Title: Characterization of the Recombinant MutY Homolog, an Adenine DNA Glycosylase, from Yeast Schizosaccharomyces pombe
Abstract: The mutY homolog (SpMYH) gene from a cDNA library of Schizosaccharomyces pombeencodes a protein of 461 amino acids that displays 28 and 31% identity to Escherichia coli MutY and human MutY homolog (MYH), respectively. Expressed SpMYH is able to complement an E. coli mutY mutant to reduce the mutation rate. Similar to E. coli MutY protein, purified recombinant SpMYH expressed in E. coli has adenine DNA glycosylase and apurinic/apyrimidinic lyase activities on A/G- and A/7,8-dihydro-8-oxoguanine (8-oxoG)-containing DNA. However, both enzymes have different salt requirements and slightly different substrate specificities. SpMYH has greater glycosylase activity on 2-aminopurine/G and A/2-aminopurine but weaker activity on A/C thanE. coli MutY. Both enzymes also have different substrate binding affinity and catalytic parameters. Although SpMYH has great affinity to A/8-oxoG-containing DNA as MutY, the binding affinity to A/G-containing DNA is substantially lower for SpMYH than MutY. SpMYH has similar reactivity to both A/G- and A/8-oxoG-containing DNA; however, MutY cleaves A/G-containing DNA about 3-fold more efficiently than it does A/8-oxoG-containing DNA. Thus, SpMYH is the functional eukaryotic MutY homolog responsible for reduction of 8-oxoG mutational effect. The mutY homolog (SpMYH) gene from a cDNA library of Schizosaccharomyces pombeencodes a protein of 461 amino acids that displays 28 and 31% identity to Escherichia coli MutY and human MutY homolog (MYH), respectively. Expressed SpMYH is able to complement an E. coli mutY mutant to reduce the mutation rate. Similar to E. coli MutY protein, purified recombinant SpMYH expressed in E. coli has adenine DNA glycosylase and apurinic/apyrimidinic lyase activities on A/G- and A/7,8-dihydro-8-oxoguanine (8-oxoG)-containing DNA. However, both enzymes have different salt requirements and slightly different substrate specificities. SpMYH has greater glycosylase activity on 2-aminopurine/G and A/2-aminopurine but weaker activity on A/C thanE. coli MutY. Both enzymes also have different substrate binding affinity and catalytic parameters. Although SpMYH has great affinity to A/8-oxoG-containing DNA as MutY, the binding affinity to A/G-containing DNA is substantially lower for SpMYH than MutY. SpMYH has similar reactivity to both A/G- and A/8-oxoG-containing DNA; however, MutY cleaves A/G-containing DNA about 3-fold more efficiently than it does A/8-oxoG-containing DNA. Thus, SpMYH is the functional eukaryotic MutY homolog responsible for reduction of 8-oxoG mutational effect. 7,8-dihydro-8-oxoguanine MutY homolog apurinic/apyrimidinic inosine nebularine 2-aminopurine kilobase(s) polymerase chain reaction isopropyl-1-thio-β-d-galactopyranoside 8-oxoG glycosylase helix-hairpin-helix. Cellular and organism aging have been correlated with accumulated DNA damage (1Ames B.N. Gold L.S. Mutat. Res. 1991; 250: 3-16Crossref PubMed Scopus (681) Google Scholar, 2Kasai H. Nishimura S. Sies H. Oxidative Stress: Oxidants and Antioxidants. Academic Press, London1991: 99-116Google Scholar). Oxygen is metabolized inside the cell by a series of one-electron reductions with the generation of reactive and potentially damaging intermediates called reactive oxygen species (3Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Oxoford University Press, New York1989Google Scholar). The frequency of oxidative damage to DNA has been estimated at 104 lesions/cell/day in humans (4Fraga C.G. Shigenaga M.K. Park J.-W. Degan P. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4533-4537Crossref PubMed Scopus (985) Google Scholar). 8-Oxo-7,8-dihydrodeoxyguanine (8-oxoG or GO1) is one of the most stable products of oxidative DNA damage. The formation of GO in DNA, if not repaired, can lead to misincorporation of A opposite to the GO lesion and result in G:C to T:A transversions (5Moriya M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1122-1126Crossref PubMed Scopus (431) Google Scholar, 6Moriya M. Ou C. Bodepudi V. Johnson F. Takeshita M. Grollman A.P. Mutat. Res. 1991; 254: 281-288Crossref PubMed Scopus (325) Google Scholar, 7Wood M.L. Dizdaroglu M. Gajewski E. Essigmann J.M. Biochemistry. 1990; 29: 7024-7032Crossref PubMed Scopus (691) Google Scholar, 8Cheng K.C. Cahill D.S. Kasai H. Nishimura S. Loeb L.A. J. Biol. Chem. 1991; 267: 166-172Abstract Full Text PDF Google Scholar). InEscherichia coli, a family of enzymes, MutY, MutM, and MutT, is involved in defending against the mutagenic effects of GO lesions (9Michaels M.L. Miller J.H. J. Bacteriol. 1992; 174: 6321-6325Crossref PubMed Scopus (609) Google Scholar, 10Tchou J. Grollman A.P. Mutat. 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Chem. 1994; 269: 18814-18820Abstract Full Text PDF PubMed Google Scholar). TheE. coli MutY is an adenine glycosylase that is responsible for the correction of A/GO as well as A/G and A/C mismatches (9Michaels M.L. Miller J.H. J. Bacteriol. 1992; 174: 6321-6325Crossref PubMed Scopus (609) Google Scholar,17Au K.G. Cabrera M. Miller J.H. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9163-9166Crossref PubMed Scopus (116) Google Scholar, 18Lu A-L. Chang D.-Y. Genetics. 1988; 118: 593-600Crossref PubMed Google Scholar, 19Lu A-L. Chang D.-Y. Cell. 1988; 54: 805-812Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 20Michaels M.L. Cruz C. Grollman A.P. Miller J.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7022-7025Crossref PubMed Scopus (548) Google Scholar, 21Radicella J.P. Clark E.A. Fox M.S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9674-9678Crossref PubMed Scopus (97) Google Scholar, 22Su S.-S. Lahue R.S. Au K.G. Modrich P. J. Biol. Chem. 1988; 263: 6829-6835Abstract Full Text PDF PubMed Google Scholar). MutY removes misincorporated adenines paired with GO lesions and reduces the GO mutational effects. Recent results show that MutY and the N-terminal catalytic domain can be trapped in a stable covalent enzyme-DNA intermediate in the presence of sodium borohydride (23Lu A-L. Yuen D.S. Cillo J. J. Biol. Chem. 1996; 271: 24138-24143Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 24Manuel R.C. Lloyd R.S. Biochemistry. 1997; 36: 11140-11152Crossref PubMed Scopus (78) Google Scholar, 25Gogos A. Cillo J. Clarke N.D. Lu A-L. Biochemistry. 1996; 35: 16665-16671Crossref PubMed Scopus (82) Google Scholar) and support the hypothesis that MutY contains both DNA glycosylase and AP lyase activities. MutY homologous (MYH) activities have been identified in human HeLa (26Yeh Y.-C. Chang D.-Y. Masin J. Lu A-L. J. Biol. Chem. 1991; 266: 6480-6484Abstract Full Text PDF PubMed Google Scholar) and calf thymus (27McGoldrick J.P. Yeh Y.-C. Solomon M. Essigmann J.M. Lu A-L. Mol. Cell. Biol. 1995; 15: 989-996Crossref PubMed Google Scholar) extracts. Both human and calf MYH systems share similar features with the E. coli mutY-dependent pathway: mismatch specificities to A/G, A/C, and A/GO, and cleavage of the A but not G strand. Recently, a human cDNA of putative hMYH was cloned and its open reading frame predicts a 60-kDa protein (28Slupska M.M. Baikalov C. Luther W.M. Chiang J.-H. Wei Y.-F. Miller J.H. J. Bacteriol. 1996; 178: 3885-3892Crossref PubMed Scopus (327) Google Scholar), which is in good agreement with the size of a band detected in HeLa extracts with MutY antibodies (27McGoldrick J.P. Yeh Y.-C. Solomon M. Essigmann J.M. Lu A-L. Mol. Cell. Biol. 1995; 15: 989-996Crossref PubMed Google Scholar). hMYH shares high homology with the E. coli MutY protein (28Slupska M.M. Baikalov C. Luther W.M. Chiang J.-H. Wei Y.-F. Miller J.H. J. Bacteriol. 1996; 178: 3885-3892Crossref PubMed Scopus (327) Google Scholar). However, no enzyme activity has been reported for the protein encoded by this open reading frame. Here, we report the cloning and expression of the MYH gene from Schizosaccharomyces pombe. Expression of SpMYH suppresses the spontaneous mutation rate of E. coli mutY mutant strains. Like E. coli MutY, purified recombinant SpMYH has both adenine glycosylase and AP lyase activities on A/G and A/GO mismatches. Defined oligonucleotides containing various purines were used to examine the substrate specificity. SpMYH has slightly different substrate specificity from that of E. coli MutY protein. SpMYH has greater glycosylase activity on 2-aminopurine (2AP)/G and A/2AP but weaker activity on A/C than doesE. coli MutY. Both enzymes also have different substrate binding affinity and catalytic parameters. These results suggest that SpMYH is a functional eukaryotic homolog of the bacterial MutY. The high homology of MutY homologs among different organisms suggests important roles in their cellular functions. According to the published genomic sequence of S. pombe (accession no.Z69240), the putative mutY homolog (MYH) sequence contains two introns and codes for a 461-residue protein. To clone theS. pombe MYH gene, we synthesized two PCR primers, Chang 219 (5′-GGAGATATACATATGTCGGATTCAAATCATTC-3′) and Chang 220 (5′-GCAGCCGGATCCTTAGCACTCTGCTTTCGT-3′). Chang 219 and Chang 220 anneal at the first six and last six codons of the predicted coding sequence for SpMYH, respectively. DNA prepared from an S. pombe cDNA library in pGADGH (kindly provided by D. Beach, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) was used as a template for the PCR reactions. PCR reactions (100 μl) contained 20 ng of plasmid DNA, 100 pmol of each primer, 1.5 mmMgCl2, and 5 units of Pfu DNA polymerase (Stratagene). The reactions were performed as follows: 95 °C (1.0 min), 58 °C (1.5 min) and 72 °C (1.5 min) for 30 cycles and a final cycle with extension time at 72 °C for 4 min. The 1.4-kb PCR product was purified using the Wizard PCR clean-up kit (Promega), digested with NdeI and BamHI, ligated intoNdeI- and BamHI-digested pET11a, and transformed into DH5α cells. Sequence analysis of one such clone (pSP11a-2.3) revealed that it contained several mutations. To obtain a mutation-free SpMYH clone, the 1.4-kb PCR product was labeled and used as a probe to screen the S. pombe cDNA library in pGADGH by colony hybridization. Out of 6 × 106 colonies screened, two contained theSpMYH sequence. Subsequent restriction and PCR analysis indicated that the MYH cDNA was within a 1.6-kbBamHI fragment in one of the clones. This fragment was isolated and transferred to pUC19 to generate pSPMYH19. Based on the restriction map of pSPMYH19, the 1.1-kb EcoRI fragment containing the C-terminal domain of SpMYH and 21 base pairs of pUC19 sequence was isolated and ligated with the 5.76-kb fragment of pSP11a-2.3 that contained the vector pET11a and the N-terminal portion of SpMYH to yield the plasmid pSPMYH11a-4. The SpMYHsequence of pSPMYH11a-4 (accession no. AF053340) was exactly the same as the predicted sequence for the S. pombe MYH cDNA. The recombinant expressed a 52-kDa protein in GBE943(DE3) (lacIp4000(LacIq)lacZp4008(Lac L8)srlC-300::Tn10λ−IN(rrD-rrnE)1micA68::Tn10Kan) cells following induction with 0.4 mmisopropyl-1-thio-β-d-galactopyranoside (IPTG). The expression host, GBE943 with λDE3 lysogen, was constructed according to the procedures described by Invitrogen. Independent overnight cultures of each strain were grown to an A 590 of 0.7 in LB medium containing 50 mg/ml ampicillin when necessary. After 2.5 h of induction by the addition of 0.1 mm IPTG, 0.1 ml of cells from each culture was plated onto LB agar containing 0.1 mg/ml rifampicin. The cell titer of each culture was determined by plating a 10−6 dilution onto LB agar. The ratio of Rifrcells to total cells was the mutation rate. Eighteen liters of E. coliGBE943/De3 cells harboring overproduction plasmid pSPMYH11a-4 were cultured to an A 590 of 0.7 in LB broth containing 50 mg/ml ampicillin at 37 °C. The cells were induced by adding IPTG to 0.4 mm and cultured overnight at 28 °C and harvested by centrifugation. All column chromatography was conducted in a Waters 650 FPLC system at 4 °C, and centrifugation was done at 16,5000 × g for 30 min. Cells (54 g of cell paste) were resuspended in 200 ml of buffer A (20 mmpotassium phosphate (pH 7.4), 1 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride) containing 50 mm KCl and disrupted with a bead beater (Biospec Products, Bartlesville, OK) using 0.1-mm glass beads. The cell debris was removed by centrifugation, and the supernatant was treated with 5% streptomycin sulfate. After stirring for 30 min, the solution was centrifugated, and the supernatant was collected as fraction I (595 ml). Ammonium sulfate (134 g) was added to fraction I, and the protein was precipitated for 1 h. After centrifugation, 126 g of ammonium sulfate was added to the supernatant and the protein pellets collected by centrifugation were resuspended in 50 ml of buffer A containing 50 mm KCl and dialyzed against two changes of 3 liters of the same buffer for 4 h each. The dialyzed protein sample was diluted 2-fold with buffer A containing 50 mmKCl as fraction II (140 ml). Fraction II was loaded onto a 50-ml phosphocellulose column, which had been equilibrated with buffer A containing 0.05 m KCl. After washing with 100 ml of equilibration buffer, proteins were eluted with a 400-ml linear gradient of KCl (0.05–0.6 m) in buffer A. Fractions that eluted between 0.2 and 0.4 m KCl were pooled (fraction III, 50 ml). Fraction III was loaded onto a 30-ml hydroxylapatite column equilibrated with buffer B (0.01 m potassium phosphate (pH 7.4), 10 mm KCl, 0.5 mm dithiothreitol, 0.1 mm EDTA, 0.1 mm phenylmethylsulfonyl fluoride). After washing with 60 ml of equilibration buffer, the flow-through and early elution fractions were pooled and dialyzed against buffer A containing 0.05 m KCl and 10% (v/v) glycerol for 2 h (fraction IV, 53 ml). Fraction IV was loaded onto a 5-ml heparin-agarose column equilibrated with buffer A containing 0.05m KCl and 10% glycerol. After washing with 10 ml of equilibration buffer, the column was developed with a 50-ml linear gradient of KCl (0.05–0.6 m) in buffer A with 10% glycerol. Fractions containing the MYH nicking activity, which eluted between 0.15 and 0.3 m KCl, were pooled and dialyzed against 2 liters of buffer A containing 0.05 m KCl and 10% glycerol to yield fraction V (78 ml). Fraction V was then applied to a 1-ml Mono S column that had been equilibrated in buffer A containing 0.05 m KCl and 10% glycerol. After washing with 20 ml of equilibration buffer, the column was eluted with a step gradient of 0.1, 0.3, and 0.5 m KCl in 20 ml of buffer A each with 10% glycerol. Fractions containing the MYH nicking activity, which eluted between 0.1 and 0.3 m KCl, were pooled (fraction VI, 8.5 ml), divided into small aliquots, and stored at −80 °C. Cleavage of A/G-containing 44-mer DNA was assayed during the purification of the recombinant SpMYH enzyme. One unit of activity is defined as that resulting in cleavage of 0.018 fmol of labeled DNA in 30 min at 30 °C. Protein concentration was determined by the Bradford method (29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214435) Google Scholar). Oligonucleotides of 19-mer and 40-mer containing base mismatches were labeled as described by Luet al. (30Lu A-L. Tsai-Wu J.-J. Cillo J. J. Biol. Chem. 1995; 270: 23582-23588Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The nicking activity of SpMYH, which is the combined action of the glycosylase and AP lyase activities, was assayed similarly as described (31Tsai-Wu J.-J. Liu H.-F. Lu A-L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8779-8783Crossref PubMed Scopus (130) Google Scholar). The standard reaction mixture contained 10 mm Tris-HCl (pH 7.6), 0.5 mmdithiothreitol, 0.5 mm EDTA, 1.45% glycerol, 50 μg/ml bovine serum albumin, and 1.8 fmol of labeled DNA in a total volume of 20 μl. SpMYH protein, diluted in a buffer containing 20 mm potassium phosphate (pH 7.4), 1.5 mmdithiothreitol, 0.1 mm EDTA, 50 mm KCl, 200 μg/ml bovine serum albumin, and 50% glycerol, was added to the reaction mixture and incubated at 30 °C for 30 min. The reaction products were analyzed on 8 or 14% polyacrylamide DNA sequencing gels. Kinetic analyses were performed using a concentration range of DNA substrates with 0.4 nm SpMYH. Following autoradiography, bands corresponding to cleavage products and intact DNA were excised from the gel and quantified by liquid scintillation counting.K m and V max values were obtained from results of three experiments by a computer-fitted curve generated by the Enzfitter program (32Leatherbarrow R.J. Enzfitter: A Non-linear Regression Analysis Program for IBM PC. Elsevier Science Publishers BV, Amsterdam1987Google Scholar). The binding of SpMYH to various oligonucleotides was assayed by gel retardation. 3′ end-labeled 44-bp or 20-bp oligonucleotides (1.8 fmol) were incubated with various concentrations of SpMYH in a 20-μl binding buffer containing 10 mm Tris-HCl (pH 7.6), 0.5 mm dithiothreitol, 40 mm NaCl, 5 mm EDTA, 1.15% glycerol, 50 μg/ml bovine serum albumin, and 5 ng of poly(dI-dC) at 30 °C for 30 min. Protein-DNA complexes were analyzed on 8% polyacrylamide gels in 50 mm Tris borate (pH 8.3) and 1 mm EDTA as described previously (19Lu A-L. Chang D.-Y. Cell. 1988; 54: 805-812Abstract Full Text PDF PubMed Scopus (63) Google Scholar). The apparent dissociation constants (K d values) of SpMYH and DNA were determined using a range of protein concentrations. Following autoradiography, bands corresponding to enzyme-bound and free DNA were excised from the gel and quantified by liquid scintillation counting. K dvalues were obtained from results of three experiments by a computer-fitted curve generated by the Enzfitter program (32Leatherbarrow R.J. Enzfitter: A Non-linear Regression Analysis Program for IBM PC. Elsevier Science Publishers BV, Amsterdam1987Google Scholar). Reactions were carried out as described in the SpMYH cleavage assay except that the reactions were performed in the presence of NaBH4. A NaBH4 stock solution was freshly prepared immediately prior to use. After incubation at 30 °C for 30 min, SDS dye was added to the samples, which were heated at 90 °C for 2 min and separated on a 12% polyacrylamide gel in the presence of SDS according to Laemmli (33Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206620) Google Scholar), and the gel was dried and autoradiographed. The SpMYH gene encodes a protein of 461 amino acid residues that displays 28 and 31% identity and 59 and 62% conservation to the E. coli MutY and human MYH, respectively, by comparison using an ALIGN program. The eukaryotic MYH sequences contain extra amino acid stretches at the N- and C-terminal regions as compared with the bacterial MutY sequences. The amino acid sequences of the N-terminal part of the SpMYH share significant homology to other DNA glycosylases including E. coli endonuclease III (endo III) (Fig. 1). Two regions of highest similarity shared among these enzymes can be identified in the known three-dimensional structure of E. coli endo III (34Kuo C.-F. McRee D.E. Fisher C.L. O'Haandley S.F. Cunningham R.P. Tainer J.A. Science. 1992; 258: 434-440Crossref PubMed Scopus (277) Google Scholar, 35Thayer M.M. Ahern H. Xing D. Cunningham R.P. Tainer J.A. EMBO J. 1995; 14: 4108-4120Crossref PubMed Scopus (435) Google Scholar): the helix-hairpin-helix domain (residues 147–181 of SpMYH) and the iron-sulfur domain (residues 213–242 of SpMYH). There are also distinct residues (shaded in gray boxes in Fig. 1) in the MutY family that are different from that of endo III family. Interestingly, when these residues are placed on the structure of E. coli endo III, they are located at the edge of the cleft between the iron-sulfur domain and the six antiparallel α-helices. To demonstrate that the open reading frame of putativeSpMYH gene encodes a functional MYH protein, we have expressed this Sp-cDNA under the control of the T7 promoter in pET11a in E. coli. E. coli GBE943 (mutY) harboring the plasmid pSPMYH11a-4 were induced to express the SpMYH protein by addition of IPTG to the growth medium, and the mutation rate was measured. As shown in Table I,mutY mutant (GBE943/DE3) exhibited a 40-fold higher mutation rate than the wild-type E. coli. GBE943/DE3 cells expressing SpMYH had mutation rates almost as low as the wild-type cell, whereas the vector (pET11a) alone had no effect on the mutation rate (Table I). Expression of SpMYH in wild-type E. coli cells caused a slightly lower mutation rate than cells with vector alone (Table I).Table IMutation rate of E. coli mutY mutant expressing MYH from S. pombeStrainMutation rate RifR colonies/108 cells-FoldGBE791/DE3 (wt)1.81GBE791/DE3/pET11a1.50.8GBE791/DE3/pSPMYH11a-40.60.3GBE943/DE3 (mutY)76.242GBE943/DE3/pET11a53.230GBE943/DE3/pSPMYH11a-45.23 Open table in a new tab To further demonstrate that the putative SpMYH encodes a functional MYH protein, we purified the recombinant SpMYH from the overproducing E. coli GBE943(DE3) strain harboring the plasmid pSPMYH11a-4. The SpMYH protein was purified by ammonium sulfate precipitation and phosphocellulose, hydroxylapatite, heparin-agarose, and Mono S chromatographic separation. We recovered 19 mg of SpMYH protein from 54 g of cell paste with about an 18-fold increase in specific activity. The purity of the products at different stages of the procedure is illustrated in Fig. 2. As judged on a 10% SDS-polyacrylamide gel, the protein was purified to a very high degree. The mobility of SpMYH in the gel matched the predicted size (52 kDa). SpMYH appears as a monomer because it was eluted at a molecular mass of about 45 kDa through a gel-filtration column (Superose 12) (data not shown). When SpMYH was initially assayed in the MutY buffer (20 mm Tris-HCl, pH 7.6, 1 mm dithiothreitol, 1 mm EDTA, 80 mm NaCl, and 2.9% glycerol), there was little cleavage activity (Fig. 3, lane 4). This prompted us to find the optimal conditions for SpMYH. The SpMYH glycosylase activity was reduced by adding 40 mm NaCl to the reaction buffer (Fig. 3, lane 8) and was abolished at NaCl concentration higher than 80 mm (Fig. 3, lanes 9 and 10). SpMYH cleavage activity was not inhibited by 8 mm EDTA (Fig. 3, lane 13) but was abolished by 32 mm EDTA (Fig. 3, lane 15). The effects of salt and EDTA on the SpMYH binding to 20-mer DNA containing an A/G mismatch were similar to that on the SpMYH cleavage except 40 mm NaCl had little effect on binding (data not shown). Therefore, the low SpMYH activity in MutY buffer is due to the presence of 80 mm NaCl. As shown in Fig. 4, SpMYH can cleave a 20-mer oligonucleotide containing an A/G mismatch in a dose-dependent manner. Heating the samples at 90 °C for 2 min before loading to the sequencing gel enhanced the cleavage activity (Fig. 4, compare A and B). However, further treatment of the products with piperidine at 90 °C (a condition promotes β-elimination) did not significantly increase the extent of cleavage (Fig. 4, compare B and C). Thus, SpMYH does contain intrinsic AP lyase activity, although it is not strictly coupled to the glycosylase activity. These properties of SpMYH are similar to the E. coli MutY protein, which contains both DNA glycosylase and AP lyase activities. If SpMYH has an AP lyase activity and uses the similar mechanism like MutY (23Lu A-L. Yuen D.S. Cillo J. J. Biol. Chem. 1996; 271: 24138-24143Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 24Manuel R.C. Lloyd R.S. Biochemistry. 1997; 36: 11140-11152Crossref PubMed Scopus (78) Google Scholar, 25Gogos A. Cillo J. Clarke N.D. Lu A-L. Biochemistry. 1996; 35: 16665-16671Crossref PubMed Scopus (82) Google Scholar), an imino intermediate should be reduced by NaBH4 to form a stable covalent protein-DNA complex. Thus, DNA containing an A/G mismatch was incubated with SpMYH in the presence of different concentrations of NaBH4. As shown in Fig. 5, SpMYH can be trapped in two covalently linked protein-DNA complexes as a doublet in the presence of NaBH4 although not as efficiently as MutY. The reason for the formation of two complexes is not quite clear. The optimal trapping concentration of NaBH4 is about 20–30 mm (Fig. 5 A) (less trapping is observed at 10 mmNaBH4 with data not shown). The covalent complexes of SpMYH and DNA migrated slower than the MutY-DNA complex due to the larger size of SpMYH protein (the molecular mass of SpMYH is 52 kDa and that of MutY is 39 kDa). At 100 mm NaBH4, E. coli MutY can be trapped efficiently but the trapping ability of SpMYH is minimal (data not shown). The effect of NaBH4 is consistent with the inhibition of SpMYH cleavage activity by 80 mm NaCl that is present in the MutY reaction (Fig. 3). In the presence of 30 mm NaBH4, DNA containing an A/G mismatch was tested in the trapping assay with increasing amount of SpMYH protein (Fig. 5 B). At enzyme:DNA molar ratios ranging from 10 (Fig. 5 B, lane 3) to 320 (Fig. 5 B, lane 8), covalent complexes were detected. Because Schiff's base formation is an important criteria for the class of DNA glycosylase with AP lyase activity, our results strongly suggest that SpMYH protein possesses AP lyase activity. We have shown that E. coli MutY can cleave several mismatches with different efficiencies (30Lu A-L. Tsai-Wu J.-J. Cillo J. J. Biol. Chem. 1995; 270: 23582-23588Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). To compare the SpMYH with MutY, we tested the glycosylase activity on different mismatches. As shown in Fig. 6, both enzymes cleave A/G, N/G, and A/GO in a similar order and have very weak or no cleavage on I/G, A/I, C/GO, and C/G. However, SpMYH has greater activity on 2AP/G and A/2AP but weaker activity on A/C than E. coli MutY. Because SpMYH and MutY have different glycosylase activities on different substrates, we then determined the apparent dissociation constants (K d) of SpMYH from different mismatches. The apparent dissociation constants (K dvalues) of SpMYH and DNAs were determined using a range of protein concentrations with a fixed DNA concentration (90 pm). Representative autoradiograms of the binding assays and the corresponding binding curves for SpMYH to A/G- and A/GO-containing 44-mer DNA are shown in Fig. 7. When the concentration of SpMYH protein was higher than 50 nm, an extra slower migrating complex was observed in binding assays with A/G-44 DNA (Fig. 7 A). SpMYH binding is saturated below 70% of A/GO-44 DNA at the highest MutY concentration tested (Fig. 7 D). As shown in Table II, SpMYH has great affinity to A/GO-containing DNA as does MutY, but the binding affinity to A/G-containing DNA is substantially lower for SpMYH than MutY. The difference in the binding affinity of SpMYH with A/G-44 and A/GO-44 is 155-fold and with A/G-20 and A/GO-20 is 700-fold. The difference in the binding affinity of MutY with A/G-44 and A/GO-44 is 13-fold and with A/G-20 and A/GO-20 is 80-fold. SpMYH has higher nonspecific binding to 44-mer homoduplex than MutY. Therefore, SpMYH has only a 4–7-fold higher binding affinity to A/G mismatch than C/G pair, whereas MutY has a 70–175-fold higher binding affinity to A/G mismatch than C/G pair. As shown above, SpMYH has greater cleavage activity on 2AP/G and A/2AP than MutY; however, the binding affinities of SpMYH to these two mismatches were not greater than that of MutY.Table IIApparent dissociation constant (Kd) of SpMYH and E. coli MutY for mismatch-containing DNADNA duplexSpMYH K dMutYK dnmnmA/G-4419.1 ± 2.51.8 ± 0.3A/GO-440.123 ± 0.0150.141 ± 0.008C/G-4487.4 ± 10.1315 ± 49A/G-20168 ± 265.3 ± 0.5A/GO-200.239 ± 0.0150.066 ± 0.013C/G-20715 ± 89370 ± 802AP/G-20163 ± 1012 ± 3A/2AP-20812 ± 9894 ± 14Binding constants for MutY are derived from Luet al. (30Lu A-L. Tsai-Wu J.-J. Cillo J. J. Biol. Chem. 1995; 270: 23582-23588Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Open table in a new tab Binding constants for MutY are derived from Luet al. (30Lu A-L. Tsai-Wu J.-J. Cillo J. J. Biol. Chem. 1995; 270: 23582-23588Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The cleavage efficiencies of 20-mer oligonucleotide containing A/G or A/GO by SpMYH and MutY were compared (Table III). As measured at 0.4 nm protein concentration, the K m values for SpMYH on both substrates were slightly higher than that of MutY. The turnover number (K cat) for SpMYH on an A/G 20-mer is 2 times lower than that of the MutY. SpMYH has similar reactivity (K cat/K m) to both A/G- and A/GO-containing DNA; however, MutY cleaves A/G-containing DNA about 3-fold more efficiently than it does A/GO-containing DNA. When 44-mer DNA substrates were tested for cleavage, SpMYH also displays similar specificity constants (K cat/K m) with both A/G and A/GO mismatches. The specificity constants of SpMYH with 44-mer is 3–4 times higher than with 20-mer DNA substrates.Table IIIKinetic parameters of SpMYH and E. coli MutYEnzymeDNAK mV maxk catk cat·