Proc. Nati. Acad. Sci. USA
Vol. 89, pp. 8779-8783, September 1992 Biochemistry
Escherichia coli MutY protein has both N-glycosylase and apurinic/apyrimidinic endonuclease activities on AoC and AoG mispairs JYY-JIH TSAI-WU, HsIN-FEI Liu, AND A-LIEN Lu* Department of Biological Chemistry, School of Medicine, University of Maryland, Baltimore, MD 21201
Communicated by Maurice S. Fox, June 19, 1992 (received for review February 4, 1992)
The homology between MutY and endonuclease III suggested that MutY may be an iron-sulfur protein containing a [4Fe-4S]2+ cluster (19). The MutY protein has been purified to near homogeneity by Modrich and coworkers (24). They showed that MutY is an AoG-specific adenine glycosylase but lacks detectable AP endonuclease activity. However, tightly associated AoG mismatch-specific binding and nicking activities that are MutYdependent have been identified in E. coli extracts (18, 25). When mapped on a suitable substrate, the nicks occur at the first phosphodiester bond 3' to and the second phosphodiester bond 5' to the mismatched adenine. In this paper we show that the glycosylase and the 3' AP endonuclease activities are catalyzed by a single MutY protein. Purified MutY, an iron-sulfur protein, can recognize and nick DNA containing AoG or AoC mismatches. However, DNA with an AoC mismatch is a much weaker substrate than DNA with an AoG mismatch.
ABSTRACT In Escherichia coli the mutY (or micA)dependent DNA mismatch repair pathway can convert AoG and AoC mismatches to C-G and G-C base pairs, respectively, through a short repair-tract mechanism. The MutY protein has been purified to near homogeneity from an E. coli overproducer strain. Purified MutY has been shown to contain both N-glycosylase and 3' apurinic/apyrimidinic (AP) endonuclease activities. The N-glycosylase removes the mispaired adenines of AoG and AoC mismatches, and the AP endonuclease acts on the first phosphodiester bond 3' to the AP sites. The N-glycosylase and the nicking (combined N-glycosylase and AP endonuclease) activities copurifiled through multiple chromatographic steps without a change in relative specific activities. Furthermore, both N-glycosylase and AP endonuclease activities can be recovered by renaturation of a single polypeptide band from an SDS/polyacrylamide gel. Renaturation required the presence of iron and sulfide. These findings suggest that the MutY protein, like endonuclease HI, is an iron-sulfur protein. DNA fragments with AoC mismatches were 20-fold less active than DNA with AoG mispairs as a substrate for purified MutY.
MATERIALS AND METHODS
Among all eight possible base mismatches, AoG mismatch represents a special type of base pairing. Structural analyses showed A-G can form hydrogen bonds with three possible conformations: A(anti)-G(anti), A(anti)-G(syn), and A(syn)G(anti) (1-4). AoG mispairs have been shown to occur frequently as replication errors (5) and also can arise from genetic recombination between two closely related sequences. There are two pathways capable of repairing AoG mismatches in Escherichia coli. One is dependent on ATP, dam methylation, and mutHLSU gene functions (6-13). The other is dependent on mutY(or micA) and polA functions but not the above three factors (14-17). The dam-dependent pathway can repair some AoG mismatches to either C0G or A-T base pairs depending on the state of methylation. The mutY (or micA)-dependent pathway acts on AoG mismatches to restore C-G base pairs exclusively (15, 17). In the absence of ATP, extracts from mutY (or micA) mutants cannot bind, nick, or repair heteroduplex DNA containing AoG mismatches (14, 18). The defect in AoG mismatch repair of mutY, a mutator, suggests that this repair process is responsible for the reduction of C-G-to-A-T transversions. The mutY (or micA) mutants are also defective in repairing AoC mismatches in transfected A heteroduplex DNAs (16). Recent cloning and sequence analyses showed mutY and micA are different alleles of one gene (18, 19). The mutY gene encodes a 39.1-kDa protein with homology to E. coli endonuclease III, which has both glycosylase and 3' apurinic/apyrimidinic (AP) endonuclease activities (20-22). The AP endonuclease activity of endonuclease III is the result of the enzyme catalyzing a 3-elimination reaction (23).
DNA Heteroduplexes. The DNA heteroduplexes used in the enzyme activity assay were constructed by annealing two sets of complementary oligonucleotides (40-mer or 116-mer) to generate a single base mismatch at a defined position (18, 26, 27). The annealed heteroduplexes were radioactively labeled by filling in the 3' end with the Klenow fragment of DNA polymerase I in the presence of [a-32P]dCTP or [a-32P]dATP (28). To label the 5' end of the heteroduplexes, the oligonucleotides were labeled with T4 polynucleotide kinase and [y-32P]ATP before annealing to the complementary strands. Enzyme Assays. The endonuclease nicking activity was assayed as described (25). The glycosylase activity was monitored by adding piperidine to the nicking reaction to a final concentration of 1 M. After a 30-min incubation at 900C, the reaction products were analyzed in 8% polyacrylamide DNA sequencing gels. MutY Protein Purification. MutY protein was purified as described with some modifications (25). E. coli strain JM109 harboring a MutY overexpression plasmid pJTW1O-12 J.J.T.-W. and A-L.L., unpublished results) was grown at 370C in a 100-liter fermentor. The expression of MutY protein was induced by the addition of 1 mM isopropyl f3-D-thiogalactoside to the culture at an A59o of 0.6. The cells were harvested 3 hr later. The cell paste was stored at -800C until use. All column chromatography was conducted in a Water's 650 FPLC system at 40C and centrifugation was at 16,500 X g for 30 min. Cells (47 g) were resuspended in 120 ml of buffer T [50 mM Tris-HCl, pH 7.6/0.1 mM EDTA/0.5 mM dithiothreitol (DTT)/0.1 mM phenylmethylsulfonyl fluoride (PMSF)] and disrupted with a bead beater (Biospec Products,
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.
Abbreviations: AP, apurinic/apyrimidinic; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride. *To whom reprint requests should be addressed. 8779
8780
Bartlesville, OK) using 0.1-mm glass beads. The cell debris removed by centrifugation, and the supernatant was treated with 5% streptomycin sulfate. After stirring for 30 min, the solution was centrifuged, and the supernatant was collected as fraction I (235 ml). Ammonium sulfate (162 g) was added to fraction I, and the proteins were precipitated overnight at 40C. After centrifugation, the protein pellets were resuspended in 12 ml of buffer T and dialyzed against two changes of 1 liter ofbuffer T. The dialyzed protein sample was then diluted 4-fold with buffer A (20 mM potassium phosphate, pH 7.4/0.5 mM DTT/0.1 mM EDTA/0.1 mM PMSF) containing 0.05 M KCl as fraction II (100 ml) and applied to a 30-ml phosphocellulose column equilibrated with buffer A containing 0.05 M KC1. After washing with 60 ml of equilibration buffer, proteins were eluted with a 300-ml linear gradient of KCl (0.05-0.5 M) in buffer A. Fractions containing the AoG-specific nicking activity were pooled (fraction III, 66.7 ml) and loaded onto a 20-ml hydroxylapatite column equilibrated with 10 mM potassium phosphate (pH 7.4), 10 mM KCl, 0.5 mM DTT, 0.1 mM EDTA, and 0.1 mM PMSF. After washing with 40 ml of equilibration buffer, the flowthrough fractions were pooled (fraction IV, 63 ml) and dialyzed against buffer A containing 0.05 M KCl and 10% (vol/vol) glycerol. The sample was then applied to a 5-ml heparin-agarose column equilibrated with buffer A containing 0.05 M KCl and 10% glycerol. After washing with 10 ml of equilibration buffer, the column was developed with a 50-ml linear gradient of KCl (0.1-0.6 M) in buffer A containing 10%1 glycerol. Fractions containing the AoG-specific nicking activity, which eluted at about 0.38 M KCl, were pooled (fraction V, 17 ml). Fraction V was dialyzed overnight against 1 liter ofbufferA containing 0.05 M KCl and 10% glycerol and applied to a 1-ml Mono S FPLC column equilibrated with buffer S (50 mM potassium phosphate, pH 7.5/1 mM DTT/ 0.1 mM EDTA/0.1 mM PMSF/10%o glycerol) containing 0.1 M NaCl. The column was washed with 10 ml of equilibration buffer and developed with 20-ml linear gradient of NaCl (0.1-0.4 M) in buffer S. MutY activity appeared at two peaks: one was eluted at the washing step (fraction VI-1, 10 ml) and the other was eluted at 0.2 M NaCl (fraction VI-2, 7.5 ml). Fraction VI was divided into small aliquots and stored at -80°C or dialyzed against buffer A containing 50%o glycerol and stored at -20°C. Denaturatlon and Renaturaffon of MutY Protein. The denaturation and renaturation of MutY protein were performed according to Hager and Burgess (29) with some modifications. Fraction VI-1 (150 pZg) was applied to a 12% polyacrylamide gel containing SDS (30). After electrophoresis, the gel was stained with ice-cold 0.25 M KCl for 5 min and destained with ice-cold distilled water until clear white bands appeared. The MutY protein band was excised, transferred to a siliconized glass tube, and further destained with distilled water. The protein was eluted into 4 ml of elution buffer (50 mM Tris HCl, pH 7.8/0.1% SDS/0.15 M NaCl/0.1 mg of bovine serum albumin per ml) by rotating the tube overnight at 4°C. After removing the gel pieces, the eluted protein was concentrated by precipitation with 4 vol of ice-cold acetone. The protein pellet was redissolved in 400 A1 of 6 M guanidine hydrochloride in dialysis buffer (50 mM Tris HCl, pH 7.6/ 0.15 M NaCl/20%o glycerol/0.1 mg of bovine serum albumin per mi/10 mM 2-mercaptoethanol/0.1 mM ammonium sulfide/0.1 mM ferrous ammonium sulfate). Renaturation was carried out by gradually removing guanidine hydrochloride through dialysis against 400 ml of dialysis buffer. Renaturation samples (40 ;4) were taken at different time points to check for nicking and glycosylase activities of MutY. Other Materials and Methods. The column matrixes were purchased from different companies: phosphocellulose P-1l was from Whatman, hydroxylapatite was from Bio-Rad, and heparin-agarose was from Bethesda Research Laboratories. was
Proc. NatL Acad. Sci. USA 89 (1992)
Biochemistry: Tsai-Wu et al.
Mono S HR 5/5 column was purchased from Pharmacia. Protein concentration was determined according to the method of Bradford (31) using bovine serum albumin as the standard. The 32P-labeled nucleotides were obtained from DuPont/NEN.
RESULTS Purification of MutY Protein. Previously, MutY protein was partially purified from E. coli strain M5248 through three chromatographic steps: phosphocellulose, hydroxylapatite (25), and heparin-agarose (18). Fraction V from this preparation (18) contained both AoG-specific binding and nicking activities. In this paper, an AoG-specific nicking assay was used to purify MutY protein from a strain harboring the overproducing plasmid pJTW1O-12 (J.-J.T.-W. and A-L.L., unpublished results). A similar purification scheme was applied except that 45% ammonium sulfate was used to precipitate proteins from fraction I (rather than 35%) and an extra Mono S column chromatography step was added after heparin-agarose chromatography (see Materials and Methods). We recovered 28 mg of MutY protein (fractions VI-1 and VI-2; Table 1) from 47 g of overproducing cell paste with about a 16-fold increase in specific activity. Compared with the specific activities of AoG nicking in a crude extract from wild-type cells, this overproducer strain yielded about 1000fold more MutY protein. The Mono S column (1 ml) was overloaded and that resulted in two separated peaks (fractions VI-1 and VI-2). When less protein was applied to the column, only peak 2 was observed (data not shown). The protein compositions in fractions I-VI are shown in Fig. 1. Electrophoresis employed an SDS/12% polyacrylamide gel. In fraction V, MutY (migating as a 36-kDa protein) was purified to >98% homogeneity, and in fractions VI-1 and VI-2, MutY was purified to >99%o homogeneity. Mismatch Nicking Sp ty of MutY Protin. Radicella et al. (16) showed that micA mutants failed to correct both AOG and AoC mismatches in a A heteroduplex assay. While AoG mismatches were repaired well by the MutY-dependent pathway, there was no evidence that cell extracts from wild-type E. coli could act on AoC mismatches by an in vitro repair assay (14, 15). Furthermore, partially purified MutY from wild-type E. coli could not bind or nick AoC-containing DNA (25). Using our concentrated pure MutY protein, we again checked its mismatch specificity. AoG-containing DNAs were the best substrates (Fig. 2, lanes 1 and 3). No detectable nicking activity was found when DNA heteroduplexes containing ToG, ToC, ToT, CoC, AoA, and GoG mispairs were used as substrates (data not shown). However, DNA heteroduplexes containing an A4C or CoA mismatch were nicked after incubation with fraction V (Fig. 2, lanes 2 and 4). As Table 1. Purification of MutY from an overproducing E. coli strain
Protein,
Specific activity,
Recovery, % Fraction mg Step units/mg I 369,000 (100) Streptomycin sulfate 2867 1010 II Ammonium sulfate 43 455,000 111 2,720,000 28 III Phosphoceilulose 21 60 IV 3,717,000 Hydroxylapatite 39 4,615,000 17 V Heparin agarose 12 VI-1 23 Mono S 5,566,000 5 6,600,000 3 VI-2 Mono S Protein connation was measured by the Bradford assay (31), and activity was assayed with a 3' end-labeled 120-mer DNA duplex with AoG mismatch as substrate. One unit of nicking activity is defined as that resulting in cleavage of 1% (0.018 finol) of the labeled DNA in 30 min under the assay conditions.
Biochemistry: Tsai-Wu et al. kDa
1
3
2
4
Proc. Natl. Acad. Sci. USA 89 (1992) 5
6
7
111_-
71_-44
_*
29_18_ 15FIG. 1. SDS/polyacrylamide gel analysis of MutY fractions. The proteins were separated on a 12% polyacrylamide gel in the presence of SDS according to Laemmli (30) and stained with Coomassie blue. Lanes 1-7 are fractions 1 (52 pg), II (40 pg), III (8.3 pg), IV (6.7 Pg), V (5.8 ,ug), VI-1 (5.8 pg), and VI-2 (5.3 kg), respectively. Molecular mass standards (Bethesda Research Laboratories) were run in a parallel lane.
determined by densitometry of the autoradiograph, the nicking activity of MutY on an AoG substrate was 20-fold greater than that on an AoC substrate (Fig. 2, lane 1 vs. lane 2 and lane 3 vs. lane 4). Interestingly, MutY was found to nick only the dA strand but not the dC strand of AoC substrate (Fig. 2, lanes 4 and 5). These results are consistent with the AoC to G-C repair in vivo (16). When the MutY protein purified from wild-type cells was incubated with an AoC heteroduplex DNA at a higher protein concentration, a weak nicking activity was detected (data not shown). Therefore, MutY can act not only on AoG mismatches but also on AoC mismatches, but with lower activity. Copurification of A°G-Specific Nicking and Glycosylase Activities. By using an AoG substrate labeled at the 5' end of the Fraction
V
+
_*
0 0
~0
4o
%..
--
4c*
Vol. 89, pp. 8779-8783, September 1992 Biochemistry
Escherichia coli MutY protein has both N-glycosylase and apurinic/apyrimidinic endonuclease activities on AoC and AoG mispairs JYY-JIH TSAI-WU, HsIN-FEI Liu, AND A-LIEN Lu* Department of Biological Chemistry, School of Medicine, University of Maryland, Baltimore, MD 21201
Communicated by Maurice S. Fox, June 19, 1992 (received for review February 4, 1992)
The homology between MutY and endonuclease III suggested that MutY may be an iron-sulfur protein containing a [4Fe-4S]2+ cluster (19). The MutY protein has been purified to near homogeneity by Modrich and coworkers (24). They showed that MutY is an AoG-specific adenine glycosylase but lacks detectable AP endonuclease activity. However, tightly associated AoG mismatch-specific binding and nicking activities that are MutYdependent have been identified in E. coli extracts (18, 25). When mapped on a suitable substrate, the nicks occur at the first phosphodiester bond 3' to and the second phosphodiester bond 5' to the mismatched adenine. In this paper we show that the glycosylase and the 3' AP endonuclease activities are catalyzed by a single MutY protein. Purified MutY, an iron-sulfur protein, can recognize and nick DNA containing AoG or AoC mismatches. However, DNA with an AoC mismatch is a much weaker substrate than DNA with an AoG mismatch.
ABSTRACT In Escherichia coli the mutY (or micA)dependent DNA mismatch repair pathway can convert AoG and AoC mismatches to C-G and G-C base pairs, respectively, through a short repair-tract mechanism. The MutY protein has been purified to near homogeneity from an E. coli overproducer strain. Purified MutY has been shown to contain both N-glycosylase and 3' apurinic/apyrimidinic (AP) endonuclease activities. The N-glycosylase removes the mispaired adenines of AoG and AoC mismatches, and the AP endonuclease acts on the first phosphodiester bond 3' to the AP sites. The N-glycosylase and the nicking (combined N-glycosylase and AP endonuclease) activities copurifiled through multiple chromatographic steps without a change in relative specific activities. Furthermore, both N-glycosylase and AP endonuclease activities can be recovered by renaturation of a single polypeptide band from an SDS/polyacrylamide gel. Renaturation required the presence of iron and sulfide. These findings suggest that the MutY protein, like endonuclease HI, is an iron-sulfur protein. DNA fragments with AoC mismatches were 20-fold less active than DNA with AoG mispairs as a substrate for purified MutY.
MATERIALS AND METHODS
Among all eight possible base mismatches, AoG mismatch represents a special type of base pairing. Structural analyses showed A-G can form hydrogen bonds with three possible conformations: A(anti)-G(anti), A(anti)-G(syn), and A(syn)G(anti) (1-4). AoG mispairs have been shown to occur frequently as replication errors (5) and also can arise from genetic recombination between two closely related sequences. There are two pathways capable of repairing AoG mismatches in Escherichia coli. One is dependent on ATP, dam methylation, and mutHLSU gene functions (6-13). The other is dependent on mutY(or micA) and polA functions but not the above three factors (14-17). The dam-dependent pathway can repair some AoG mismatches to either C0G or A-T base pairs depending on the state of methylation. The mutY (or micA)-dependent pathway acts on AoG mismatches to restore C-G base pairs exclusively (15, 17). In the absence of ATP, extracts from mutY (or micA) mutants cannot bind, nick, or repair heteroduplex DNA containing AoG mismatches (14, 18). The defect in AoG mismatch repair of mutY, a mutator, suggests that this repair process is responsible for the reduction of C-G-to-A-T transversions. The mutY (or micA) mutants are also defective in repairing AoC mismatches in transfected A heteroduplex DNAs (16). Recent cloning and sequence analyses showed mutY and micA are different alleles of one gene (18, 19). The mutY gene encodes a 39.1-kDa protein with homology to E. coli endonuclease III, which has both glycosylase and 3' apurinic/apyrimidinic (AP) endonuclease activities (20-22). The AP endonuclease activity of endonuclease III is the result of the enzyme catalyzing a 3-elimination reaction (23).
DNA Heteroduplexes. The DNA heteroduplexes used in the enzyme activity assay were constructed by annealing two sets of complementary oligonucleotides (40-mer or 116-mer) to generate a single base mismatch at a defined position (18, 26, 27). The annealed heteroduplexes were radioactively labeled by filling in the 3' end with the Klenow fragment of DNA polymerase I in the presence of [a-32P]dCTP or [a-32P]dATP (28). To label the 5' end of the heteroduplexes, the oligonucleotides were labeled with T4 polynucleotide kinase and [y-32P]ATP before annealing to the complementary strands. Enzyme Assays. The endonuclease nicking activity was assayed as described (25). The glycosylase activity was monitored by adding piperidine to the nicking reaction to a final concentration of 1 M. After a 30-min incubation at 900C, the reaction products were analyzed in 8% polyacrylamide DNA sequencing gels. MutY Protein Purification. MutY protein was purified as described with some modifications (25). E. coli strain JM109 harboring a MutY overexpression plasmid pJTW1O-12 J.J.T.-W. and A-L.L., unpublished results) was grown at 370C in a 100-liter fermentor. The expression of MutY protein was induced by the addition of 1 mM isopropyl f3-D-thiogalactoside to the culture at an A59o of 0.6. The cells were harvested 3 hr later. The cell paste was stored at -800C until use. All column chromatography was conducted in a Water's 650 FPLC system at 40C and centrifugation was at 16,500 X g for 30 min. Cells (47 g) were resuspended in 120 ml of buffer T [50 mM Tris-HCl, pH 7.6/0.1 mM EDTA/0.5 mM dithiothreitol (DTT)/0.1 mM phenylmethylsulfonyl fluoride (PMSF)] and disrupted with a bead beater (Biospec Products,
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.
Abbreviations: AP, apurinic/apyrimidinic; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride. *To whom reprint requests should be addressed. 8779
8780
Bartlesville, OK) using 0.1-mm glass beads. The cell debris removed by centrifugation, and the supernatant was treated with 5% streptomycin sulfate. After stirring for 30 min, the solution was centrifuged, and the supernatant was collected as fraction I (235 ml). Ammonium sulfate (162 g) was added to fraction I, and the proteins were precipitated overnight at 40C. After centrifugation, the protein pellets were resuspended in 12 ml of buffer T and dialyzed against two changes of 1 liter ofbuffer T. The dialyzed protein sample was then diluted 4-fold with buffer A (20 mM potassium phosphate, pH 7.4/0.5 mM DTT/0.1 mM EDTA/0.1 mM PMSF) containing 0.05 M KCl as fraction II (100 ml) and applied to a 30-ml phosphocellulose column equilibrated with buffer A containing 0.05 M KC1. After washing with 60 ml of equilibration buffer, proteins were eluted with a 300-ml linear gradient of KCl (0.05-0.5 M) in buffer A. Fractions containing the AoG-specific nicking activity were pooled (fraction III, 66.7 ml) and loaded onto a 20-ml hydroxylapatite column equilibrated with 10 mM potassium phosphate (pH 7.4), 10 mM KCl, 0.5 mM DTT, 0.1 mM EDTA, and 0.1 mM PMSF. After washing with 40 ml of equilibration buffer, the flowthrough fractions were pooled (fraction IV, 63 ml) and dialyzed against buffer A containing 0.05 M KCl and 10% (vol/vol) glycerol. The sample was then applied to a 5-ml heparin-agarose column equilibrated with buffer A containing 0.05 M KCl and 10% glycerol. After washing with 10 ml of equilibration buffer, the column was developed with a 50-ml linear gradient of KCl (0.1-0.6 M) in buffer A containing 10%1 glycerol. Fractions containing the AoG-specific nicking activity, which eluted at about 0.38 M KCl, were pooled (fraction V, 17 ml). Fraction V was dialyzed overnight against 1 liter ofbufferA containing 0.05 M KCl and 10% glycerol and applied to a 1-ml Mono S FPLC column equilibrated with buffer S (50 mM potassium phosphate, pH 7.5/1 mM DTT/ 0.1 mM EDTA/0.1 mM PMSF/10%o glycerol) containing 0.1 M NaCl. The column was washed with 10 ml of equilibration buffer and developed with 20-ml linear gradient of NaCl (0.1-0.4 M) in buffer S. MutY activity appeared at two peaks: one was eluted at the washing step (fraction VI-1, 10 ml) and the other was eluted at 0.2 M NaCl (fraction VI-2, 7.5 ml). Fraction VI was divided into small aliquots and stored at -80°C or dialyzed against buffer A containing 50%o glycerol and stored at -20°C. Denaturatlon and Renaturaffon of MutY Protein. The denaturation and renaturation of MutY protein were performed according to Hager and Burgess (29) with some modifications. Fraction VI-1 (150 pZg) was applied to a 12% polyacrylamide gel containing SDS (30). After electrophoresis, the gel was stained with ice-cold 0.25 M KCl for 5 min and destained with ice-cold distilled water until clear white bands appeared. The MutY protein band was excised, transferred to a siliconized glass tube, and further destained with distilled water. The protein was eluted into 4 ml of elution buffer (50 mM Tris HCl, pH 7.8/0.1% SDS/0.15 M NaCl/0.1 mg of bovine serum albumin per ml) by rotating the tube overnight at 4°C. After removing the gel pieces, the eluted protein was concentrated by precipitation with 4 vol of ice-cold acetone. The protein pellet was redissolved in 400 A1 of 6 M guanidine hydrochloride in dialysis buffer (50 mM Tris HCl, pH 7.6/ 0.15 M NaCl/20%o glycerol/0.1 mg of bovine serum albumin per mi/10 mM 2-mercaptoethanol/0.1 mM ammonium sulfide/0.1 mM ferrous ammonium sulfate). Renaturation was carried out by gradually removing guanidine hydrochloride through dialysis against 400 ml of dialysis buffer. Renaturation samples (40 ;4) were taken at different time points to check for nicking and glycosylase activities of MutY. Other Materials and Methods. The column matrixes were purchased from different companies: phosphocellulose P-1l was from Whatman, hydroxylapatite was from Bio-Rad, and heparin-agarose was from Bethesda Research Laboratories. was
Proc. NatL Acad. Sci. USA 89 (1992)
Biochemistry: Tsai-Wu et al.
Mono S HR 5/5 column was purchased from Pharmacia. Protein concentration was determined according to the method of Bradford (31) using bovine serum albumin as the standard. The 32P-labeled nucleotides were obtained from DuPont/NEN.
RESULTS Purification of MutY Protein. Previously, MutY protein was partially purified from E. coli strain M5248 through three chromatographic steps: phosphocellulose, hydroxylapatite (25), and heparin-agarose (18). Fraction V from this preparation (18) contained both AoG-specific binding and nicking activities. In this paper, an AoG-specific nicking assay was used to purify MutY protein from a strain harboring the overproducing plasmid pJTW1O-12 (J.-J.T.-W. and A-L.L., unpublished results). A similar purification scheme was applied except that 45% ammonium sulfate was used to precipitate proteins from fraction I (rather than 35%) and an extra Mono S column chromatography step was added after heparin-agarose chromatography (see Materials and Methods). We recovered 28 mg of MutY protein (fractions VI-1 and VI-2; Table 1) from 47 g of overproducing cell paste with about a 16-fold increase in specific activity. Compared with the specific activities of AoG nicking in a crude extract from wild-type cells, this overproducer strain yielded about 1000fold more MutY protein. The Mono S column (1 ml) was overloaded and that resulted in two separated peaks (fractions VI-1 and VI-2). When less protein was applied to the column, only peak 2 was observed (data not shown). The protein compositions in fractions I-VI are shown in Fig. 1. Electrophoresis employed an SDS/12% polyacrylamide gel. In fraction V, MutY (migating as a 36-kDa protein) was purified to >98% homogeneity, and in fractions VI-1 and VI-2, MutY was purified to >99%o homogeneity. Mismatch Nicking Sp ty of MutY Protin. Radicella et al. (16) showed that micA mutants failed to correct both AOG and AoC mismatches in a A heteroduplex assay. While AoG mismatches were repaired well by the MutY-dependent pathway, there was no evidence that cell extracts from wild-type E. coli could act on AoC mismatches by an in vitro repair assay (14, 15). Furthermore, partially purified MutY from wild-type E. coli could not bind or nick AoC-containing DNA (25). Using our concentrated pure MutY protein, we again checked its mismatch specificity. AoG-containing DNAs were the best substrates (Fig. 2, lanes 1 and 3). No detectable nicking activity was found when DNA heteroduplexes containing ToG, ToC, ToT, CoC, AoA, and GoG mispairs were used as substrates (data not shown). However, DNA heteroduplexes containing an A4C or CoA mismatch were nicked after incubation with fraction V (Fig. 2, lanes 2 and 4). As Table 1. Purification of MutY from an overproducing E. coli strain
Protein,
Specific activity,
Recovery, % Fraction mg Step units/mg I 369,000 (100) Streptomycin sulfate 2867 1010 II Ammonium sulfate 43 455,000 111 2,720,000 28 III Phosphoceilulose 21 60 IV 3,717,000 Hydroxylapatite 39 4,615,000 17 V Heparin agarose 12 VI-1 23 Mono S 5,566,000 5 6,600,000 3 VI-2 Mono S Protein connation was measured by the Bradford assay (31), and activity was assayed with a 3' end-labeled 120-mer DNA duplex with AoG mismatch as substrate. One unit of nicking activity is defined as that resulting in cleavage of 1% (0.018 finol) of the labeled DNA in 30 min under the assay conditions.
Biochemistry: Tsai-Wu et al. kDa
1
3
2
4
Proc. Natl. Acad. Sci. USA 89 (1992) 5
6
7
111_-
71_-44
_*
29_18_ 15FIG. 1. SDS/polyacrylamide gel analysis of MutY fractions. The proteins were separated on a 12% polyacrylamide gel in the presence of SDS according to Laemmli (30) and stained with Coomassie blue. Lanes 1-7 are fractions 1 (52 pg), II (40 pg), III (8.3 pg), IV (6.7 Pg), V (5.8 ,ug), VI-1 (5.8 pg), and VI-2 (5.3 kg), respectively. Molecular mass standards (Bethesda Research Laboratories) were run in a parallel lane.
determined by densitometry of the autoradiograph, the nicking activity of MutY on an AoG substrate was 20-fold greater than that on an AoC substrate (Fig. 2, lane 1 vs. lane 2 and lane 3 vs. lane 4). Interestingly, MutY was found to nick only the dA strand but not the dC strand of AoC substrate (Fig. 2, lanes 4 and 5). These results are consistent with the AoC to G-C repair in vivo (16). When the MutY protein purified from wild-type cells was incubated with an AoC heteroduplex DNA at a higher protein concentration, a weak nicking activity was detected (data not shown). Therefore, MutY can act not only on AoG mismatches but also on AoC mismatches, but with lower activity. Copurification of A°G-Specific Nicking and Glycosylase Activities. By using an AoG substrate labeled at the 5' end of the Fraction
V
+
_*
0 0
~0
4o
%..
--
4c*
