Flipping Duplex DNA Inside Out

The Escherichia coli MutY adenine glycosylase plays a critical role in repairing mismatches in DNA between adenine and the oxidatively damaged guanine base 8-oxoguanine. Crystallographic studies of the catalytic core domain of MutY show that the scissile adenine is extruded from the DNA helix to be bound in the active site of the enzyme (Guan, Y., Manuel, R. C., Arvai, A. S., Parikh, S. S., Mol, C. D., Miller, J. H., Lloyd, S., and Tainer, J. A. (1998) Nat. Struct. Biol. 5, 1058–1064). However, the structural and mechanistic bases for the recognition of the 8-oxoguanine remain poorly understood. In experiments using a single-stranded 8-bromoguanine-containing synthetic oligodeoxyribonucleotide alone and in a duplex construct mismatched to an adenine, we observed UV cross-linking between MutY and the 8-bromoguanine probe. We further observed enhanced cross-linking in the single strand experiments, suggesting that neither the duplex context nor the mismatch with adenine is required for recognition of the 8-oxoguanine moiety. Stopped-flow fluorescence studies using 2-aminopurine-containing oligodeoxyribonucleotides further revealed the sequential extrusion of the 8-oxoguanine at 108 s−1followed by the adenine at 16 s−1. A protein isomerization step following base flipping at 1.9 s−1 was also observed and is postulated to provide additional stabilization of the extruded adenine thereby facilitating its capture by the active site for excision. The Escherichia coli MutY adenine glycosylase plays a critical role in repairing mismatches in DNA between adenine and the oxidatively damaged guanine base 8-oxoguanine. Crystallographic studies of the catalytic core domain of MutY show that the scissile adenine is extruded from the DNA helix to be bound in the active site of the enzyme (Guan, Y., Manuel, R. C., Arvai, A. S., Parikh, S. S., Mol, C. D., Miller, J. H., Lloyd, S., and Tainer, J. A. (1998) Nat. Struct. Biol. 5, 1058–1064). However, the structural and mechanistic bases for the recognition of the 8-oxoguanine remain poorly understood. In experiments using a single-stranded 8-bromoguanine-containing synthetic oligodeoxyribonucleotide alone and in a duplex construct mismatched to an adenine, we observed UV cross-linking between MutY and the 8-bromoguanine probe. We further observed enhanced cross-linking in the single strand experiments, suggesting that neither the duplex context nor the mismatch with adenine is required for recognition of the 8-oxoguanine moiety. Stopped-flow fluorescence studies using 2-aminopurine-containing oligodeoxyribonucleotides further revealed the sequential extrusion of the 8-oxoguanine at 108 s−1followed by the adenine at 16 s−1. A protein isomerization step following base flipping at 1.9 s−1 was also observed and is postulated to provide additional stabilization of the extruded adenine thereby facilitating its capture by the active site for excision. The effect of cellular damages by reactive oxygen species in carcinogenesis and aging is well documented (1Ames B.N. Shigenaga M.K. Hagen T.M. Biochim. Biophys. Acta. 1995; 1271: 165-170Crossref PubMed Scopus (338) Google Scholar, 2Shigenaga M.K. Hagen T.M. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10771-10778Crossref PubMed Scopus (1818) Google Scholar, 3Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5381) Google Scholar). Even in the absence of external oxidative stress, normal metabolic processes produce oxidative damages to DNA (4Beckman K.B. Ames B.N. J. Biol. Chem. 1997; 272: 19633-19636Abstract Full Text Full Text PDF PubMed Scopus (839) Google Scholar, 5Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4299) Google Scholar, 6Demple B. Harrison L. Annu. Rev. Biochem. 1994; 63: 915-948Crossref PubMed Scopus (1289) Google Scholar) requiring repair. Oxidative damage of DNA bases alters their base pairing properties (5Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4299) Google Scholar, 7Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.1995: 159-160Google Scholar) thereby interfering with replication and transcription (8Verdine G.L. Bruner S.D. Chem. Biol. 1997; 4: 329-334Abstract Full Text PDF PubMed Scopus (93) Google Scholar). A predominant lesion found in DNA exposed to reactive oxygen species is 8-oxoguanine, which is especially deleterious due to its ability to form a stable Hoogsteen base pair with adenine in addition to the canonical Watson-Crick base pair with cytosine (9Lipscomb L.A. Peek M.E. Morningstar M.L. Verghis S.M. Miller E.M. Rich A. Essigmann J.M. Williams L.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 719-723Crossref PubMed Scopus (219) Google Scholar, 10Gannett P.M. Sura T.P. Chem. Res. Toxicol. 1993; 6: 690-700Crossref PubMed Scopus (52) Google Scholar). The facile by DNA polymerase misincorporation of an adenine across from the 8-oxoguanine (11Haracska L. Yu S.L. Johnson R.E. Prakash L. Prakash S. Nat. Genet. 2000; 25: 458-461Crossref PubMed Scopus (301) Google Scholar) results in a mutagenic adenine:8-oxoguanine mismatch, a site where further replication prior to repair would lead to C → A or G → T transversions. In Escherichia coli, the MutY adenine glycosylase (MutY) 1The abbreviations used are: MutYE. coli MutY adenine glycosylaseP2-aminopurineB8-bromoguanineO8-oxoguaninettopbbottom1The abbreviations used are: MutYE. coli MutY adenine glycosylaseP2-aminopurineB8-bromoguanineO8-oxoguaninettopbbottom plays a critical role in preventing mutations stemming from oxidative damages to DNA by excising the adenine from the adenine:8-oxoguanine mismatch. E. coli MutY adenine glycosylase 2-aminopurine 8-bromoguanine 8-oxoguanine top bottom E. coli MutY adenine glycosylase 2-aminopurine 8-bromoguanine 8-oxoguanine top bottom Like all DNA-nucleotide-modifying enzymes, including DNA methylases, base-excision repair glycosylases, and endonucleases (12Cheng X. Blumenthal R.M. Structure. 1996; 4: 639-645Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 13Lloyd R.S. Cheng X. Biopolymers. 1997; 44: 139-151Crossref PubMed Google Scholar, 14Roberts R.J. Cheng X. Annu. Rev. Biochem. 1998; 67: 181-198Crossref PubMed Scopus (306) Google Scholar, 15Hornby D.P. Ford G.C. Curr. Opin. Biotechnol. 1998; 9: 354-358Crossref PubMed Scopus (19) Google Scholar), MutY faces the 2-fold task of recognizing and accessing chemical moieties on DNA bases hidden within the double helix of duplex DNA. These enzymes have evolved an elegantly simple strategy for exposing their targets by rotating the phosphodiester bonds surrounding the nucleotide, causing the target base to be flipped out of the DNA helix (16Lau A.Y. Scharer O.D. Samson L. Verdine G.L. Ellenberger T. Cell. 1998; 95: 249-258Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 17Morikawa K. Shirakawa M. Mutat. Res. 2000; 460: 257-275Crossref PubMed Scopus (26) Google Scholar, 18O'Gara M. Horton J.R. Roberts R.J. Cheng X. Nat. Struct. Biol. 1998; 5: 872-877Crossref PubMed Scopus (106) Google Scholar, 19Parikh S.S. Mol C.D. Slupphaug G. Bharati S. Krokan H.E. Tainer J.A. EMBO J. 1998; 17: 5214-5226Crossref PubMed Scopus (416) Google Scholar, 20Hosfield D.J. Guan Y. Haas B.J. Cunningham R.P. Tainer J.A. Cell. 1999; 98: 397-408Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 21Slupphaug G. Mol C.D. Kavli B. Arvai A.S. Krokan H.E. Tainer J.A. Nature. 1996; 384: 87-92Crossref PubMed Scopus (483) Google Scholar). Using the E. coli uracil-DNA glycosylase, we have recently reported the first kinetic mechanism of a base-flipping enzyme, defining for the first time the correct temporal sequence of events at the enzyme active site during catalysis (22Wong, I., Lundquist, A. J., Bernards, A. S., and Mosbaugh, D. W. (2002) J. Biol. Chem. 277, in pressGoogle Scholar). Following initial nonspecific binding, the DNA backbone is scanned to locate the site. A protein-induced distortion of the DNA helix at the target site causes the target nucleotide to become mobile, capable of rapid and reversible extrusion from the DNA duplex. The efficient capture of the extruded base, however, requires a protein conformation change, which inserts the side chain of Leu191 into the DNA duplex at the site vacated by the extruded base. This ratchet-like movement by the protein prevents the flipped-out base from returning into the DNA duplex, thereby driving it into the active site of the enzyme to be excised. Crystallographic studies on the catalytic core domain of MutY reveals an active site binding pocket for an extruded adenine (23Guan Y. Manuel R.C. Arvai A.S. Parikh S.S. Mol C.D. Miller J.H. Lloyd S. Tainer J.A. Nat. Struct. Biol. 1998; 5: 1058-1064Crossref PubMed Scopus (294) Google Scholar), suggesting that MutY also uses a similar base-flipping reaction mechanism. However, MutY is unique among base-excision repair enzymes in recognizing a mismatch between a damaged 8-oxoguanine and a normal adenine while exclusively catalyzing the removal of the undamaged base (24Chmiel N.H. Golinelli M.P. Francis A.W. David S.S. Nucleic Acids Res. 2001; 29: 553-564Crossref PubMed Scopus (77) Google Scholar, 25Lu A.L. Methods Mol. Biol. 2000; 152: 3-16PubMed Google Scholar, 26Porello S.L. Leyes A.E. David S.S. Biochemistry. 1998; 37: 14756-14764Crossref PubMed Scopus (175) Google Scholar). Unlike other base-flipping enzymes, the recognition target of MutY must extend beyond the adenine being excised; thus, the characteristic base-flipping reaction mechanism where the extruded base is also the site of the chemical reactions cannot fully explain the mode of target recognition by MutY. Biochemical evidence shows that the C-terminal domain missing from the crystal structure of the catalytic core domain is required for 8-oxoguanine recognition (27Gogos A. Cillo J. Clarke N.D. Lu A.L. Biochemistry. 1996; 35: 16665-16671Crossref PubMed Scopus (82) Google Scholar, 28Li X. Lu A.L. Nucleic Acids Res. 2000; 28: 4593-4603Crossref PubMed Scopus (15) Google Scholar, 29Noll D.M. Gogos A. Granek J.A. Clarke N.D. Biochemistry. 1999; 38: 6374-6379Crossref PubMed Scopus (112) Google Scholar). NMR studies of this missing domain reveal significant structural homologies with the known structure of MutT (29Noll D.M. Gogos A. Granek J.A. Clarke N.D. Biochemistry. 1999; 38: 6374-6379Crossref PubMed Scopus (112) Google Scholar, 30House P.G. Volk D.E. Thiviyanathan V. Manuel R.C. Luxon B.A. Gorenstein D.G. Lloyd R.S. Prog. Nucleic Acids Res. Mol. Biol. 2001; 68: 349-364Crossref PubMed Google Scholar, 31Volk D.E. House P.G. Thiviyanathan V. Luxon B.A. Zhang S. Lloyd R.S. Gorenstein D.G. Biochemistry. 2000; 39: 7331-7336Crossref PubMed Scopus (49) Google Scholar), an enzyme that hydrolyzes deoxy-8-oxoguanosine 5′-triphosphate. As MutT uses a binding pocket to recognize the 8-oxoguanine portion of its substrate (32Abeygunawardana C. Weber D.J. Gittis A.G. Frick D.N. Lin J. Miller A.F. Bessman M.J. Mildvan A.S. Biochemistry. 1995; 34: 14997-15005Crossref PubMed Scopus (92) Google Scholar), the presence of an analogous pocket has been postulated for the C-terminal domain of MutY (29Noll D.M. Gogos A. Granek J.A. Clarke N.D. Biochemistry. 1999; 38: 6374-6379Crossref PubMed Scopus (112) Google Scholar, 30House P.G. Volk D.E. Thiviyanathan V. Manuel R.C. Luxon B.A. Gorenstein D.G. Lloyd R.S. Prog. Nucleic Acids Res. Mol. Biol. 2001; 68: 349-364Crossref PubMed Google Scholar). If such a cleft were to exist, then the recognition of an 8-oxoguanine by MutY would likely require the flipping of the non-scissile 8-oxoguanine out of the DNA helix in addition to the expected extrusion of the adenine for excision. However, no direct biochemical or structural evidence currently exists for such a double base-flipping mechanism. Here we report the results of UV cross-linking experiments using 8-bromoguanine-containing oligodeoxyribonucleotides to probe the topological nature of the structural contact between MutY and 8-oxoguanine. In addition, we also show results of real time stopped-flow fluorimetric detection of both 8-oxoguanine and adenine base flipping. These results will be discussed in the context of the existing structural and mechanistic models of base-flipping enzymes to provide direct insight into the mechanism of target recognition and selectivity by MutY. Ultrapure grade urea was obtained from USB Corp. (Cleveland, OH). SigmaUltra grade HEPES and its sodium salt were obtained from Sigma. Spectroscopic grade glycerol was obtained from Aldrich. Except as noted, all buffers were made with reagent grade chemicals and Milli-Q Plus (Millipore, Bedford, MA) purified distilled-deionized water. MutY Storage Buffer contained 50 mm HEPES-NaOH (pH 7.5), 500 mm NaCl, and 50% spectroscopic grade glycerol. Buffer Y contained 5 mmHEPES-NaOH (pH 7.5), 20 mm Tris-HCl (pH 7.5), 50 mm NaCl, and 5% spectroscopic grade glycerol. All buffer stock solutions were filtered through 0.2-μm polyethersulfone filters (Nalgene, Rochester, NY). Synthetic oligodeoxyribonucleotides were either synthesized by Gene Link (Hawthorne, NY) or by the Central Laboratory Services of the Center for Gene Research, Oregon State University and further purified by urea-polyacrylamide gel electrophoresis and electroelution as described in Bao et al. (33Bao K.K. Skalka A.M. Wong I. J. Biol. Chem. 2002; 277: 12089-12098Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). Concentrations were determined spectrophotometrically using extinction coefficients calculated according to Cantor et al. (34Cantor C.R. Warshaw M.M. Shapiro H. Biopolymers. 1970; 9: 1059-1077Crossref PubMed Scopus (878) Google Scholar). The 21-nucleotide-long single-stranded oligodeoxyribonucleotides are listed in Table Iwith their nucleotide sequences. Oligodeoxyribonucleotides were 5′-32P-labeled using T4 polynucleotide kinase (USB Corp.) and [γ-32P]ATP (3000 Ci/mmol, Amersham Biosciences) as described previously in Bao et al. (33Bao K.K. Skalka A.M. Wong I. J. Biol. Chem. 2002; 277: 12089-12098Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar).Table IOligodeoxyribonucleotide substratest1-aOligodeoxyribonucleotides are designated as either t for the adenine-bearing “top” strand or b for the 8-oxoguanine-containing “bottom” strand followed by the 5′–3′ sequence of the central triplet. Duplex constructs are designated by their component top and bottom strands conjoined by a colon. 5′-32P-Labeled sequences are denoted by an asterisk (*) in the text. (top) strand tXYZ5′ CAT CAG AAC XYZ TCA TCG TTA tAAT5′ strand Oligodeoxyribonucleotides are designated as either t for the adenine-bearing “top” strand or b for the 8-oxoguanine-containing “bottom” strand followed by the 5′–3′ sequence of the central triplet. Duplex constructs are designated by their component top and bottom strands conjoined by a colon. 5′-32P-Labeled sequences are denoted by an asterisk (*) in the text. in a The E. coli MutY was purified from E. the which contained a of the obtained by of E. coli between the and the of MutY was purified by a from S.L. Williams S.D. H. M.L. David S.S. J. Chem. 1996; Scopus Google Scholar, R.C. Lloyd R.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) using a of on a and on a The MutY was determined spectrophotometrically using an extinction of MutY an to between and and was in MutY Storage Buffer at MutY and DNA in Buffer Y were at 20 for in a 5 mm a to and into gel were from DNA by on a gel and using a of 8-bromoguanine-containing DNA prior to UV was out by a using experiments were using a State at a with a were by of MutY with of 5′-32P-labeled duplex DNA substrate and were with of The were then to for to the backbone of the DNA at the site to that were from the by electrophoresis on a urea Following with a the of DNA substrate and were using Biosciences) as described in Bao et al. (33Bao K.K. Skalka A.M. Wong I. J. Biol. Chem. 2002; 277: 12089-12098Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar) and Wong et al. (22Wong, I., Lundquist, A. J., Bernards, A. S., and Mosbaugh, D. W. (2002) J. Biol. Chem. 277, in pressGoogle Scholar). time with enzyme in substrate were to a single time fluorescence were using a stopped-flow with a were at a with a The 2-aminopurine was at and its fluorescence was at using a were by to a of the of the where the time required a of a was at the would then a of to for the structural between MutY and 8-oxoguanine, we synthesized the with a 8-bromoguanine for the 8-oxoguanine Table experiments were out with either 5′-32P-labeled single-stranded or the duplex an mismatch. adenine with a in the of to a adenine:8-oxoguanine mismatched duplex, the of the 8-bromoguanine in cross-linking at for were by gel shows the results of experiments out at MutY and which active site experiments binding of substrate DNA A with MutY with either single-stranded or duplex with the of The of cross-linking was due to the of cross-linking was observed with at however, we to at the to damage to the DNA and protein as well as to nonspecific DNA experiments with and or no MutY and show cross-linking nor prior to also that cross-linking to the single-stranded was by the addition of while the cross-linking of duplex was by the addition of These results show binding of the single-stranded and UV cross-linking direct contact between MutY and the 8-bromoguanine during In a duplex DNA substrate where this base is hidden in the DNA such contact either from the of a of the enzyme into the or by the of the base from the helix into a binding pocket on the enzyme the of cross-linking with the duplex construct alone evidence of flipping of the However, the of cross-linking with the single-stranded where the structural of the duplex and the scissile adenine are that the recognition of the guanine requires neither the prior binding of duplex DNA nor the extrusion of In addition, the cross-linking observed with single-stranded DNA that the site of cross-linking on MutY is to a 8-bromoguanine hidden within the of duplex DNA. contact were to with a protein side chain into the DNA duplex to probe for the then cross-linking would be expected with the single-stranded construct where the by the of the duplex is it is that the cross-linking with single-stranded in have binding of the 8-bromoguanine by the adenine binding we this to be due to the presence of and in the sequence of results the of a 8-oxoguanine binding pocket on the enzyme as on the structural to MutT (29Noll D.M. Gogos A. Granek J.A. Clarke N.D. Biochemistry. 1999; 38: 6374-6379Crossref PubMed Scopus (112) Google Scholar, 30House P.G. Volk D.E. Thiviyanathan V. Manuel R.C. Luxon B.A. Gorenstein D.G. Lloyd R.S. Prog. Nucleic Acids Res. Mol. Biol. 2001; 68: 349-364Crossref PubMed Google Scholar, 31Volk D.E. House P.G. Thiviyanathan V. Luxon B.A. Zhang S. Lloyd R.S. Gorenstein D.G. Biochemistry. 2000; 39: 7331-7336Crossref PubMed Scopus (49) Google Scholar). the extrusion of 8-oxoguanine from the DNA duplex, we synthesized an mismatched substrate with the base 2-aminopurine to the 8-oxoguanine The of of 2-aminopurine from R. Biochemistry. 2001; PubMed Scopus Google Scholar) has been in studies of enzymes (22Wong, I., Lundquist, A. J., Bernards, A. S., and Mosbaugh, D. W. (2002) J. Biol. Chem. 277, in pressGoogle Scholar, R. J.M. Biochemistry. 1994; PubMed Scopus (102) Google Scholar, D.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar, J.M. Biochemistry. 1999; 38: PubMed Scopus Google Scholar, R.P. S.S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). we expected to an of the fluorescence of the 2-aminopurine probe in to the of extrusion of its 8-oxoguanine. a duplex with the 2-aminopurine probe to the scissile adenine was also synthesized to adenine flipping. adenine at 500 MutY and DNA time for the duplex and and the normal substrate of the a for for all at the active site of s−1 in with reported S.L. Leyes A.E. David S.S. Biochemistry. 1998; 37: 14756-14764Crossref PubMed Scopus (175) Google Scholar, 29Noll D.M. Gogos A. Granek J.A. Clarke N.D. Biochemistry. 1999; 38: 6374-6379Crossref PubMed Scopus (112) Google Scholar), the of as substrate in kinetic shows the real time fluorescence of the 2-aminopurine-containing within of with 500 MutY in a stopped-flow this time we observed a in fluorescence with a of 108 s−1. the 2-aminopurine probe to the 8-oxoguanine, this 8-oxoguanine base flipping at 108 s−1 reaction In a similar using a substrate where the was to adenine flipping the observed fluorescence and with shows stopped-flow for adenine flipping 8-oxoguanine flipping and a The time for to adenine flipping with an initial followed by with the initial of the time shows that the initial observed with adenine flipping with 8-oxoguanine that the extrusion of the adenine 8-oxoguanine flipping. The in fluorescence further a for adenine extrusion with an initial extrusion of the adenine with an of 16 s−1 followed by a step at 1.9 s−1. In a using a duplex a base no fluorescence was bottom The of the 1.9 s−1 a for this However, an of s−1 would out this step as being excision. In the base-flipping kinetic mechanism of the E. coli uracil-DNA glycosylase, a step following base flipping to the of a side chain of the enzyme into the DNA helix to the vacated by the extruded base (22Wong, I., Lundquist, A. J., Bernards, A. S., and Mosbaugh, D. W. (2002) J. Biol. Chem. 277, in pressGoogle Scholar). The isomerization additional stabilization for the conformation of the extruded base by preventing its into the DNA double the 1.9 s−1 likely a similar isomerization step in the reaction mechanism of MutY to the capture of the extruded adenine in for excision. were observed of the of the 2-aminopurine to 8-oxoguanine or adenine flipping with the sequential mechanism in 4: 8-oxoguanine extrusion adenine flipping and isomerization step this stabilization the of the flipped conformation where the scissile adenine is by the active site of the requiring the 8-oxoguanine to be flipped the adenine, the enzyme that to it are for excision. the stabilization by the isomerization step following the extrusion of both bases of the mismatch that base both 8-oxoguanine and adenine bases become with in target is thereby as step of the mechanism to the catalysis while the In addition, is in for a scissile the of the 8-oxoguanine and the adenine the double base-flipping mechanism a for the where the 8-oxoguanine is first the 8-oxoguanine of this mismatched base pair is by the conformation of its as in structural studies T. Biochemistry. 1994; PubMed Scopus Google Scholar), and biochemical evidence further that MutY recognize the conformation of the 8-oxoguanine base in this context G. Miller J.H. M. Johnson Biochemistry. 1996; 35: PubMed Scopus Google Scholar). MutY likely of this in its the DNA backbone to the double base-flipping mechanism of an a would the of the by the to and base in the duplex DNA. The reaction reported observed at DNA and 500 The of the and for step must the of studies currently results that the reaction used were However, the and of the 2-aminopurine-containing substrate pair and 8-oxoguanine flipping. In addition, by the temporal sequence of the substrate recognition to we were to the of the mechanistic between the catalytic for substrate These stopped-flow studies provide direct kinetic detection of structural by in their temporal to the mechanistic by which this of enzymes R.S. Cheng X. Biopolymers. 1997; 44: 139-151Crossref PubMed Google Scholar) and to a hidden We A. for in the oligodeoxyribonucleotides and A. and for critical of the and