Proc. Nati. Acad. Sci. USA Vol. 88, pp. 6839-6842, August 1991
Biochemistry
Human cells contain protein specifically binding to a single 1,N6-ethenoadenine in a DNA fragment (vinyl chloride/site-spedfic carcinogen adduct/DNA nicking/mismatch recognition)
B. RYDBERG, M. K. DOSANJH,
AND
B. SINGER*
Donner Laboratory, Cell and Molecular Biology Division, Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720
Communicated by H. Fraenkel-Conrat, May 15, 1991 A human DNA binding protein has been ABSTRACT characterized from cell-free extracts of liver, placenta, and cultured cells. This protein, apparent molecular mass =35 kDa, to our knowledge, does not resemble other proteins reported to bind to carcinogen-modified DNA. The probe used for characterization was a 25-base oligonucleotide containing a single site-specifically placed 1,NM-ethenoadenine (MA), a product of vinyl chloride metabolism. When annealed to form an eA-T or EA-C pair, a strong affinity to the protein was observed, with a binding constant of -1 x 109 M-l. In contrast, very little binding was found with an eAJA pair and none was found with an eAG pair. This suggests protein recognition of a specific structural alteration. Other defined probes with alkyl adducts did not bind. In addition, the human cell extracts and a rat liver extract were found to nick specifically at the 5' side of the EA adduct, which could indicate a possible associated repair activity.
1,N6-Ethenoadenine (EA) is formed in DNA by the metabolism of vinyl chloride, a known human and rodent carcinogen (1, 2). The stable metabolite chloroacetaldehyde is bifunctional and forms etheno rings between the amino group and adjacent endo nitrogen of adenine, guanine, and cytosine (3). EA, which is highly fluorescent, has been identified in organs of rats and mice given vinyl chloride or related carcinogens, such as ethyl carbamate and acrylonitrile (4-7). Although the specific effects of -A in animals are not known, replication of EA in polynucleotides in vitro results in a low level of transversions and transitions (8-10). The eA-containing sequence used in the present work has been found to be mutagenic in Escherichia coli, albeit at a low frequency, causing a variety of mutations including both transitions and transversions (A. K. Basu and J. M. Essigmann, personal communication). Physical studies using two-dimensional NMR of eAST and EANG pairs in a 9-base oligonucleotide suggest two configurations, both of which lead to structural distortion (11, 12). Mammalian binding proteins have been described that specifically bind to base mismatches (13, 14) or to DNA damaged by physical (15-19) and chemical agents (15, 20, 21). It has been suggested that such binding proteins may play a role in DNA repair, as has been demonstrated for prokaryotes (22). The ability to detect specific proteins complexing to known chemical adducts in DNA has been greatly facilitated by using gel-retardation assays (13, 15, 21). We now report a human DNA binding protein that recognizes EA-T or EA-C in a defined oligonucleotide.
MATERIALS AND METHODS Materials. The 25-base oligonucleotide 5'-CCGCT(eA)GCGGGTACCGAGCTCGAAT-3', was a gift from A. K. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Basu and J. M. Essigmann (Massachusetts Institute of Technology). The same oligonucleotide with adenine in place of EA, the complementary 25-mer oligonucleotides with any base opposite the eA adduct, and the 5-mer 5'-CCGCT-3' were synthesized using the phosphoramidite method. HeLa cells were obtained from R. Goth-Goldstein (Lawrence Berkeley Laboratory, Berkeley, CA) and human transformed hepatocyte cells were from M. McCall (Lawrence Berkeley Laboratory). Frozen human placenta was a gift from J. Cleaver (University of California San Francisco), human liver samples were from F. P. Guengerich (Vanderbilt University School of Medicine, Nashville, TN), and rat liver samples were from J. A. Swenberg (University of North Carolina, Chapel Hill, NC). Preparation of Cell-Free Extracts. Frozen pellets from cultured cells and frozen tissue samples were processed essentially as described by Jiricny et al. (13) except that the procedure was scaled down to 0.5 g of packed cells or tissue and the final centrifugation was carried out in a 100.2 rotor using a Beckman TL-100 ultracentrifuge at 100,000 rpm for 75 min. The crude extracts (pH 7.5) had a final concentration of 20% (vol/vol) glycerol. The extraction buffer for the livers contained the following protease inhibitors: leupeptin (2 ,ug/ml), chymostatin (2 Ag/ml), and pepstatin A (2 ,ug/ml) (Sigma). All extracts were frozen in small samples and stored at -70TC, where they appeared stable for at least 3 months. In some cases, the extracts were 3-fold purified and concentrated by using the fraction precipitated at 35-50%o saturated ammonium sulfate. The precipitate was suspended in 25 mM Hepes-KOH, pH 7.8/0.5 mM EDTA/0.5 mM dithiothreitol/10%o glycerol/0.5 mM phenylmethylsulfonyl fluoride/1 mM benzamidine and dialyzed against the same buffer at 40C for 6-12 hr. Protein concentrations in extracts were determined using the Bradford assay (23). Band-Shift Assay. Oligonucleotides were 5'-end-labeled with 32p using polynucleotide kinase and then annealed to the complementary strand in 150 mM Tris'HCl, pH 7.8/30 mM MgCl2 as described (24). The binding reaction was carried out in 25 mM Hepes-KOH, pH 7.8/0.5 mM EDTA/0.5 mM dithiothreitol/150 mM NaCl/10%o glycerol/1 Ag of poly (dldC) and up to 5 dul of cell-free extract in a final volume of 20 gl. Unlabeled double-stranded competitor oligonucleotides were added when necessary and 10-40 fmol of the 32P-labeled oligonucleotide was added last. After 30 min at room temperature (unless otherwise stated), 5-10 ul of the reaction mixture was electrophoresed on a nondenaturing 6% polyacrylamide gel using TBE buffer (0.09 M Tris borate, pH 8.0/2 mM EDTA) at 10 V/cm for 75 min. After drying, the gel was autoradiographed. Quantitation of the relative amount of oligonucleotide bound to the binding protein was obtained by densitometer scanning and integration using a Hoefer model GS300 densitometer connected to a Hoefer model GS350 data system. Abbreviation: MA, 1,N6-ethenoadenine. *To whom reprint requests should be addressed.
6839
6840
Biochemistry: Rydberg et al.
Proc. Natl. Acad Sci. USA 88 (1991)
The sensitivity of the binding factor to proteinase K (100
,ug/ml) or RNase A (20 A&g/ml) was determined by prein-
cubation of the cell extract in binding buffer for 40 min at 370C with the respective enzyme. Determination of the Binding Constant K. The binding constant was determined by the method of Muller (25) and as used by Donahue et al. (21). Briefly, a low concentration [TJ] of 32P-labeled probe and a sufficient amount of cell extract to give a fraction of probe bound (b) close to 0.5 were used. While keeping these factors constant, various concentrations of unlabeled probe were added as competitor to determine the concentration [Ij] that gives rise to a 50% inhibition of the bound 32P-labeled probe. The binding constant K was then calculated using the equation by Muller (25)
4g protein
2
_-
j[Tj]}.
Nicking Assay. The cell-free extracts were incubated with the 32P-labeled probe as described above except that NaCl was omitted and incubation was at 370C for 1-3 hr. The samples were heat denatured and electrophoresed on a denaturing 12% polyacrylamide gel as described (24) at 50 V/cm for 1 hr. The oligomer 5'-CCGCT-3' was 32P-labeled at the 5' end and used as a marker. Gel Filtration. A Sephacryl S-200 column (0.7 x 28 cm) was equilibrated with 25 mM Hepes-KOH, pH 7.8/0.5 mM EDTA/0.5 mM dithiothreitol/0.5 M NaCl/10% glycerol. HeLa cell extract, concentrated by ammonium sulfate precipitation and dialyzed against the running buffer, was eluted at 3 ml/hr. Fractions were assayed for the binding protein and quantitated by scanning densitometry as described. The column was calibrated with bovine serum albumin (67 kDa) and RNase A (13.7 kDa). The peak of the binding protein and the calibration peaks were symmetrical, and the elution volume at the half-peak area was calculated and plotted against the logarithm of the molecular weight.
RESULTS Human DNA Binding Protein That Recognizes eA. The presence of binding proteins in cell-free extracts from whole cells was tested using the technique of gel retardation. Most experiments were carried out using a 25-mer oligonucleotide containing the adduct EA at position 6 from the 5' end (Fig. 1). This oligonucleotide was annealed to the complementary oligonucleotide to produce an EA-T pair. Typical results using HeLa cell extracts are shown in Fig. 2. In addition to nonspecific retarded bands near the top of the gel, a characteristic band, indicated by an arrow, appears with increasing amounts of extract (lanes 1-5). This band can be competed out using 5-20 times the amount of nonlabeled eA-Tcontaining oligonucleotide (lanes 6-8) but is unaffected by the same amounts of oligonucleotide lacking the EA adduct (lanes 9-11). This indicates that the band is specific for the 5'-CCGCT*GCG 3'-GGCGATCGC
.2 4 8
-
EA SPECIFIC BAND -_
Wh.i. a
4.
.1i
FREE PROBE _ 1
1/K = (1 - 1.5b + 0.5b2)'{[hI -
10 10 10 10 10 10 10 - .2 .48
4
cA-T pmole A-T pmole
2
9 10 11
3 4 5 '6 7 8
FIG. 2. DNA binding protein specific for MA in extracts from HeLa cells. The EA T-containing labeled 25-mer probe (0.04 pmol) was incubated with increasing amounts of cell-free extract (lanes 1-5). Above the figure is indicated the total amount of protein added per assay. Unlabeled competitor 25-mer oligomer containing the EA-T pair (lanes 6-8) or a normal A-T pair in the same position (lanes 9-11) was included in the incubation mixture in the amounts shown. -, None added.
eA T-containing oligonucleotide. Similar results were also obtained using extracts from a human transformed hepatocyte cell line (data not shown). The binding factor is a protein, since proteinase K abolished the delayed band, whereas RNase A was without effect. Cell extracts were also prepared from human livers from six individuals (five males, 20-45 years; 1 female, 49 years) obtained postmortem and a human placenta from a normal delivery. All human samples tested this way contained the eA-specific DNA binding protein (Fig. 3). The amounts of the binding protein appeared to be similar in all liver samples, within a factor of 2 as determined by scanning densitometry. Extracts from E. coli (noninduced) and Saccharomyces cerevisiae were also tested. Under the experimental conditions used for human cell extracts, no EA-specific band could be detected. However, a weak eA-specific band, migrating at a different position, was seen using extracts from rat liver (data not shown). Characteristics of the Human Binding Protein. The binding to EA-containing oligomers required the presence of a cation in the incubation mixture. Near optimum binding was ob-
I
HUMAN LIVERS 2 3 4 5 6
G
Biochemistry
Human cells contain protein specifically binding to a single 1,N6-ethenoadenine in a DNA fragment (vinyl chloride/site-spedfic carcinogen adduct/DNA nicking/mismatch recognition)
B. RYDBERG, M. K. DOSANJH,
AND
B. SINGER*
Donner Laboratory, Cell and Molecular Biology Division, Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720
Communicated by H. Fraenkel-Conrat, May 15, 1991 A human DNA binding protein has been ABSTRACT characterized from cell-free extracts of liver, placenta, and cultured cells. This protein, apparent molecular mass =35 kDa, to our knowledge, does not resemble other proteins reported to bind to carcinogen-modified DNA. The probe used for characterization was a 25-base oligonucleotide containing a single site-specifically placed 1,NM-ethenoadenine (MA), a product of vinyl chloride metabolism. When annealed to form an eA-T or EA-C pair, a strong affinity to the protein was observed, with a binding constant of -1 x 109 M-l. In contrast, very little binding was found with an eAJA pair and none was found with an eAG pair. This suggests protein recognition of a specific structural alteration. Other defined probes with alkyl adducts did not bind. In addition, the human cell extracts and a rat liver extract were found to nick specifically at the 5' side of the EA adduct, which could indicate a possible associated repair activity.
1,N6-Ethenoadenine (EA) is formed in DNA by the metabolism of vinyl chloride, a known human and rodent carcinogen (1, 2). The stable metabolite chloroacetaldehyde is bifunctional and forms etheno rings between the amino group and adjacent endo nitrogen of adenine, guanine, and cytosine (3). EA, which is highly fluorescent, has been identified in organs of rats and mice given vinyl chloride or related carcinogens, such as ethyl carbamate and acrylonitrile (4-7). Although the specific effects of -A in animals are not known, replication of EA in polynucleotides in vitro results in a low level of transversions and transitions (8-10). The eA-containing sequence used in the present work has been found to be mutagenic in Escherichia coli, albeit at a low frequency, causing a variety of mutations including both transitions and transversions (A. K. Basu and J. M. Essigmann, personal communication). Physical studies using two-dimensional NMR of eAST and EANG pairs in a 9-base oligonucleotide suggest two configurations, both of which lead to structural distortion (11, 12). Mammalian binding proteins have been described that specifically bind to base mismatches (13, 14) or to DNA damaged by physical (15-19) and chemical agents (15, 20, 21). It has been suggested that such binding proteins may play a role in DNA repair, as has been demonstrated for prokaryotes (22). The ability to detect specific proteins complexing to known chemical adducts in DNA has been greatly facilitated by using gel-retardation assays (13, 15, 21). We now report a human DNA binding protein that recognizes EA-T or EA-C in a defined oligonucleotide.
MATERIALS AND METHODS Materials. The 25-base oligonucleotide 5'-CCGCT(eA)GCGGGTACCGAGCTCGAAT-3', was a gift from A. K. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Basu and J. M. Essigmann (Massachusetts Institute of Technology). The same oligonucleotide with adenine in place of EA, the complementary 25-mer oligonucleotides with any base opposite the eA adduct, and the 5-mer 5'-CCGCT-3' were synthesized using the phosphoramidite method. HeLa cells were obtained from R. Goth-Goldstein (Lawrence Berkeley Laboratory, Berkeley, CA) and human transformed hepatocyte cells were from M. McCall (Lawrence Berkeley Laboratory). Frozen human placenta was a gift from J. Cleaver (University of California San Francisco), human liver samples were from F. P. Guengerich (Vanderbilt University School of Medicine, Nashville, TN), and rat liver samples were from J. A. Swenberg (University of North Carolina, Chapel Hill, NC). Preparation of Cell-Free Extracts. Frozen pellets from cultured cells and frozen tissue samples were processed essentially as described by Jiricny et al. (13) except that the procedure was scaled down to 0.5 g of packed cells or tissue and the final centrifugation was carried out in a 100.2 rotor using a Beckman TL-100 ultracentrifuge at 100,000 rpm for 75 min. The crude extracts (pH 7.5) had a final concentration of 20% (vol/vol) glycerol. The extraction buffer for the livers contained the following protease inhibitors: leupeptin (2 ,ug/ml), chymostatin (2 Ag/ml), and pepstatin A (2 ,ug/ml) (Sigma). All extracts were frozen in small samples and stored at -70TC, where they appeared stable for at least 3 months. In some cases, the extracts were 3-fold purified and concentrated by using the fraction precipitated at 35-50%o saturated ammonium sulfate. The precipitate was suspended in 25 mM Hepes-KOH, pH 7.8/0.5 mM EDTA/0.5 mM dithiothreitol/10%o glycerol/0.5 mM phenylmethylsulfonyl fluoride/1 mM benzamidine and dialyzed against the same buffer at 40C for 6-12 hr. Protein concentrations in extracts were determined using the Bradford assay (23). Band-Shift Assay. Oligonucleotides were 5'-end-labeled with 32p using polynucleotide kinase and then annealed to the complementary strand in 150 mM Tris'HCl, pH 7.8/30 mM MgCl2 as described (24). The binding reaction was carried out in 25 mM Hepes-KOH, pH 7.8/0.5 mM EDTA/0.5 mM dithiothreitol/150 mM NaCl/10%o glycerol/1 Ag of poly (dldC) and up to 5 dul of cell-free extract in a final volume of 20 gl. Unlabeled double-stranded competitor oligonucleotides were added when necessary and 10-40 fmol of the 32P-labeled oligonucleotide was added last. After 30 min at room temperature (unless otherwise stated), 5-10 ul of the reaction mixture was electrophoresed on a nondenaturing 6% polyacrylamide gel using TBE buffer (0.09 M Tris borate, pH 8.0/2 mM EDTA) at 10 V/cm for 75 min. After drying, the gel was autoradiographed. Quantitation of the relative amount of oligonucleotide bound to the binding protein was obtained by densitometer scanning and integration using a Hoefer model GS300 densitometer connected to a Hoefer model GS350 data system. Abbreviation: MA, 1,N6-ethenoadenine. *To whom reprint requests should be addressed.
6839
6840
Biochemistry: Rydberg et al.
Proc. Natl. Acad Sci. USA 88 (1991)
The sensitivity of the binding factor to proteinase K (100
,ug/ml) or RNase A (20 A&g/ml) was determined by prein-
cubation of the cell extract in binding buffer for 40 min at 370C with the respective enzyme. Determination of the Binding Constant K. The binding constant was determined by the method of Muller (25) and as used by Donahue et al. (21). Briefly, a low concentration [TJ] of 32P-labeled probe and a sufficient amount of cell extract to give a fraction of probe bound (b) close to 0.5 were used. While keeping these factors constant, various concentrations of unlabeled probe were added as competitor to determine the concentration [Ij] that gives rise to a 50% inhibition of the bound 32P-labeled probe. The binding constant K was then calculated using the equation by Muller (25)
4g protein
2
_-
j[Tj]}.
Nicking Assay. The cell-free extracts were incubated with the 32P-labeled probe as described above except that NaCl was omitted and incubation was at 370C for 1-3 hr. The samples were heat denatured and electrophoresed on a denaturing 12% polyacrylamide gel as described (24) at 50 V/cm for 1 hr. The oligomer 5'-CCGCT-3' was 32P-labeled at the 5' end and used as a marker. Gel Filtration. A Sephacryl S-200 column (0.7 x 28 cm) was equilibrated with 25 mM Hepes-KOH, pH 7.8/0.5 mM EDTA/0.5 mM dithiothreitol/0.5 M NaCl/10% glycerol. HeLa cell extract, concentrated by ammonium sulfate precipitation and dialyzed against the running buffer, was eluted at 3 ml/hr. Fractions were assayed for the binding protein and quantitated by scanning densitometry as described. The column was calibrated with bovine serum albumin (67 kDa) and RNase A (13.7 kDa). The peak of the binding protein and the calibration peaks were symmetrical, and the elution volume at the half-peak area was calculated and plotted against the logarithm of the molecular weight.
RESULTS Human DNA Binding Protein That Recognizes eA. The presence of binding proteins in cell-free extracts from whole cells was tested using the technique of gel retardation. Most experiments were carried out using a 25-mer oligonucleotide containing the adduct EA at position 6 from the 5' end (Fig. 1). This oligonucleotide was annealed to the complementary oligonucleotide to produce an EA-T pair. Typical results using HeLa cell extracts are shown in Fig. 2. In addition to nonspecific retarded bands near the top of the gel, a characteristic band, indicated by an arrow, appears with increasing amounts of extract (lanes 1-5). This band can be competed out using 5-20 times the amount of nonlabeled eA-Tcontaining oligonucleotide (lanes 6-8) but is unaffected by the same amounts of oligonucleotide lacking the EA adduct (lanes 9-11). This indicates that the band is specific for the 5'-CCGCT*GCG 3'-GGCGATCGC
.2 4 8
-
EA SPECIFIC BAND -_
Wh.i. a
4.
.1i
FREE PROBE _ 1
1/K = (1 - 1.5b + 0.5b2)'{[hI -
10 10 10 10 10 10 10 - .2 .48
4
cA-T pmole A-T pmole
2
9 10 11
3 4 5 '6 7 8
FIG. 2. DNA binding protein specific for MA in extracts from HeLa cells. The EA T-containing labeled 25-mer probe (0.04 pmol) was incubated with increasing amounts of cell-free extract (lanes 1-5). Above the figure is indicated the total amount of protein added per assay. Unlabeled competitor 25-mer oligomer containing the EA-T pair (lanes 6-8) or a normal A-T pair in the same position (lanes 9-11) was included in the incubation mixture in the amounts shown. -, None added.
eA T-containing oligonucleotide. Similar results were also obtained using extracts from a human transformed hepatocyte cell line (data not shown). The binding factor is a protein, since proteinase K abolished the delayed band, whereas RNase A was without effect. Cell extracts were also prepared from human livers from six individuals (five males, 20-45 years; 1 female, 49 years) obtained postmortem and a human placenta from a normal delivery. All human samples tested this way contained the eA-specific DNA binding protein (Fig. 3). The amounts of the binding protein appeared to be similar in all liver samples, within a factor of 2 as determined by scanning densitometry. Extracts from E. coli (noninduced) and Saccharomyces cerevisiae were also tested. Under the experimental conditions used for human cell extracts, no EA-specific band could be detected. However, a weak eA-specific band, migrating at a different position, was seen using extracts from rat liver (data not shown). Characteristics of the Human Binding Protein. The binding to EA-containing oligomers required the presence of a cation in the incubation mixture. Near optimum binding was ob-
I
HUMAN LIVERS 2 3 4 5 6
G
