The EMBO Journal vol. 10 no. 1 2 pp.3913 - 3921, 1991

Complementation of DNA repair in xeroderma pigmentosum group A cell extracts by a protein with affinity for damaged DNA Peter Robins, Christopher J.Jones, Maureen Biggerstaff, Tomas Lindahl and Richard D.Wood Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts EN6 3LD, UK Communicated by T.Lindahl

Complementation group A of xeroderma pigmentosum (XP) represents one of the most prevalent and serious forms of this cancer-prone disorder. Because of a marked defect in DNA excision repair, cells from individuals with XP-A are hypersensitive to the toxic and mutagenic effects of ultraviolet light and many chemical agents. We report here the isolation of the XP-A DNA repair protein by complementation of cell extracts from a repairdefective human XP-A cell line. XP-A protein purified from calf thymus migrates on denaturing gel electrophoresis as a doublet of 40 and 42 kilodaltons. The XP-A protein binds preferentially to ultraviolet lightirradiated DNA, with a preference for damaged over nondamaged nucleotides of 103. This strongly suggests that the XP-A protein plays a direct role in the recognition of and incision at lesions in DNA. We further show that this protein corresponds to the product encoded by a recently isolated gene that can restore excision repair to XP-A cells. Thus, excision repair of plasmid DNA by cell extracts sufficiently resembles genomic repair in cells to reveal accurately the repair defect in an inherited disease. The general approach described here can be extended to the identification and isolation of other human DNA -

repair proteins.

Key words: DNA binding protein/DNA repair/human/UV damage/xeroderma pigmentosum

Introduction DNA excision repair is the process responsible for eliminating most ultraviolet radiation (UV) damage from DNA, as well as base alterations caused by a variety of chemical mutagens. One way to gain an understanding of this process in mammalian cells is to delineate the biochemical basis of defects in DNA excision repair that are associated with certain inherited human syndromes and with repair-defective mutant cell lines isolated in the laboratory. Comprehension of the basis of these defects should give powerful insights into the normal process of DNA excision repair in mammalian cells. Cell lines derived from patients with xeroderma pigmentosum (XP) are an essential tool in such investigations. Individuals with this autosomal, recessively inherited syndrome are subject to a high incidence of sunlight-induced skin disorders, including cancers (Cleaver and Kraemer, 1989). Cells from XP patients exhibit reduced levels of DNA repair synthesis in response to ultraviolet light (D Oxford University Press

and many chemical mutagens. Cell fusion studies with cultured skin fibroblasts currently define seven repair complementation groups (XP-A to XP-G) and a 'variant' form (de Weerd-Kastelein et al., 1972; Hoeijmakers and Bootsma, 1990). Cells from most or all of these XP complementation groups appear to have a defect in the initial stages of repair that lead to incision at the site of DNA damage. The different complementation groups represent separate proteins, some of which are believed to act together in a complex to repair damaged DNA. Although DNA sequence features suggest possible activities for a few of these gene products, no functions have been firmly established and proteins have yet to be isolated in a purified, active form in sufficient quantities for study. Ultimately, it will be necessary to reconstruct excision repair in vitro with purified components. To facilitate functional studies, we have developed a cellfree system that can mediate DNA excision repair in extracts from mammalian cells. Repair is carried out by enzymes in gently prepared soluble extracts from human cell lines and assessed by monitoring the introduction of short patches of nucleotides into damaged circular plasmid DNA (Wood et al., 1988). Using this approach we and others have shown that cell extracts can carry out repair synthesis in DNA damaged by ultraviolet light, psoralens and platinating agents (Wood et al., 1988; Wood, 1989; Hansson and Wood, 1989; Sibghat-Ullah et al., 1989; Sibghat-Ullah and Sancar, 1990) and that this repair synthesis is localized to sites of DNA damage (Hansson et al., 1989). Extracts from all investigated excision-deficient complementation groups of XP are deficient in repair of damaged circular DNA (Wood et al., 1988; Hansson et al., 1990, 1991). Furthermore, mixing of cell extracts from different complementation groups can lead to reconstitution of the DNA repair activity (Wood et al., 1988). This feature suggests that the biochemical complementation approach can be used to isolate repair proteins. XP complementation group A is one of the most severe forms of the disease, and represents approximately onequarter of all patients tested. There is good evidence that XP-A cells are defective in introducing incisions into damaged DNA (Fornace et al., 1976; Zelle and Lohman, 1979; Erixon and Ahnstrom, 1979; de Jonge et al., 1985; Hansson et al., 1990; Thielmann et al., 1991). Based on our previous observations that mixing of extracts from different XP cell lines could lead to complementation of repair synthesis, we have undertaken purification of a protein from mammalian cells that can complement XP-A cell

extracts. Results Covalently closed circular plasmid DNA was freed from traces of nicked DNA and irradiated with UV light. In order to provide an internal control, the irradiated DNA was mixed 3913

P.Robins et al.

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3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 GM2249 G M2485 GM1953 GM2250 GM2344 GM2345 GM2252 (XP-D) (normal) (XP-A) (XP-A) (XP-A) (XP-B) (XP-C)

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Fig. 1. Fractionation of mammalian cell extracts to yield complementing protein that specifically corrects repair synthesis by XP-A cell extracts. All reactions included 125 /tg of cell extract protein from the indicated cell lines. The upper panel shows the ethidium bromide stained gel, while the lower panel shows the autoradiograph of the dried gel. The position of nonirradiated plasmid DNA is indicated by '-' and UV-irradiated plasmid by '+'. First lane in each group of three (lanes 1, 4, 7, 10, 13, 16 and 19): cell extract only. Second lane in each group of three (lanes 2, 5, 8, 11, 14, 17 and 20): cell extract plus 6 Mg XP-A correcting fraction from HeLa cells, purified by adsorption to phosphocellulose, ammonium sulphate precipitation, gel filtration on an AcA44 column and chromatography on heparin-agarose and CM Sepharose. Third lane in each group of three (lanes 3, 6, 9, 12, 15, 18 and 21): cell extract plus 0.1 g of cyclobutane pyrimidine dimer DNA glycosylase (UV endonuclease) from Micrococcus luteus.

with nonirradiated DNA in the form of a related plasmid of slightly larger size. The mixture of the two plasmid DNAs was incubated with a cell extract (Manley et al., 1983) of a human lymphoblastoid cell line in the presence of a buffer containing the four deoxyribonucleoside triphosphates, [01-32P]dATP, ATP and an ATP regenerating system. Under the experimental conditions employed, pyrimidine dimers are removed by excision repair. DNA repair synthesis in the UV-irradiated plasmid was measured by recovery of the plasmid DNAs from the reaction mixture, followed by linearization of the DNAs by restriction enzyme treatment, separation of the two DNA molecules by agarose gel electrophoresis, autoradiography and scintillation counting of excised bands. As shown previously (Wood et al., 1988), repair in vitro of UV-irradiated DNA is observed with cell extracts of lines established from normal individuals, but not with lines of XP origin. Mixing of cell extracts from two XP cell lines of different genetic complementation groups restores the repair signal. In the present work, we have employed a single XP cell extract and complemented it by adding fractionated cellular protein. Isolation of XP-A protein by complementation in vitro Extracts from the human cell line HeLa were fractionated by different methods to establish suitable initial steps for the purification. At each stage, protein fractions were mixed with whole cell extract from XP-A cells and the mixture was assessed for the ability to promote in vitro DNA repair synthesis in damaged DNA. Activity that could stimulate damage-dependent repair synthesis by XP-A extracts was found to bind to phosphocellulose and could be precipitated with ammonium sulphate at 45% saturation. Upon gel filtration, XP-A complementing activity eluted within a somewhat broad peak between 30 and 60 kilodaltons (kDa) apparent molecular mass. Chromatography of this material on heparin-agarose and CM Sepharose yielded a protein fraction that specifically stimulated repair synthesis when

3914

added to cell extracts of lines derived from three unrelated XP-A donors (Figure 1). The same fraction did not complement XP-B, XP-C or XP-D cell extracts, but damagedependent DNA synthesis by these extracts could be initiated by addition of UV endonuclease from Micrococcus luteus (Figure 1) or UvrABC enzyme from Escherichia coli (Hansson et al., 1990). Extracts from the normal human lymphoblastoid cell line GM1953 were also found to yield XP-A complementing activity after fractionation. However, if fractionation was carried out starting with extracts prepared from XP-A lymphoid cell lines, XP-A specific stimulating activity was not detected at any stage (data not shown). In further experiments, repair was analysed using a plasmid DNA molecule containing a site-specifically placed acetylaminofluorene deoxyguanosine adduct (Hansson et al., 1989). It was found that the fraction used in Figure 1 restored a normal pattern of repair synthesis to XP-A extracts, localized at the site of the adduct (J.Hansson, M.Munn, D.Rupp and R.Wood, unpublished data). These experiments indicated that it was possible to purify an XP-A complementing activity from human cells in culture. However, since it was clear that large amounts of cells would be required to produce sufficient protein for study, we investigated the suitability of calf thymus tissue as an alternative source. Fractionation of extracts from fresh calf thymus on phosphocellulose and by ammonium sulphate precipitation yielded active starting material. XP-A complementing protein was subsequently purified by sequential chromatographic steps as outlined in Table I. An example of a repair assay for XP-A protein during the sixth step of the procedure is shown in Figure 2. Synthesis in both damaged and nondamaged plasmids is monitored together in each reaction, and it is clear that the XP-A complementing fractions enhance synthesis specifically in damaged DNA. Incorporation of radioactive material in nondamaged DNA remains low in all reactions, providing that scrupulous care is given to the preparation of closed

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domination of the complementation reaction by the factors that nonspecifically stimulated repair synthesis. From step IV, the XP-A correcting activity was separated from the stimulatory factor. In the final two steps, (FPLC Mono Q and Mono S chromatography), it was apparent from analysis by SDS -PAGE that two bands of relative molecular mass 40 and 42 kDa co-eluted with XP-A correcting activity (Figure 3). In the most purified fractions these comprised -95% of the protein visible by silver staining. Evidence is presented below showing that the two bands are closely related and probably derived from the same polypeptide. As expected, in assays similar to those shown in Figure 1 the most purified XP-A fractions from calf thymus could only complement XP-A extracts, and not extracts made from cell lines of other XP groups. The data in Table I show that the XP-A protein is present only at very low abundance in calf thymus cells. However, 3915

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purification by gel filtration.

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DNA binding The phenotype of XP-A cells suggests that the XP-A protein is involved in recognition of DNA lesions or incision of damaged DNA. Purified XP-A protein (Fraction VII) was therefore examined for a possible DNA binding activity. 32P-labelled DNA was prepared from double-stranded, nondamaged linear plasmid, and then either irradiated with various UV fluences, or heat-denatured to give singlestranded DNA. These probes were incubated in solution with XP-A protein at 30°C, and the extent of binding was assessed by measuring the retention of DNA -protein complexes on nitrocellulose filters. As shown in Figure 4A, XP-A protein was able to bind to all three types of DNA to various extents. A given amount of protein bound more readily to doublestranded than to single-stranded DNA. Significantly, more XP-A protein bound to irradiated double-stranded DNA than to nonirradiated DNA. The extent of binding of XP-A protein was dependent on the KCl concentration in the binding mixture. At only 25 mM KCl, the observed difference in binding between nonirradiated and irradiated DNA was minimal. A KCl concentration of 50 mM gave an ionic strength allowing a substantial fraction of probe binding while retaining a clearly apparent difference in binding of irradiated versus nonirradiated DNA. Binding did not require addition of Mg2+ ion, but inclusion of 5 mM Mg2+ increased the overall extent of binding; addition of 1 mM ATP to the binding buffer had no effect (data not shown). Standard conditions of 50 mM KCl, 5 mM MgCl2 and no ATP were

therefore chosen. The irradiated DNA indicated in Figure 4A received 9 kJ/m2 of UV. At this fluence, formation of cyclobutane pyrimidine dimers is at photochemical equilibrium with 15% of all thymines dimerized. In order to estimate the affinity for irradiated over nonirradiated DNA, we assessed -

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the amounts of XP-A protein in calf thymus and human lymphoblastoid cells appear to be similar. Although it was not possible to quantify the XP-A protein directly in crude cell extracts from thymus, we estimate that Fraction VII of the XP-A protein was 500 000-fold purified, allowing for 5-fold purification by phosphocellulose and a further 3-fold -

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fluence, kJ/m2 Fig. 4. Binding of XP-A protein to DNA. A. 1 ng of 32P-labelled DNA, either double-stranded (U), single-stranded (0) or doublestranded and UV-irradiated with 9 kJ/m2 (0), was incubated with purified (1 ng/jl) XP-A protein (fraction VII). DNA bound to protein was collected on nitrocellulose filters. In parallel control experiments, human single stranded binding protein HSSB (Kenny et al., 1990) was tested with the same probes. 5 ng of HSSB bound 70% of the singlestranded probe to a filter but could bind only 10-15% of irradiated or nonirradiated double-stranded probe DNA. B. Fluence-response for binding of XP-A protein to damaged DNA. Double-stranded DNA was irradiated with increasing UV fluences and incubated with 6 ng of purified XP-A protein. Points at 0 and 9 kJ/m2 represent the average of six and four experiments respectively (bars show range); other points show the average of two experiments. C. Transformation of the data from Figure 4B to give El', a quantity proportional to the number of molecules bound per fragment (at lower fluences).

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temperature (0 C) Fig. 5. Heat-inactivation of XP-A protein. 1 U of purified XP-A protein (fraction VII) was heated for 5 min at the indicated temperature in extract buffer containing 200 pg/ml BSA before addition to a reaction mixture which included 100 itg extract protein from GM2345 XP-A cells. Synthesis in irradiated DNA is shown by closed symbols and synthesis in unirradiated DNA by open symbols. Circles indicate reactions including XP-A protein, squares show a control with XP-A cell extract but without XP-A protein.

binding of purified XP-A protein to probes containing fewer sites of damage. Our measurements of lesion induction in plasmid DNA using the same UV source give -0.73 cyclobutane pyrimidine dimers per 1000 bp per 100 J/m2 (Wood, 1989). The next most frequent photoproducts are (6-4) pyrimidine dimers, which occur at -0.24 adducts per 1000 bp per 100 J/m2 so that there is a total of about one UV lesion per 1000 bp at 100 J/m2. As anticipated, the amount of probe bound was fluencedependent (Figure 4B). To interpret such binding data it is necessary to take account of the fraction of probe that remains unbound. A Poisson transformation of the data gives a quantity Ev which is proportional to the average number v of protein molecules bound per DNA fragment, and the probability e that a bound fragment is retained on the filter (Mazur and Grossman, 1991). The curve will saturate when e v is equal to the filter efficiency ( - 0.7 in this case). From this transformation (Figure 4C) it is apparent that the number of XP-A molecules bound to irradiated DNA is doubled over that in nonirradiated DNA by a fluence in the range of 100 J/m2. Since the nondamaged probe fragments (average size 1870 bp) bind about half as much XP-A protein as probes containing two UV dimers per fragment, the preference of XP-A protein for binding damaged versus nondamaged nucleotides can be estimated at - 1000-fold. Most damagedependent repair synthesis in cell extracts and in cells appears to occur at (6-4) photoproducts (Wood, 1989; Sibghat-Ullah and Sancar, 1990); if most of the observed binding of XPA protein occurs at (6-4) photoproducts, the discrimination factor for these sites is several-fold larger than 103. More precise measurements will be possible with substrates containing site-specifically located lesions. Because the XP-A protein appears to be one of the components involved in introducing incisions into damaged DNA, it was important to test purified XP-A protein for such an activity. Fraction VII XP-A protein (26 U) was incubated in 25 AI of standard repair synthesis reaction mixture (including 50 mM KCl, 5 mrM Mg2+ and 2 mM ATP) with

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250 ng of either nondamaged closed circular pHM 14 DNA or UV-irradiated (450 J/m2) closed circular pBluescript KS +, or with 500 ng closed circular single-stranded M 13mpl 8. Analysis by agarose gel electrophoresis showed that no single-strand breaks were produced by XP-A protein in any of these molecules after a 1.5 h incubation at 37°C (not shown). It would have been possible to detect a few percent nicking of any of the substrates. This result is not

3917

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Figure 7. A. Immunoblot across the XP-A purification scheme using an antipeptide antibody. Marker lanes (M) contain 925 Bq of 14C-labelled protein markers (Amersham). The remaining lanes contain protein from the same fractions shown in Figure 3: fraction I (60 pg); fraction II (23 ptg); fraction III (13 Ag); fraction IV (5 jig); fraction V (0.8 pg); fraction VI (0.04 jg); fraction VII (0.003 Ag). Protein samples were electrophoresed on a 10% polyacrylamide gel, transferred to an Immobilon P PVDF membrane and incubated with rabbit antibody raised against peptide 1 (see text) and then with 1251-labelled anti-rabbit antibody before exposure to X-ray film. A cross-reacting band of -35 kDa appeared after step IV but eluted separately from XP-A complementing activity during step V. B. Immunoblot of fraction VII using two anti-peptide antibodies. Lane 1, fraction VII (0.003 ytg) blotted with antibody 1. M, size markers. Lane 2, fraction VII (0.009 jig) blotted with antibody against the carboxy-terminal peptide 2. Before blotting the membrane was separated through the centre of the marker lane. Each half was incubated with the appropriate serum and the membrane was realigned before exposure to X-ray film.

unexpected, since the XP-A protein probably works in concert with other polypeptides to incise DNA.

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Stability of the XP-A protein The heat stability of the XP-A protein was investigated by incubating aliquots of purified fraction VII at various temperatures and then assaying for activity. Heating was carried out in the presence of 200 /.g/ml bovine serum albumin (BSA) to minimize nonspecific adsorption to surfaces. Heating of 1 U of protein for 5 min at 40°C or lower was not detrimental, but heating at 56°C inactivated all detectable complementing activity. The inactivation took place across a 16°C temperature range (Figure 5). The broadness of this inactivation profile may reflect the nature of the assay, which assesses complementation of a whole cell extract. It seems likely that the cell extract contains components which can aid in refolding partially denatured proteins. Intact cells are probably even more efficient in this refolding process, since it has been reported that microinjection of partially purified protein fractions can complement in vivo repair synthesis in XP-A fibroblast cell lines even after the fractions are boiled in buffers containing SDS (Hoeijmakers et al., 1990).

Fig. 8. Immunoblot showing binding and elution of the XP-A protein doublet from double-stranded DNA-cellulose. 1 ml of fraction VI XP-A protein (L) was loaded onto a 0.1 ml column of DNA cellulose at 0.08 M KCI and eluted stepwise with buffer containing the indicated KCI concentrations (molar). The first lane contained 14C-labelled molecular weight markers (M). In addition, the XP-A protein doublet could not be separated on a column of UV-irradiated double-stranded DNA -cellulose (data not shown).

Relationship to the protein product of a cloned human gene Tanaka et al. (1990) recently isolated a human gene which can restore UV resistance to XP-A fibroblast cell lines when transferred into their genome. Mutations in this gene have been identified in cell lines established from XP-A patients (Satokata et al., 1990). The cDNA is predicted to encode a protein of 273 amino acids with a molecular mass of 31 kDa. It was clearly of interest to establish the relationship of this predicted protein to our purified XP-A correcting fraction, which contained a protein doublet of apparent molecular mass 40 and 42 kDa. For this purpose, antibodies were prepared against peptides deduced from the cDNA sequence given by Tanaka et al. (1990). 'Peptide 1' corresponds to amino acids 32-46 of this sequence. Figure 6 shows a parallel complementation

activity assay, SDS -polyacrylamide gel and immunoblot with protein fractions from the Mono S column used in step VII of the purification. XP-A complementing activity clearly co-elutes with the protein doublet of 40/42 kDa, and both of these bands are recognized by an antibody raised against peptide 1. This strongly suggests that the XP-A protein purified by in vitro complementation corresponds to the protein encoded by the genetically derived XP-A gene. Since both polypeptides in the most purifed fraction were recognized by antiserum against peptide 1, it was of interest to follow their occurrence across the purification scheme for complementing activity (Figure 7A). From step II, both bands were detectable by immunoblotting. The lower band is stronger in all fractions, and the upper band appears with increasing intensity as the purification progresses. It appears that either the slower migrating form is derived from the

3918

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faster during the purification procedure, or that the less abundant upper band is more active in complementation and is preferentially enriched during purification. Both forms bind to single-stranded and to double-stranded DNA -cellulose, and both are eluted from double-stranded DNA -cellulose by about the same KCl concentration (Figure 8). The existence of two polypeptides that copurify and are immunologically related suggests that one is derived from the other either by limited proteolysis, postranslational modification, partial protein unfolding on the gel or possibly alternative splicing of the transcript. Experiments have been performed to examine several of these alternatives. First, both bands appeared even after extensive boiling in buffer with SDS and reducing agents, so that one is unlikely to be a partially unfolded conformer of the other. Their apparent difference in molecular mass of 2 kDa could be explained by proteolysis of 10-20 amino acids from one end of the slower migrating form, giving rise to the faster migrating form. An antibody raised against a peptide corresponding to the predicted carboxy-terminus of the protein was found to recognize both bands, ruling out C-terminal cleavage (Fig 7B). In an initial attempt to derive N-terminal peptide sequence from our most purified protein fraction, lack of cleavage in the sequencing reactions suggested N-terminal blockage of both forms of the protein. Finally, treatment of purified XP-A protein in either native or denatured form with bacterial alkaline phosphatase, calf intestinal phosphatase or potato acid phosphatase did not alter the migration of either XP-A protein band (data not shown). It thus does not appear that differential modification by phosphorylation is the source of the doublet. We cannot directly comment on alternative splicing. Two mRNAs of 1.1 and 1.3 kb have been observed in human cells, but these can be explained by the presence of appropriately spaced alternative polyadenylation sites in the transcript (Tanaka et al., 1990). Two protein bands of 40 and 42 kDa were apparently also detected in normal human cells after immunoprecipitation using a polyclonal antibody against recombinant protein (Tanaka et al., 1990). -

Discussion Complementation in vitro by a purified DNA excision repair protein In the work reported here it has been possible for the first time to isolate a mammalian DNA excision repair protein by in vitro complementation of a repair-defective cell extract. The approach has allowed purification of the XP-A protein in active form without subjecting the protein to denaturing conditions, and has permitted functional studies to be undertaken. Significantly, the XP-A protein has been found to bind preferentially to damaged DNA, strongly suggesting that it plays a role in the lesion recognition step of DNA excision repair. We have further shown that this XP-A complementing protein corresponds to the product encoded by a recently isolated gene that can restore excision repair to cells derived from group A of xeroderma pigmentosum. This correspondence is of considerable importance, since it shows that excision repair of plasmid DNA by soluble cell extracts has enough features in common with genomic repair in cells to reveal accurately the repair defect in an inherited human

disease. This encourages the use of the same approach for the isolation and study of other XP proteins. At present, very little is known about the XP-C, XP-E, XP-F and XP-G proteins, and the corresponding cDNAs have not been cloned. Additionally, in vitro complementation should be a widely applicable method to assay for the activity of proteins expressed from cloned excision repair genes. Function of the XP-A protein Experiments with cells in culture clearly demonstrate that the XP-A protein plays a central role in the repair of many types of lesions and that XP-A cells are defective in introducing incisions into damaged DNA (Cleaver and Kraemer, 1989). The purified XP-A protein is shown here to restore repair synthesis to XP-A cell extracts in the absence of semiconservative DNA replication, transcription or translation. Therefore it appears that the XP-A protein is a component of the machinery that introduces nicks into damaged DNA, rather than a regulatory factor for other genes. The argument for a role of XP-A protein in incision at lesions is considerably reinforced by its DNA binding properties. The protein binds preferentially to damaged DNA, with a discrimination factor for UV lesions of - 103. Interestingly, this is the same order of magnitude of damaged site discrimination as found for the E.coli UvrA protein (Seeberg and Steinum, 1982; Van Houten et al., 1987; Mazur and Grossman, 1991). Neither XP-A protein nor UvrA can introduce nicks into DNA on its own. A further parallel is the presence of zinc finger motifs in both UvrA and XP-A (Tanaka et al., 1990). Those in UvrA are thought to be essential for DNA binding (Myles and Sancar, 1991; Claassen and Grossman, 1991). However, there are substantial differences between the two proteins. For one, UvrA is a 104 kDa protein with several apparent domains. Further, UvrA binds DNA as a dimer, and formation of this dimer is ATP-dependent. However, no ATP binding sites are obvious from the translated XP-A cDNA sequence, and we have found that ATP does not significantly affect binding of purified XP-A to DNA. In experiments to examine nicking of DNA with purified UvrA, B, and C proteins, partially purified XP-A protein was unable to substitute for any one of the three Uvr polypeptides (L.Grossman and R.Wood,

unpublished observations). To achieve the high discrimination between damaged and nondamaged sites that is required for an efficient repair mechanism, other factors are needed. In E. coli , the UvrB and UvrC proteins provide the added specificity (BertrandBurggraf et al., 1991). No doubt the same is true in mammalian cells, and there are many candidates for such factors, including additional XP and other repair gene products. The XP-A protein has been repeatedly suggested to function as a factor that assists in the disassembly of chromatin, to promote accessibility for incision nucleases (Mortelmans et al., 1976; Kano and Fujiwara, 1983; Parrish and Lambert, 1990). In our experiments, the DNA is introduced into repair reactions as purified plasmid circles, and so the involvement of a mechanism of chromatin disassembly in repair seems unnecessary. Although limited formation of nucleosome-sized structures can take place in 'Manley'-type whole cell extracts (Hough et al., 1982), we have not found that extensive packaging into chromatin takes 3919

P.Robins et al.

place in the extracts. Chromatin assembly in extracts can be assessed by monitoring the degree of supercoiling in purified molecules after incubation (Smith and Stillman, 1991), and we observe only a small amount of supercoiling after purification of closed circular plasmid DNA from a repair synthesis incubation (unpublished observations). Additionally, repair synthesis is detectable at early times, and increases essentially linearly during the first 1 -2 h of incubation, so there is no evidence for a lag phase associated with chromatin disassembly (Wood and Robins, 1989). A recent careful study of the conditions required for nucleosome assembly in Manley extracts (Banerjee and Cantor, 1990) also shows that only a very small amount of assembly takes place under repair reaction conditions. It therefore appears unlikely that the function of the XP-A protein is to act as a chromatin disassembly factor. By microinjection of protein fractions into XP-A fibroblasts and monitoring complementation by restoration of DNA repair synthesis, a protein fraction from calf thymus has been obtained with characteristics very similar to the one described here, containing two major polypeptides of -40 and 42 kDa (A.Eker, W.Vermeulen, J.Hoeijmakers and D.Bootsma, personal communication). Microinjection of material eluted from the 40-45 kDa region of a twodimensional polyacrylamide gel has also been independently found to correct defective DNA repair synthesis in XP-A cells (M.Yamaizumi, personal communication). Materials and methods Cell lines and extracts

Epstein-Barr virus immortalized human lymphoblastoid cell lines were obtained from the NIGMS Human Mutant Cell Repository (Coriell Institute, Camden, NJ, USA). The XP-A cell lines used were GM2344 (derived from patient XP1WI, a 42 month old Caucasian male); GM2250 (from patient XP12BE, a 10 year old Caucasian female); and GM2345 (from patient XP20S, an 8 year old Oriental female). Cells were cultured in RPMI 1640 medium supplemented with 15 % fetal calf serum and antibiotics. All cell cultures were checked regularly and found to be free from contamination with Mycoplasma. Whole cell extracts were made according to the method of Manley et al. (1983) with minor modifications, as previously described (Wood et al., 1988; Hansson et al., 1991). For each preparation, 1-2 litre cultures of cells in late exponential growth phase (5-8 x 105 cells/ml) were used. After preparation, the cell extracts were immediately frozen and stored at -800C. Plasmid DNA Plasmids pBluescript KS+ (Stratagene) and pHM14 (Rydberg et al., 1990) were grown in Ecoli host strain JM109 [F' traD36 laclq A(lacZ)MJ5 proABlrecAl endAl gyrA96 thi A(lac-proAB) hsdRl 7 (rK - mK+) supE44 relAl ]. Purified pBluescript KS+ DNA was UV-irradiated with 450 J/m2. This irradiated plasmid (2961 bp) and nonirradiated pHM14 (3740 bp) were each treated separately with Nth protein purified to homogeneity from E. coli strain XN99 cI857/pHIT1 (Asahara et al., 1989). Closed circular DNA was re-isolated from sucrose gradients as described (Wood et al., 1988;

Biggerstaff et al., 1991).

In vitro repair reactions Standard 50 Al reaction mixtures contained 300 ng each of UV-irradiated plasmid pBluescript KS+ and nondamaged plasmid pHM14, 45 mM HEPES-KOH (pH 7.8), 70 mM KCI, 7.5 mM MgCl2, 0.9 mM dithiothreitol (DTT), 0.4 mM EDTA, 2 mM ATP, 20 AM each of dGTP, dCTP and TTP, 8 ztM dATP, 74 kBq of [ac-32P]dATP (111 TBq/mmol), 40 mM phosphocreatine (di-Tris salt), 2.5 jig creatine phosphokinase (type I, Sigma), 3.4% glycerol, 18 ytg BSA and 125 jig of XP-A whole cell extract protein. Reactions were incubated for 3 h at 30°C. Plasmid DNA was purified from the reaction mixtures, linearized with BamHI and electrophoresed overnight on a 1% agarose gel containing 0.5 tg/ml ethidium bromide. Data were collected by autoradiography, densitometry and scintillation counting of excised DNA bands (Wood et al., 1988).

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Purification of XP-A correcting protein from calf thymus Calf thymus glands were removed immediately after slaughter and packed in ice. All subsequent operations were performed at 0-4°C. After removal of fat and connective tissue, each thymus gland was cut into several pieces and suspended in an equal volume (w/v) of buffer A (50 mM Tris-HCI, pH 7.5, 1 mM EDTA, 0.5 mM DTT) containing 100 mM NaCI. This initial buffer was supplemented with protease inhibitors (Sigma) as follows: 0.5 mM phenylmethylsulphonylfluoride, 0.025 trypsin inhibitor units/ml aprotinin and 5 jig/ml each of leupeptin, pepstatin A and chymostatin. The tissue suspension was homogenized for 3 x 30 s in a Waring blender and then stirred slowly for 1 h before centrifugation at 8000 g for 30 min. The supematant was removed and adjusted to 20 mM NaCl by addition of buffer A. A 1 litre slurry (150 g dry weight in buffer A) of phosphocellulose P11 (Whatman) was added to the protein solution for each kg calf thymus tissue, and the suspension stirred slowly for 1 h before centrifugation at 500 g for 5 min. The supernatant was discarded and the slurry resuspended in 1 litre buffer A containing 500 mM NaCl and stirred slowly for 1 h before centrifugation at 5000 g for 5 min. Crystalline ammonium sulphate was added to the supernatant to 45% saturation and stirred for 30 min. After centrifugation at 15 000 g for 30 min the pellets were resuspended in buffer B (25 mM HEPES-KOH, pH 7.8, 1 mM EDTA, 10% glycerol, 2 mM DTT) containing 1 M KCl to give - 40 mg/ml protein in 30 ml. The protein solution was centrifuged at 15 000 g for 30 min and the supernatant applied to a column (25 x 1500 mm) of Ultragel AcA44 (IBF Biotechnics). 3.8 kBq of [methyl-14C] methylated-BSA (1 MBq/mg, New England Nuclear) was loaded with the sample as an internal marker that could be followed by scintillation counting of aliquots from the eluted fractions. Protein fractions eluting from the column with an apparent molecular mass between 30 kDa and 60 kDa were pooled and dialysed extensively against buffer B containing 100 mM KCI. The protein solution was applied to a 30 ml column of heparin -agarose (Gibco BRL) and washed with buffer B containing 100 mM KCl. Active protein was then eluted with buffer B containing 400 mM KCI. Eluted protein derived from 4 kg of calf thymus tissue was dialysed against several changes of buffer C (25 mM HEPES-KOH, pH 6.8, 1 mM EDTA, 10% glycerol, 2 mM DTT) containing 80 mM KCl, before applying to an S Sepharose (Pharmacia) column (5 ml bed). After washing the column with the same buffer, protein was eluted with a linear gradient of 80-500 mM KCl in buffer C (total 80 ml). CM Sepharose could alternatively be used in step IV; after washing the column with buffer C containing 125 mM KCl, active protein was eluted with buffer C containing 250 mM KCI. The active fractions were pooled and dialysed against buffer D (25 mM HEPES-KOH, pH 7.8, 0.2 mM EDTA, 500 mM KCl, 5% glycerol, 2 mM DTT) containing 1 mM potassium phosphate, before applying to a column with a 15 ml bed volume of hydroxylapatite Bio-Gel HT (Bio-Rad). After washing the column with the same buffer, and buffer D containing 25 mM potassium phosphate, the active protein was eluted with buffer D containing 60 mM

phosphate. The protein was then dialysed against buffer B containing 100 mM KCI, before applying to an FPLC Mono Q HR 5/5 column (Pharmacia). Protein was eluted with a linear gradient of 100-400 mM KCI in buffer B (total 25 ml). The active fractions were pooled and 3 volumes of buffer C were added, to reduce the KCl concentration to below 100 mM KCl, before applying to an FPLC Mono S HR 5/5 column (Pharmacia). Protein was eluted with a linear gradient of 100-600 mM KCl in buffer C (total 25 ml). Active fractions (0.5 ml each) were frozen at -80°C. Protein could be frozen at -80°C in buffer containing 10% glycerol between purification steps. Repeated freezing and thawing led to a very gradual loss of complementing activity. One unit of XP-A complementing activity is defined as the amount of protein required to increase UV-dependent repair synthesis by a factor of two over the UV-dependent synthesis seen with XP-A extract alone. If X = (fmol dAMP incorporated into UV-irradiated plasmid - fmol dAMP incorporated into nondamaged plasmid) in a reaction with XP-A extract only, and Y = (fmol dAMP incorporated into UV-irradiated plasmid fmol dAMP incorporated into nondamaged plasmid) in a reaction with XP-A extract and complementing protein fraction, then the units of complementing activity in the protein fraction are given by U = (Y - X) X. The activity of a given fraction is best determined by titration. -

DNA binding Measurement of binding of DNA -protein complexes to nitrocellulose filters was carried out essentially according to Ausubel et al. (1989). DNA for binding was prepared by filling in the ends of EcoRI-digested plasmidpHM14 with E. coli DNA polymerase I (Klenow fragment) in the presence of [cl-32P]dATP. This yields one fragment of 2961 bp and one of 779 bp in an equimolar ratio. Unincorporated label was removed on a Sephadex G25 column, and the DNA was phenol extracted, ethanol precipitated and

DNA repair with XP-A protein resuspended in 10 mM Tris-HCl pH 8.0, 1 mM EDTA. The labelled DNA was irradiated as required with 254 nm (peak) UV light in small droplets in a Petri dish on ice at a flux of 1 W/m2. Binding reactions contained 1 ng of DNA (10 000 c.p.m.) in 50 11 of 20 mM HEPES-KOH pH 7.7, 50 mM KCI, 5 mM MgCl2, 1 mM DTT, 100 ,ug/ml BSA, 10% glycerol and binding protein fraction as indicated. After incubation for 25 min at 30°C, the 50 ul samples were applied to alkali-pretreated cellulose nitrate filters (Millipore HAWP 02500, 0.45 um pore size) and rinsed twice with 0.5 ml of binding buffer without BSA. The filters were counted in vials containing 5 ml Picofluor and compared with the total counts in a reaction under identical quenching conditions. To obtain a quantity proportional to the number of protein molecules bound per fragment, data was transformed according to the equation Eu = -ln[(total - sample)l(total - blank)] as described by Mazur and Grossman (1991). The number of counts bound to a filter in a reaction without XP-A protein is given by blank, and this was - 9% of the total counts for double-stranded DNA and - 2% of the total for single-stranded probes. The data utilized in this transformation is the sum of the binding curves generated by the two EcoRI fragments of pHM14. Since the value of Ev is small at low fluences, it is proportional to the fraction of probe bound by protein to the filter. Calculations using the arithmetic average of probe size therefore yield a reasonable approximation.

Antibodies and immunoblotting Peptide 1 (NH2-RQRALMLRQARLAAR-COOH) corresponding to amino acids 32-46 of the deduced XPAC polypeptide (Tanaka et al., 1990) was selected on the basis of its predicted hydrophilicity, surface probability, antigenic index (as given by a computer search) and suitability for coupling to carrier protein. Peptide 2 (NH2-RTCTMCGHELTYE-COOH) corresponds to amino acids 260 -271 and represents the C-terminus of the protein (minus two residues), with an additional arginine at the N-terminus of the peptide to aid in coupling. Rabbit polyclonal antibodies were raised against peptides coupled to bovine thyroglobulin (T1001, Sigma) with glutaraldehyde. Antisera were affinity-purified on columns containing peptide coupled to BSA (Sigma RIA grade) linked to activated Sepharose, and then dialysed against phosphate-buffered saline A (PBS) before storage at -80°C (Harlow and Lane, 1989). Protein samples were separated on 0.75 mm, 10% SDS-polyacrylamide minigels and transferred onto Immobilon P PVDF membrane (Millipore) in 25 mM Tris-HCI, pH 8.3 containing 0.2 M glycine and 20% methanol. Following transfer the membranes were washed with PBS and then blocked with 5 % nonfat milk in PBS containing 0.02% sodium azide for 1 h. After washing three times with 0.1% NP40 in PBS the membranes were incubated for 24 h in a 1/50 dilution of purified antibody in PBS containing 10% fetal calf serum, 1% BSA, 0.1% NP40 and 0.02% sodium azide. Membranes were then washed in 1 % NP40 in PBS, then PBS alone to remove detergent and incubated for 24 h with 37 kBq of '25I-labelled donkey anti-rabbit IgG (Amersham) in PBS containing 10% fetal calf serum and 0.02% sodium azide. After further washing (as above) the blots were dried and placed against X-ray film.

Acknowledgements We are grateful to S.Keyse, D.Coverley, P.Vaughan and J.Hansson for advice and some experimental material; to L.Grossman and D.Szymkowski for productive discussions; to A.Tomkinson, E.Roberts and G.Daly for help with preparation of calf thymus extracts; to N.O'Reilly and G.Evan (ICRF Peptide Synthesis Unit) and D.Pappin (ICRF Peptide Sequencing Unit); to R.Peat for growth of cell lines and to J.Nicholson for photography. We appreciate discussions with J.Hoeijmakers, A.Eker, D.Bootsma, K.Tanaka and M.Yamaizumi.

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Received on August 7, 1991; revised on September 4, 1991

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(1989) Biochemistry, 28, 4444-4449. Ausubel,F.M., Brent,R., Kingston,R.E., Moore,D.D., Seidman,J.G., Smith,J.A. and Struhl,K. (1989) Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley Interscience, New York, pp. 12.8.1 - 12.8.9. Banerjee,S. and Cantor,C.R. (1990) Mol. Cell Biol., 10, 2863-2873. Bertrand-Burggraf,E., Selby,C.P., Hearst,J.E. and Sancar,A. (1991) J. Mol. Biol., 219, 27-36. Biggerstaff,M., Robins,P., Coverley,D. and Wood,R.D. (1991) Mutation Res., 254, 217-224. Claassen,L.A. and Grossman,L. (1991)J. Biol. Chem., 266, 11388-11394. Cleaver,J.E. and Kraemer,K.H. (1989) In Scriver,C.R., Beaudet,A.L.,

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Complementation of DNA repair in xeroderma pigmentosum group A cell extracts by a protein with affinity for damaged DNA.

Complementation group A of xeroderma pigmentosum (XP) represents one of the most prevalent and serious forms of this cancer-prone disorder. Because of...
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