by nitrocellulose filter binding assays. Reaction mixtures (50 p,l) contained 50 mM Tris-HCl, pH 8.0, 1 mM DTI', 10% (v/v) glycerol, 0.1 m g / m l BSA, 5 mM MgCI 2, 150 mM KCI, 70/~M poly (dI-dC), and 10 fmoles (molecules) of PM2 [3H]DNA irradiated at 200 J / m 2 with a General Electric germicidal lamp. Mixtures were incubated for 15 rain at 30°C then diluted with 2 ml TMK150 (50 mM Tris-HCl, pH 8.0, 5 mM MgCI 2, 150 mM KCI) and immediately filtered through nitrocellulose filters (Schleicher
and Schuell) pre-equilibrated in TMK150. Filters were rinsed once with 2 ml TMK150, dried, and then the bound DNA was determined by scintillation counting. The number of protein-DNA complexes per DNA molecule was calculated assuming a Poisson distribution of binding sites (Kuhnlein et al., 1976) and corrected for nonspecific binding in control reactions containing unirradiated DNA. One unit of binding protein forms one fmole UV-specific protein-DNA complexes. The KCI and poly(dl-dC) concentrations were chosen to mask nonspecific binding by activities present in cruder fractions. Under standard assay conditions, the DNA-binding activity in purifiied fractions was essentially completely damage-dependent. Gel mobility shift assays were performed essentially as described (Chu and Chang, 1988). Briefly, the 32p-labeled 40 bp fragment described above was UV-irradiated at 9 k J / m 2. Binding reactions (10/~!) contained approximately 0.2 ng
A
÷
4.
--
UV
---- Band 2 - - - . Band 1
- - - Unbound DNA Fig. 1. Gel-mobilityshift assay of XP-E and normal fibroblastextracts. Bindingassayswere performedas described in the text with approximately2 × 105 cell equivalentsof whole-cellextract from the indicated strains. Under these conditions,the DNA-damage binding protein forms two electrophoreticallydistinctcomplexes,indicatedas Band 1 and Band 2. (A) Extracts were assayedwith UV-irradiated probe DNA.(B) Extracts were assayedwith irradiatedor unirradiatedprobe DNA as indicated.
52
[32P]DNA with 10% (v/v) glycerol, 0.35 mM poly(dl-dC), 0.1 mg/ml BSA, 5 mM MgCI 2, 60 mM KCI and 50 mM Tris-HCl, pH 8.0. The reactions were incubated 15 min at 30°C, electrophoresed on 5% polyacrylamide gels in TBE, and the gels were dried and autoradiographed. Mono S chromotography Protein from whole cell extracts was precipitated with 0.472 g/ml of (NH4)2SO4 on ice, collected by centrifugation for 15 rain at 12000 x g, and resuspended in PD buffer (10 mM KPO 4, pH 7.5, 1 mM DTr). Samples were diluted with PD buffer to a conductivity of < 8 mS and applied at 0.5 ml/min to a 0.5 × 5 cm Mono S FPLC column (Pharmacia) equilibrated in PD buffer. The column was washed with PD buffer and eluted with a linear gradient of 0-0.5 M NaC! in PD buffer. Fractions of 1 ml were collected and assayed by nitrocellulose filter binding assays. Results
Whole-cell extracts from 1 normal and 9 XP-E fibroblast strains were screened for the presence of the DNA-damage binding protein by the gel mobility shift assay (Fig. IA). Approximately equal cell equivalents of extract were assayed in each lane, so that the intensities of the shifted bands directly reflected cellular levels of binding activity. Of the 9 Japanese XP-E strains studied, 8 had roughly normal levels of DNA-damage binding activity which formed protein-DNA complexes whose electrophoretic mobilities were indistinguishable from those of complexes formed with extracts from normal cells. (The results for strains XP24KO, XP81TO and XP89TO have been independently confirmed by Kataoka and Fujiwara, 1991.) In contrast, fibroblasts from the ninth patient, XP82TO, were completely devoid of this binding activity. The bands which appeared with the XP82TO extract were not due to the DNA-damage binding protein since they had a distinct electrophoretic mobility and were formed equally well with undamaged DNA as with UV-irradiated DNA (Fig. 1B). These UV-independent bands are presumably due to other
TABLE 1 LEVELS O F D N A D A M A G E B I N D I N G P R O T E I N F R O M N O R M A L A N D XP-E CELLS Strain
Number of Cells x 10 - 8
mg protein
Binding units × 10 -3
Normal HeLa a F65
3.5 2.6
18 11
XP-E XP2RO XP3RO a XP82TO
0.22 8.4 2.2
2.4 17 6.4
< 0.05 b < 0.9 t, < 0.1 b
XP24KO XP43TO XP70TO XP80TO
0.67 1.4 2.2 0.48
1.7 2.6 l0 0.83
8.64 23.2 25.2 4.46
130 170 110 93
XP81TO XP89TO XP93TO XP95TO
0,53 2.5 2.4 2,5
2.6 10 8.5 9.1
3.47 32.7 29.3 67.7
65 130 120 270
37.8 46.6
Units per 106 cells
1l0 180 < 2.3 < 1.1 < 0.5
Transformed cell line. Lower limit of detection in this assay; no activity was detectable by either gel shift or nitrocellulose filter binding.
DNA-binding proteins present in the crude extract. The level of binding activity in the extracts was quantitated by nitrocellulose filter binding assays (Table 1), The amount of binding protein in the various XP-E strains was comparable to levels in two repair-proficient cell types, HeLa and F65. 150 units of binding protein per 106 cells is equivalent to approximately l0 s molecules per cell, so the values shown in Table 1 are in good agreement with the published estimate of Chu and Chang (1988), The notable exceptions were XP2RO and XP3RO, both previously shown to be missing the binding protein (Chu and Chang, 1988), and XP82TO. These three strains showed no detectable damage-specific DNA binding activity. The XP-E and normal fibroblast extracts were precipitated with ammonium sulfate, then chromatographed on Mono S FPLC to determine whether the binding activity seen in XP-E cells was chromatographically similar to that of the normal controls (Fig. 2). The binding protein
53 normal control F65, showed a prominent shoulder or peak of UV-specific DNA-binding activity eluting before the major peak, but no major abnormalities in chromatographic behavior were seen for any of the strains. The small peaks of UV-specific binding activity around fraction 5 in the chromatograms of the XP89TO, XP93TO and XP70TO extracts do not have gel shift activity, and may therefore be identical to a previously characterized damage binding protein specific for supercoiled DNA (Tsang and Kuhnlein, 1982). Mono S-purified DNA damage binding protein obtained from XP-E cells showed no significant differences in thermal lability from that of normal cells. The binding activity from each strain, including normal controls, showed a 3050% reduction in activity after 150-rain incubation at 54°C. In conclusion, the chromatographic profiles, electrophoretic mobilities of proteinDNA complexes, and thermal stability of binding activities suggest that the structure of the binding protein is not grossly altered in those XP-E cells in which it was detected. Discussion
Fraction Number
Fraction Number
FiB. 2. Mono S chromatosraphy profiles of DNA-damage binding activity from normal and XP-E cells, in order to simplify comparisons between extracts obtained from different numbers of cells, each chromatogram was normalized relative to the peak value for that chromatogram.The reak values (units/ml×10 -3) were as follows: HeLa, 8.67; F65,
7.70; XP24KO, 1.03; XP43TO, 8.07; XPTOTO,11.3; XP80TO, 0.637; XP81TO, 0.693; XP89TO, 6.57; XP93TO, 5.02; XP95TO, 8.48. from normal fibroblasts and from HeLa eluted reproducibly at fractions 19-20, as did the binding protein from each of the XP-E strains which contained it. The elution position of binding activity was confirmed by the gel shift assay (not shown). Strain XP82TO showed no detectable binding protein across the column profile, in agreement with the results of assays of the crude extract (not shown). Some strains, including the
Individuals with XP-E can be divided into two subclasses based on whether or not tissue culture cells derived from them contain detectable DNA-darnage binding protein. Cells from patients of the first subclass have no detectable binding activity, whereas cells from patients of the second subclass have levels comparable to those of normal, repair-proficient cells. Furthermore, the binding protein from the second subclass is indistinguishable from that of normal controls in behavior on ion-exchange chromatography, thermal stability, and electrophoretic mobility of the protein-DNA complexes formed. These results were obtained with non-transformed, diploid strains, ruling out changes of gene expression associated with the establishment of transformed cell lines as the cause of the differences seen. To date, cells or tissue from 12 XP-E patients and 20 non-XP-E individuals (including normal individuals and patients from each of the other XP groups deficient in excision repair) have been tested for the presence of DNA-damage binding
54 TABLE 2 CLINICAL AND CELLULAR CHARACTERISTICS OF XP-E PATIENTS Patient
Binding protein
UDS (% of normal) a
D o, 254 nm ( J / m 2) ~
Density at confluence b (cells/cm" × 10 -4)
MED a
XP2RO XP3RO XP82TO
No No No
40-60 70 44
2.3 ND 2.8
0.76 ND 3.2
ND ND low
XP24KO XP26KO XP43TO
Yes Yes c Yes
30-55 30-55 24
2.3 2.4 ND
0.97 ND 2.0
low normal ND
XP70TO XP80TO XP81TO
Yes Yes Yes
55 43 40
2.2 2.2 2.5
3.2 0.70 1.0
low low low
XP89TO XP93TO XP95TO
Yes Yes Yes
27 52 ND
ND ND ND
3.6 3.5 3.6
low low ND
a Data pooled from deWeerd-Kastelein et al., 1974; Fujiwara et al., 1985; Kawada et al., 1986; Kondo et al., 1988, 1989. h Culture density of fibroblasts grown to confluence as described in the text. ¢ Kataoka and Fujiwara, 1991 Abbreviations: UDS, unscheduled DNA synthesis; MED, minimal erythema dose: ND, not determined or not reported.
activity (Feldberg and Grossman, 1976; Chu and Chang, 1988, 1990; Patterson and Chu, 1989; Hirschfeld et al., 1990; Kataoka and Fujiwara, 1991; this paper). If the two related XP-E patients are counted as only one individual, cells from 2 of 11 unrelated XP-E patients show the defect, whereas all of the non-group E cells exhibit the binding activity. A X 2 analysis of these results suggests that there is a statistically significant association between an absence of binding activity and group E xeroderma pigmentosum (p < 0.05). There is no obvious correlation between the cellular and clinical phenotypes of XP-E patients and the levels of DNA damage binding protein (Table 2). Thus, a cellular deficiency of this activity is not predictive of the levels of unscheduled DNA synthesis after UV irradiation, UV hypersensitivity, fibroblast culture density at confluence, or minimal erythema dose. The age of onset of skin abnormalities such as freckles or neoplasms (Kondo et al., 1989) also does not correlate with the presence or absence of binding activity. Notably, a complete absence of this DNAdamage binding actMty results in at most only a
mild reduction in DNA-repair capacity even though the activity is abundant in normal cells (Table 1) and is further induced by DNA damage (Hirschfeld et al., 1990). This may indicate that the UV-induced lesion(s) recognized by this protein is a relatively minor component of the spectrum of lesions produced by UV irradiation. AI. ternatively, such lesions may be efficiently repaired by other DNA-repair systems with overlapping specificities. The binding protein is found in a variety of cell types and tissues [fibroblast, lymphoid, fibrosarcoma, placenta (Feldberg and Grossman, 1976; Chu and Chang, 1988, 1990; Patterson and Chu, 1989; Hirschfeld et al., 1990; Kataoka and Fujiwara, 1991)], many of which would be unlikely to experience UV irradiation in vivo. This binding activity might therefore be primarily a component of a repair pathway only peripherally engaged in repair of UV damage. The results presented here raise the question of the relationship between the DNA-damage binding protein and XP-E. Possibly, a defect in binding activity might not be the cause of the dysfunction, but might instead be only coincidentally associated with some cases of XP-E. For example, the XP-E defect might be in a gene
55
closely linked to the binding protein, in which case small chromosomal aberrations could affect both genes simultaneously. The human DNA-repair genes ERCCI, ERCC2 and XRCCI map together to bands q13.2-q13.3 of chromosome 19 (Mohrenweiser et al., 1989), raising the possibility that other DNA-repair genes might be similarly clustered. Alternatively, a defect in the DNA-damage binding protein might indeed be the cause of the disease, but the protein present in the majority of XP-E strains is altered at some domain other than its DNA-binding region. Since the protein has no observed enzymatic function (Feldberg and Grossman, 1976; S.K. and S.L., unpublished data), it seems reasonable that it interacts with other proteins in order to mediate excision repair of its target lesion(s). Many eukaryotic transcription factors provide precedents for independent DNA binding and effector domains. A third possibility is that different XP-E patients have defects in distinct genetic loci which nevertheless fail to complement one another in cell fusion experiments. Several instances of such nonallelic, noncomplementing mutations have been recently described. For example, a specific mutation at the haywire locus in Drosophila melanogaster fails to complement certain mutant alleles of /32-tubulin (Regan and Fuller, 1990). Other examples of this phenomenon include noncomplementation between certain alleles of SIR genes in yeast (Rine and Herskowitz, 1987) or between some alleles of the sqt loci in CaenorhabdMs elegans (Kusch and Edgar, 1986). In many of these cases, the lack of complementation appears to be a consequence of an intimate interaction between mutant and wild-type products of separate genetic loci. If this were the case in XP-E, physical interactions with the DNA damage binding protein could be exploited to identify the product of the other XP-E gene(s). Conclusive tests of these or other possibilities await the cloning of the structural gene for the DNA-damage binding protein and/or DNA-repair correction experiments using purified binding protein in microinjection, cell-free, or permeable-cell assay systems. We are currently carrying out such experiments.
Acknowledgements We acknowledge Dr. Seiji Kondo and Dr. Yoshisada Fujiwara for graciously providing fibroblast strains and for communicating information prior to publication. We also thank Laura Williams for critically reading the manuscript and Ann Fischer for her heroic assistance in culturing recalcitrant human cell strains. This work was supported by grants 76EV30415 from the U.S. Department of Energy and P30ES011896 from the National Institutes of Health. S.K. is the recipient of a graduate fellowship from the U.S. National Science Foundation. References Chap, C.C.-K., S.-L. Huang, H. Huang and S. Lin-Chao (1991) Cross-resistance to UV radiation of a cisplatin-resistant human cell line: overexpression of cellular factors that recognize UV-modified DNA, Mol. Ceil. Biol., 1I, 20752080. Chu, O., and E. Chang (1988) Xeroderma pigmentosum gr3up E cells lack a nuclear factor that binds to damaged DNA, Science, 242, .564-367. Chu, G., and E. Chang (1990) Cisplatin-resistant cells express increased levels of a factor that recognizes damaged DNA, Proc. Natl. Acad. $ci. (U.S.A.), 87, 3324-3327. Cleaver, J.E., and K.H. Kraemer (1989) Xeroderma pigmentosum, in: C.R. Scriver, A.L. Beaudet, W.S. Sly and D. Valle (Eds.), The Metabolic Basis of Inherited Disease, Vol. 2, McGraw-Hill, New York, pp. 2949-2971. deWeerd-Kastelein, I¢,A., W. Keijzer and D. Bootsma (1974) A third complementation group in xeroderma pigmento. sum, Mutation Res., 22, 87-91. Feldberg, R,S., and L. Grossman (1976) A DNA binding protein from human placenta specific for ultraviolet damaged DNA, Biochemistw, 15, 2402-2408. Fujiwara, Y., Y. Uehara, M. Ichihashi, Y. Yamamoto and K. Nishioka (1985) Assignment of two patients with xeroderma pigmentosum to complementation group E, Mutation Res., 145, 55-61. Hirschfeld, S., A.S. Levine, K. Ozato and M. Protic (1990) A constitutive damage specific DNA-binding protein is synthesized at higher levels in UV-irradiated primate cells, Mol. Ceil. Biol., 10, 2041-2048. Hoeijmakers, J.H.J., A.P.M. Eker, R.D. Wood and P. Robins (1990) Use of in vivo and in vitro assaysfor the characterization of mammalian excision repair and isolation of repair proteins, Mutation Res., 236, 223-238. Kataoka, H., and Y. Fujiwara (1991) UV damage-specific DNA-binding protein in xeroderma pigmentosum complementation group E, Biochem. Biophys. Res. Commun., 175, 1139-1143.
56 Kawada, A., Y. Satoh and Y. Fujiwara (1986) Xeroderma pigmentosum complementation group E: a case report, Photodermatology, 3, 233-238. Keeney, S., and S. Linn (1990) A critical review of permeabilized cell systems for studying mammalian DNA repair, Mutation Res., 236, 239-252. Koado, S., S. Fukuro, A. Mamada, A. Kawada, Y. Satoh and Y. Fujiwara (1988) Assignment of three patients to xeroderma pigmentosum complementation group E and their characteristics, J. Invest. Dermatol., 90, 152-157. Kondo, S., A. Mamada, C. Miyamoto, C.-H. Keong, Y. Satoh and Y, Fujiwara (1989) Late onset of skin cancers in two xeroderma pigmentsoum group F siblings and a review of 30 Japanese xeroderma pigmentosum patients in groups D, E and F, Photodermatology, 6, 89-95. Kuhnlein, U., E.E. Penhoet and S. Linn (1976) An altered apurinic DNA endonuclease activity in group A and group D xeroderma pigmentosum fibroblasts, Proc. Natl. Acad. Sci. (U.S.A.), 73, 1169-1173. Kusch, M., and R.S. Edgar (1986) Genetic studies of unusual loci that affect body shape of the nematode Caenorhabditis elegans and may code for cuticle structural proteins, Genetics, 113, 621-639. Mohrenweiser, H.W., A.V. Carrano, A. Fertitta, B. Perry, L.H. Thompson, J.D. Tucker and C.A. Weber (1989) Refined mapping of the three DNA repair genes, ERCCl, ERCC2, and XRCCI, on human chromosome 19, Cytogcnet. Cell Genet., 52, 11-14, Nishida, C., P. Reinhard and S. Linn (1988) DNA repair
synthesis in human fibroblasts requires DNA polymerase 6, J. Biol. Chem., 263, 501-510. Patterson, M., and G. Chu (1989) Evidence that XP cells from complementation group E are deficient in a homolog of yeast photolyase, Mol. Cell. Biol., 9, 5105-5112. Regan, C.L., and M.T. Fuller (1990) Interacting genes that affect microtubule function in Drosophila melanogaster: two classes of mutation revert the failure to complement between hay nc2 and mutations in tubulin genes, Genetics, 125, 77-90. Rine, J., and I. Herskowitz (1987) Four genes responsible for a position effect on expression from HML and HMR in Saccharomyces cereL,isiae, Genetics, 116, 9-22. Tanaka, K., I. Satokata, Z. Ogita, T. Uchida and Y. Okada (1989) Molecular cloning of a mouse DNA repair gene that complements the defect of group-A xeroderma pigmentosum, Proc. Natl. Acad. Sci. (U.S.A.), 86, 5512-5516. Tsang, S.S., and U. Kuhnlein (1982) DNA binding protein from HeLa cells that binds preferentially to supercoiled DNA damaged by ultraviolet light or N-acetoxy-N-acetyl2-aminofluorene, Biochim. Biophys. Acta, 697, 202-212. Weeda, G., R.C.A. van Ham, W. Vermeulen, D. Bootsma, A.J. van der Eb and J.HJ. Hoeijmakers (1990) A presumed DNA helicasc encoded by ERCC-3 is involved in the human repair disorders xeroderma pigmentosum and Cockayne's Syndrome, Cell, 62, 777-791. Wood, R.D., P. Robins and T. Lindahl (1988) Complementation of the xeroderma pigmentosum DNA repair defect in cell.free extracts, Cell, 53, 97-106.
49
© 1992 Elsevier Science Publishers B.V. All rights reserved 0921-8777/92/$05.00
MUTDNA 06470
Biochemical heterogeneity in xeroderma pigrnentosum complementation group E Scott K e e n e y , H a r r i s o n W e i n a n d S t u a r t L i n n Division of Biochemistry and MolecularBiology, Barker Hall, University of California, Berkeley, CA 94720 (U.S.A.) (Received 26 April 1991) (Revision received 14 June 1991) (Accepted 18 June 1991)
Keywords: DNA repair; DNA-binding proteins; Proteins, DNA-binding; UV irradiation; XP complementation group E; DNA, UV-irradiated
Summary Cells from two patients with xeroderma pigmentosum complementation group E (XP-E) have been shown to lack an activity which binds specifically to UV-irradiated DNA (Chu and Chang, 1988). We investigated the occurrence of this binding activity in cell strains from nine additional, unrelated XP-E patients and found that all but one of these strains contained normal levels of the binding protein. Furthermore, the binding activity from these XP-E strains was indistinguishable from that of normal controls in thermal stability, behavior on ion-exchange chromatography, and electrophoretic mobility of protein-DNA complexes, indicating that there were no gross structural alterations in the protein. The association of XP-E with a deficiency in DNA-damage binding protein in cells from 3 of 12 XP-E patients (compared to 0 of 20 non-XP-E controls) is statistically significant (p < 0.05), but there is no obvious correlation between the biochemical defect and the clinical or cellular characteristics of individual patients. Implications of these findings for the role of the binding protein in XP-E are discussed.
Xeroderma pigmentosum (XP) is a rare genetic disease characterized by a clinical and cellular hypersensitivity to UV irradiation (see Cleaver and Kraemer, 1989, for a recent review). Patients show dermatologic abnormalities, including thickening and hyperpigmentation of sunlight-exposed skin, and develop sunlight-induced malignancies
Correspondence: Dr. Stuart Linn, Division of Biochemistry and Molecular Biology, Barker Hall, University of California, Berkeley, CA 94720 (U.S.A.), Tel. (510) 642-7583, Fax (510) 643-5035.
at an earlier age than usual. Cells from XP patients show hypersensitivity to killing by UV irradiation, and this cellular phenotype correlates with a defect in excision repair of UV-induced DNA lesions. Individuals with XP are divided into 7 complementation groups (A through G) and an excision-proficient variant group (XP-V). Considerable effort has focused on elucidating the molecular basis of the DNA-repair defects of the various XP groups. Recently, genes which are defective in XP-A (Tanaka et al., 1989) and XP-B (Weeda et al., 1990) have been identified by molecular genetic techniques. Other approaches
50 include the use of permeabilized cells (Keeney and Linn, 1990), microinjection (Hoeijmakers et al., 1990), or an in vitro plasmid repair system (Wood et al., 1988; Hoeijmakers et al., 1990) to assay for protein factors which correct the defect of XP ceils. Alternatively, XP ceils have been screened for defects in enzymatic activities which are thought to be required for excision repair. Using the latter approach, Chu and colleagues reported that cells from two XP-E patients lack a protein which specifically binds to UV-damaged DNA (Chu and Chang, 1988). This activity is apparently identical to a protein previously identified in human placenta (Feldberg and Grossman, 1976). Several lines of evidence implicate this protein in DNA repair. First, it is absent from some DNA repair-defective XP-E cells. Second, treatment of cells in culture with the DNA-damaging agents mitomycin C or UV induce elevated levels of the DNA-damage binding activity (Hirschfeld et al., 1990). Third, tissue-culture cells which have acquired resistance to the antitumor drug cisplatin also have elevated levels of the DNAdamage binding protein, which binds to cisplatininduced DNA lesions (Chu and Chang, 1990; Chao et al., 1991). Because the original observation that XP-E cells lack the DNA.damag¢ binding protein was made for cells from consanguineous patients, designated XP2RO and XP3RO, we extended the survey to include a number of unrelated patients recently documented in Japan (Fujiwara et al., 1985; Kawada et al., 1986; Kondo et ai., 1988, 1989). We report here that a cellular deficiency in DNA-damage binding activity is not a general feature of all XP-E patients and that cells from the majority of XP-E patients express apparently normal binding protein at levels comparable to those found in repair-proficient strains. We discuss the implications of these findings for the role of the binding protein in XP-E. Materials and methods
Materials Phage PM2 ['~H] DNA was prepared as described (Kuhnlein et al., 1976), Synthetic polynucleotides were from Midland Certified Reagent
Co. For gel shift assays, the Bgll-Ncol fragment (40 bp) from the SV40 early promoter was cut from the plasmid pUC-HSO, end-labeled with the Klenow fragment of DNA polymerase I and [a-32P]dTI'P (Amersham, 3000 Ci/mmole), and purified by nondenaturing polyacry!amide gel electrophoresis. Plasmid pUC-HSO, a derivative of pUC19 containing the 200 bp HindllI-SphI fragment from the SV40 replication origin, was generously provided by Dr. M. Botchan, U.C. Berkeley. Ceil strains and culture Normal human fibroblasts (strain F65, obtained from the Naval Biomedical Research Laboratory, Oakland, CA) were cultured in Dulbecco's modified essential medium (Gibco) with 10% fetal bovine serum. XP-E fibroblast strain GM2415 (XP2RO) and lymphoblast cell line GM2450D (XP3RO) were obtained from the Human Genetic Mutant Repository, Camden, NJ. XP-E strains XP43TO, XP70TO, XP80TO, XPS1TO, XP82TO, XP89TO, XP93TO and XP95TO (Kawada et ai., 1986; Kondo et al., 1988, 1989; S. Kondo, personal communication) were graciously provided by Dr. Seiji Kondo, Tokyo Medical and Dental University. Strain XP24KO (Fujiwara et al., 1985) was generously supplied by Dr, Yoshisada Fujiwara, Kobe University School of Medicine. All XP fibroblast strains were cultured in Eagle's minimal essential medium (Whittaker) supplemented with 20% fetal bovine serum and a 2-fold concentration of essential and nonessential amino acids and vitamins. All fibroblast strains were grown in the presence of penicillin, streptomycin, and 5 mM L-glutamine at 37"C in a 5% 0 3 2 atmosphere. Lymphoblast cultures were grown in RPMI 1640 medium with 15% fetal bovine serum. Manipulations of the above cultures and media were carried out under yellow light to avoid exposure of cells to white fluorescent light. HeLa cells were grown in suspension culture in Joldik's modified Eagle's medium (Gibco) with 5% calf serum and penicillin and streptomycin. HeLa cells were collected by centrifugation and fibroblasts were harvested by scraping from the surface of culture dishes into ice-cold phosphate-buffered saline. Cell extracts were pre-
51
pared by dounce homogenization as described (Nishida et al., 1988) except that the extract was dialyzed against 10 mM PDG buffer (10 mM KPO4, pH 7.5, 1 mM D T r , 10% (v/v) glycerol), containing the protease inhibitors PMSF (1 raM), TPCK (10 /tg/ml), leupeptin (0.5 /~g/ml), and aprotinin (0.5 p,g/ml).
Assays Binding of protein to UV-irradiated DNA was assayed by nitrocellulose filter binding assays. Reaction mixtures (50 p,l) contained 50 mM Tris-HCl, pH 8.0, 1 mM DTI', 10% (v/v) glycerol, 0.1 m g / m l BSA, 5 mM MgCI 2, 150 mM KCI, 70/~M poly (dI-dC), and 10 fmoles (molecules) of PM2 [3H]DNA irradiated at 200 J / m 2 with a General Electric germicidal lamp. Mixtures were incubated for 15 rain at 30°C then diluted with 2 ml TMK150 (50 mM Tris-HCl, pH 8.0, 5 mM MgCI 2, 150 mM KCI) and immediately filtered through nitrocellulose filters (Schleicher
and Schuell) pre-equilibrated in TMK150. Filters were rinsed once with 2 ml TMK150, dried, and then the bound DNA was determined by scintillation counting. The number of protein-DNA complexes per DNA molecule was calculated assuming a Poisson distribution of binding sites (Kuhnlein et al., 1976) and corrected for nonspecific binding in control reactions containing unirradiated DNA. One unit of binding protein forms one fmole UV-specific protein-DNA complexes. The KCI and poly(dl-dC) concentrations were chosen to mask nonspecific binding by activities present in cruder fractions. Under standard assay conditions, the DNA-binding activity in purifiied fractions was essentially completely damage-dependent. Gel mobility shift assays were performed essentially as described (Chu and Chang, 1988). Briefly, the 32p-labeled 40 bp fragment described above was UV-irradiated at 9 k J / m 2. Binding reactions (10/~!) contained approximately 0.2 ng
A
÷
4.
--
UV
---- Band 2 - - - . Band 1
- - - Unbound DNA Fig. 1. Gel-mobilityshift assay of XP-E and normal fibroblastextracts. Bindingassayswere performedas described in the text with approximately2 × 105 cell equivalentsof whole-cellextract from the indicated strains. Under these conditions,the DNA-damage binding protein forms two electrophoreticallydistinctcomplexes,indicatedas Band 1 and Band 2. (A) Extracts were assayedwith UV-irradiated probe DNA.(B) Extracts were assayedwith irradiatedor unirradiatedprobe DNA as indicated.
52
[32P]DNA with 10% (v/v) glycerol, 0.35 mM poly(dl-dC), 0.1 mg/ml BSA, 5 mM MgCI 2, 60 mM KCI and 50 mM Tris-HCl, pH 8.0. The reactions were incubated 15 min at 30°C, electrophoresed on 5% polyacrylamide gels in TBE, and the gels were dried and autoradiographed. Mono S chromotography Protein from whole cell extracts was precipitated with 0.472 g/ml of (NH4)2SO4 on ice, collected by centrifugation for 15 rain at 12000 x g, and resuspended in PD buffer (10 mM KPO 4, pH 7.5, 1 mM DTr). Samples were diluted with PD buffer to a conductivity of < 8 mS and applied at 0.5 ml/min to a 0.5 × 5 cm Mono S FPLC column (Pharmacia) equilibrated in PD buffer. The column was washed with PD buffer and eluted with a linear gradient of 0-0.5 M NaC! in PD buffer. Fractions of 1 ml were collected and assayed by nitrocellulose filter binding assays. Results
Whole-cell extracts from 1 normal and 9 XP-E fibroblast strains were screened for the presence of the DNA-damage binding protein by the gel mobility shift assay (Fig. IA). Approximately equal cell equivalents of extract were assayed in each lane, so that the intensities of the shifted bands directly reflected cellular levels of binding activity. Of the 9 Japanese XP-E strains studied, 8 had roughly normal levels of DNA-damage binding activity which formed protein-DNA complexes whose electrophoretic mobilities were indistinguishable from those of complexes formed with extracts from normal cells. (The results for strains XP24KO, XP81TO and XP89TO have been independently confirmed by Kataoka and Fujiwara, 1991.) In contrast, fibroblasts from the ninth patient, XP82TO, were completely devoid of this binding activity. The bands which appeared with the XP82TO extract were not due to the DNA-damage binding protein since they had a distinct electrophoretic mobility and were formed equally well with undamaged DNA as with UV-irradiated DNA (Fig. 1B). These UV-independent bands are presumably due to other
TABLE 1 LEVELS O F D N A D A M A G E B I N D I N G P R O T E I N F R O M N O R M A L A N D XP-E CELLS Strain
Number of Cells x 10 - 8
mg protein
Binding units × 10 -3
Normal HeLa a F65
3.5 2.6
18 11
XP-E XP2RO XP3RO a XP82TO
0.22 8.4 2.2
2.4 17 6.4
< 0.05 b < 0.9 t, < 0.1 b
XP24KO XP43TO XP70TO XP80TO
0.67 1.4 2.2 0.48
1.7 2.6 l0 0.83
8.64 23.2 25.2 4.46
130 170 110 93
XP81TO XP89TO XP93TO XP95TO
0,53 2.5 2.4 2,5
2.6 10 8.5 9.1
3.47 32.7 29.3 67.7
65 130 120 270
37.8 46.6
Units per 106 cells
1l0 180 < 2.3 < 1.1 < 0.5
Transformed cell line. Lower limit of detection in this assay; no activity was detectable by either gel shift or nitrocellulose filter binding.
DNA-binding proteins present in the crude extract. The level of binding activity in the extracts was quantitated by nitrocellulose filter binding assays (Table 1), The amount of binding protein in the various XP-E strains was comparable to levels in two repair-proficient cell types, HeLa and F65. 150 units of binding protein per 106 cells is equivalent to approximately l0 s molecules per cell, so the values shown in Table 1 are in good agreement with the published estimate of Chu and Chang (1988), The notable exceptions were XP2RO and XP3RO, both previously shown to be missing the binding protein (Chu and Chang, 1988), and XP82TO. These three strains showed no detectable damage-specific DNA binding activity. The XP-E and normal fibroblast extracts were precipitated with ammonium sulfate, then chromatographed on Mono S FPLC to determine whether the binding activity seen in XP-E cells was chromatographically similar to that of the normal controls (Fig. 2). The binding protein
53 normal control F65, showed a prominent shoulder or peak of UV-specific DNA-binding activity eluting before the major peak, but no major abnormalities in chromatographic behavior were seen for any of the strains. The small peaks of UV-specific binding activity around fraction 5 in the chromatograms of the XP89TO, XP93TO and XP70TO extracts do not have gel shift activity, and may therefore be identical to a previously characterized damage binding protein specific for supercoiled DNA (Tsang and Kuhnlein, 1982). Mono S-purified DNA damage binding protein obtained from XP-E cells showed no significant differences in thermal lability from that of normal cells. The binding activity from each strain, including normal controls, showed a 3050% reduction in activity after 150-rain incubation at 54°C. In conclusion, the chromatographic profiles, electrophoretic mobilities of proteinDNA complexes, and thermal stability of binding activities suggest that the structure of the binding protein is not grossly altered in those XP-E cells in which it was detected. Discussion
Fraction Number
Fraction Number
FiB. 2. Mono S chromatosraphy profiles of DNA-damage binding activity from normal and XP-E cells, in order to simplify comparisons between extracts obtained from different numbers of cells, each chromatogram was normalized relative to the peak value for that chromatogram.The reak values (units/ml×10 -3) were as follows: HeLa, 8.67; F65,
7.70; XP24KO, 1.03; XP43TO, 8.07; XPTOTO,11.3; XP80TO, 0.637; XP81TO, 0.693; XP89TO, 6.57; XP93TO, 5.02; XP95TO, 8.48. from normal fibroblasts and from HeLa eluted reproducibly at fractions 19-20, as did the binding protein from each of the XP-E strains which contained it. The elution position of binding activity was confirmed by the gel shift assay (not shown). Strain XP82TO showed no detectable binding protein across the column profile, in agreement with the results of assays of the crude extract (not shown). Some strains, including the
Individuals with XP-E can be divided into two subclasses based on whether or not tissue culture cells derived from them contain detectable DNA-darnage binding protein. Cells from patients of the first subclass have no detectable binding activity, whereas cells from patients of the second subclass have levels comparable to those of normal, repair-proficient cells. Furthermore, the binding protein from the second subclass is indistinguishable from that of normal controls in behavior on ion-exchange chromatography, thermal stability, and electrophoretic mobility of the protein-DNA complexes formed. These results were obtained with non-transformed, diploid strains, ruling out changes of gene expression associated with the establishment of transformed cell lines as the cause of the differences seen. To date, cells or tissue from 12 XP-E patients and 20 non-XP-E individuals (including normal individuals and patients from each of the other XP groups deficient in excision repair) have been tested for the presence of DNA-damage binding
54 TABLE 2 CLINICAL AND CELLULAR CHARACTERISTICS OF XP-E PATIENTS Patient
Binding protein
UDS (% of normal) a
D o, 254 nm ( J / m 2) ~
Density at confluence b (cells/cm" × 10 -4)
MED a
XP2RO XP3RO XP82TO
No No No
40-60 70 44
2.3 ND 2.8
0.76 ND 3.2
ND ND low
XP24KO XP26KO XP43TO
Yes Yes c Yes
30-55 30-55 24
2.3 2.4 ND
0.97 ND 2.0
low normal ND
XP70TO XP80TO XP81TO
Yes Yes Yes
55 43 40
2.2 2.2 2.5
3.2 0.70 1.0
low low low
XP89TO XP93TO XP95TO
Yes Yes Yes
27 52 ND
ND ND ND
3.6 3.5 3.6
low low ND
a Data pooled from deWeerd-Kastelein et al., 1974; Fujiwara et al., 1985; Kawada et al., 1986; Kondo et al., 1988, 1989. h Culture density of fibroblasts grown to confluence as described in the text. ¢ Kataoka and Fujiwara, 1991 Abbreviations: UDS, unscheduled DNA synthesis; MED, minimal erythema dose: ND, not determined or not reported.
activity (Feldberg and Grossman, 1976; Chu and Chang, 1988, 1990; Patterson and Chu, 1989; Hirschfeld et al., 1990; Kataoka and Fujiwara, 1991; this paper). If the two related XP-E patients are counted as only one individual, cells from 2 of 11 unrelated XP-E patients show the defect, whereas all of the non-group E cells exhibit the binding activity. A X 2 analysis of these results suggests that there is a statistically significant association between an absence of binding activity and group E xeroderma pigmentosum (p < 0.05). There is no obvious correlation between the cellular and clinical phenotypes of XP-E patients and the levels of DNA damage binding protein (Table 2). Thus, a cellular deficiency of this activity is not predictive of the levels of unscheduled DNA synthesis after UV irradiation, UV hypersensitivity, fibroblast culture density at confluence, or minimal erythema dose. The age of onset of skin abnormalities such as freckles or neoplasms (Kondo et al., 1989) also does not correlate with the presence or absence of binding activity. Notably, a complete absence of this DNAdamage binding actMty results in at most only a
mild reduction in DNA-repair capacity even though the activity is abundant in normal cells (Table 1) and is further induced by DNA damage (Hirschfeld et al., 1990). This may indicate that the UV-induced lesion(s) recognized by this protein is a relatively minor component of the spectrum of lesions produced by UV irradiation. AI. ternatively, such lesions may be efficiently repaired by other DNA-repair systems with overlapping specificities. The binding protein is found in a variety of cell types and tissues [fibroblast, lymphoid, fibrosarcoma, placenta (Feldberg and Grossman, 1976; Chu and Chang, 1988, 1990; Patterson and Chu, 1989; Hirschfeld et al., 1990; Kataoka and Fujiwara, 1991)], many of which would be unlikely to experience UV irradiation in vivo. This binding activity might therefore be primarily a component of a repair pathway only peripherally engaged in repair of UV damage. The results presented here raise the question of the relationship between the DNA-damage binding protein and XP-E. Possibly, a defect in binding activity might not be the cause of the dysfunction, but might instead be only coincidentally associated with some cases of XP-E. For example, the XP-E defect might be in a gene
55
closely linked to the binding protein, in which case small chromosomal aberrations could affect both genes simultaneously. The human DNA-repair genes ERCCI, ERCC2 and XRCCI map together to bands q13.2-q13.3 of chromosome 19 (Mohrenweiser et al., 1989), raising the possibility that other DNA-repair genes might be similarly clustered. Alternatively, a defect in the DNA-damage binding protein might indeed be the cause of the disease, but the protein present in the majority of XP-E strains is altered at some domain other than its DNA-binding region. Since the protein has no observed enzymatic function (Feldberg and Grossman, 1976; S.K. and S.L., unpublished data), it seems reasonable that it interacts with other proteins in order to mediate excision repair of its target lesion(s). Many eukaryotic transcription factors provide precedents for independent DNA binding and effector domains. A third possibility is that different XP-E patients have defects in distinct genetic loci which nevertheless fail to complement one another in cell fusion experiments. Several instances of such nonallelic, noncomplementing mutations have been recently described. For example, a specific mutation at the haywire locus in Drosophila melanogaster fails to complement certain mutant alleles of /32-tubulin (Regan and Fuller, 1990). Other examples of this phenomenon include noncomplementation between certain alleles of SIR genes in yeast (Rine and Herskowitz, 1987) or between some alleles of the sqt loci in CaenorhabdMs elegans (Kusch and Edgar, 1986). In many of these cases, the lack of complementation appears to be a consequence of an intimate interaction between mutant and wild-type products of separate genetic loci. If this were the case in XP-E, physical interactions with the DNA damage binding protein could be exploited to identify the product of the other XP-E gene(s). Conclusive tests of these or other possibilities await the cloning of the structural gene for the DNA-damage binding protein and/or DNA-repair correction experiments using purified binding protein in microinjection, cell-free, or permeable-cell assay systems. We are currently carrying out such experiments.
Acknowledgements We acknowledge Dr. Seiji Kondo and Dr. Yoshisada Fujiwara for graciously providing fibroblast strains and for communicating information prior to publication. We also thank Laura Williams for critically reading the manuscript and Ann Fischer for her heroic assistance in culturing recalcitrant human cell strains. This work was supported by grants 76EV30415 from the U.S. Department of Energy and P30ES011896 from the National Institutes of Health. S.K. is the recipient of a graduate fellowship from the U.S. National Science Foundation. References Chap, C.C.-K., S.-L. Huang, H. Huang and S. Lin-Chao (1991) Cross-resistance to UV radiation of a cisplatin-resistant human cell line: overexpression of cellular factors that recognize UV-modified DNA, Mol. Ceil. Biol., 1I, 20752080. Chu, O., and E. Chang (1988) Xeroderma pigmentosum gr3up E cells lack a nuclear factor that binds to damaged DNA, Science, 242, .564-367. Chu, G., and E. Chang (1990) Cisplatin-resistant cells express increased levels of a factor that recognizes damaged DNA, Proc. Natl. Acad. $ci. (U.S.A.), 87, 3324-3327. Cleaver, J.E., and K.H. Kraemer (1989) Xeroderma pigmentosum, in: C.R. Scriver, A.L. Beaudet, W.S. Sly and D. Valle (Eds.), The Metabolic Basis of Inherited Disease, Vol. 2, McGraw-Hill, New York, pp. 2949-2971. deWeerd-Kastelein, I¢,A., W. Keijzer and D. Bootsma (1974) A third complementation group in xeroderma pigmento. sum, Mutation Res., 22, 87-91. Feldberg, R,S., and L. Grossman (1976) A DNA binding protein from human placenta specific for ultraviolet damaged DNA, Biochemistw, 15, 2402-2408. Fujiwara, Y., Y. Uehara, M. Ichihashi, Y. Yamamoto and K. Nishioka (1985) Assignment of two patients with xeroderma pigmentosum to complementation group E, Mutation Res., 145, 55-61. Hirschfeld, S., A.S. Levine, K. Ozato and M. Protic (1990) A constitutive damage specific DNA-binding protein is synthesized at higher levels in UV-irradiated primate cells, Mol. Ceil. Biol., 10, 2041-2048. Hoeijmakers, J.H.J., A.P.M. Eker, R.D. Wood and P. Robins (1990) Use of in vivo and in vitro assaysfor the characterization of mammalian excision repair and isolation of repair proteins, Mutation Res., 236, 223-238. Kataoka, H., and Y. Fujiwara (1991) UV damage-specific DNA-binding protein in xeroderma pigmentosum complementation group E, Biochem. Biophys. Res. Commun., 175, 1139-1143.
56 Kawada, A., Y. Satoh and Y. Fujiwara (1986) Xeroderma pigmentosum complementation group E: a case report, Photodermatology, 3, 233-238. Keeney, S., and S. Linn (1990) A critical review of permeabilized cell systems for studying mammalian DNA repair, Mutation Res., 236, 239-252. Koado, S., S. Fukuro, A. Mamada, A. Kawada, Y. Satoh and Y. Fujiwara (1988) Assignment of three patients to xeroderma pigmentosum complementation group E and their characteristics, J. Invest. Dermatol., 90, 152-157. Kondo, S., A. Mamada, C. Miyamoto, C.-H. Keong, Y. Satoh and Y, Fujiwara (1989) Late onset of skin cancers in two xeroderma pigmentsoum group F siblings and a review of 30 Japanese xeroderma pigmentosum patients in groups D, E and F, Photodermatology, 6, 89-95. Kuhnlein, U., E.E. Penhoet and S. Linn (1976) An altered apurinic DNA endonuclease activity in group A and group D xeroderma pigmentosum fibroblasts, Proc. Natl. Acad. Sci. (U.S.A.), 73, 1169-1173. Kusch, M., and R.S. Edgar (1986) Genetic studies of unusual loci that affect body shape of the nematode Caenorhabditis elegans and may code for cuticle structural proteins, Genetics, 113, 621-639. Mohrenweiser, H.W., A.V. Carrano, A. Fertitta, B. Perry, L.H. Thompson, J.D. Tucker and C.A. Weber (1989) Refined mapping of the three DNA repair genes, ERCCl, ERCC2, and XRCCI, on human chromosome 19, Cytogcnet. Cell Genet., 52, 11-14, Nishida, C., P. Reinhard and S. Linn (1988) DNA repair
synthesis in human fibroblasts requires DNA polymerase 6, J. Biol. Chem., 263, 501-510. Patterson, M., and G. Chu (1989) Evidence that XP cells from complementation group E are deficient in a homolog of yeast photolyase, Mol. Cell. Biol., 9, 5105-5112. Regan, C.L., and M.T. Fuller (1990) Interacting genes that affect microtubule function in Drosophila melanogaster: two classes of mutation revert the failure to complement between hay nc2 and mutations in tubulin genes, Genetics, 125, 77-90. Rine, J., and I. Herskowitz (1987) Four genes responsible for a position effect on expression from HML and HMR in Saccharomyces cereL,isiae, Genetics, 116, 9-22. Tanaka, K., I. Satokata, Z. Ogita, T. Uchida and Y. Okada (1989) Molecular cloning of a mouse DNA repair gene that complements the defect of group-A xeroderma pigmentosum, Proc. Natl. Acad. Sci. (U.S.A.), 86, 5512-5516. Tsang, S.S., and U. Kuhnlein (1982) DNA binding protein from HeLa cells that binds preferentially to supercoiled DNA damaged by ultraviolet light or N-acetoxy-N-acetyl2-aminofluorene, Biochim. Biophys. Acta, 697, 202-212. Weeda, G., R.C.A. van Ham, W. Vermeulen, D. Bootsma, A.J. van der Eb and J.HJ. Hoeijmakers (1990) A presumed DNA helicasc encoded by ERCC-3 is involved in the human repair disorders xeroderma pigmentosum and Cockayne's Syndrome, Cell, 62, 777-791. Wood, R.D., P. Robins and T. Lindahl (1988) Complementation of the xeroderma pigmentosum DNA repair defect in cell.free extracts, Cell, 53, 97-106.
