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• W2061458403 abstract "Thymine DNA glycosylase (TDG) excises thymine from G·T mispairs and removes a variety of damaged bases (X) with a preference for lesions in a CpG·X context. We recently reported that human TDG rapidly excises 5-halogenated uracils, exhibiting much greater activity for CpG·FU, CpG·ClU, and CpG·BrU than for CpG·T. Here we examine the effects of altering the CpG context on the excision activity for U, T, FU, ClU, and BrU. We show that the maximal activity (kmax) for G·X substrates depends significantly on the 5′ base pair. For example, kmax decreases by 6-, 11-, and 82-fold for TpG·ClU, GpG·ClU, and ApG·ClU, respectively, as compared with CpG·ClU. For the other G·X substrates, the 5′-neighbor effects have a similar trend but vary in magnitude. The activity for G·FU, G·ClU, and G·BrU, with any 5′-flanking pair, meets and in most cases significantly exceeds the CpG·T activity. Strikingly, human TDG activity is reduced 102.3–104.3-fold for A·X relative to G·X pairs and reduced further for A·X pairs with a 5′ pair other than C·G. The effect of altering the 5′ pair and/or the opposing base (G·X versus A·X) is greater for substrates that are larger (bromodeoxyuridine, dT) or have a more stable N-glycosidic bond (such as dT). The largest CpG context effects are observed for the excision of thymine. The potential role played by human TDG in the cytotoxic effects of ClU and BrU incorporation into DNA, which can occur under inflammatory conditions and in the cytotoxicity of FU, a widely used anticancer agent, are discussed. Thymine DNA glycosylase (TDG) excises thymine from G·T mispairs and removes a variety of damaged bases (X) with a preference for lesions in a CpG·X context. We recently reported that human TDG rapidly excises 5-halogenated uracils, exhibiting much greater activity for CpG·FU, CpG·ClU, and CpG·BrU than for CpG·T. Here we examine the effects of altering the CpG context on the excision activity for U, T, FU, ClU, and BrU. We show that the maximal activity (kmax) for G·X substrates depends significantly on the 5′ base pair. For example, kmax decreases by 6-, 11-, and 82-fold for TpG·ClU, GpG·ClU, and ApG·ClU, respectively, as compared with CpG·ClU. For the other G·X substrates, the 5′-neighbor effects have a similar trend but vary in magnitude. The activity for G·FU, G·ClU, and G·BrU, with any 5′-flanking pair, meets and in most cases significantly exceeds the CpG·T activity. Strikingly, human TDG activity is reduced 102.3–104.3-fold for A·X relative to G·X pairs and reduced further for A·X pairs with a 5′ pair other than C·G. The effect of altering the 5′ pair and/or the opposing base (G·X versus A·X) is greater for substrates that are larger (bromodeoxyuridine, dT) or have a more stable N-glycosidic bond (such as dT). The largest CpG context effects are observed for the excision of thymine. The potential role played by human TDG in the cytotoxic effects of ClU and BrU incorporation into DNA, which can occur under inflammatory conditions and in the cytotoxicity of FU, a widely used anticancer agent, are discussed. The nucleobases in DNA are subject to continuous chemical modification, generating a broad range of mutagenic and cytotoxic lesions that can lead to cancer and other diseases (1Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4325) Google Scholar, 2Loeb L.A. Christians F.C. Mutat. Res. 1996; 350: 279-286Crossref PubMed Scopus (75) Google Scholar). To counteract this inevitable damage, the cellular machinery includes systems for DNA repair (3Lindahl T. Wood R.D. Science. 1999; 286: 1897-1905Crossref PubMed Scopus (1278) Google Scholar). Damage occurring to the nucleobases is the purview of base excision repair, a pathway that is initiated by a damage-specific DNA glycosylase. These enzymes find damaged or mismatched bases within the vast expanse of normal DNA and catalyze the cleavage of the base-sugar (N-glycosidic) bond, producing an abasic or apurinic/apyrimidinic (AP) 2The abbreviations used are: APapurinic/apyrimidinicBrU5-bromouracildU2′-deoxyuridineBrdUrdbromodeoxyuridineClU5-chlorouracilFU5-fluorouracilHPLChigh pressure liquid chromatographyhTDGhuman thymine DNA glycosylasekmaxrate constant determined from single turnover kineticsMBD4methyl binding domain IVSMUG1single-strand selective monofunctional uracil DNA glycosylaseUNG2uracil DNA glycosylase.2The abbreviations used are: APapurinic/apyrimidinicBrU5-bromouracildU2′-deoxyuridineBrdUrdbromodeoxyuridineClU5-chlorouracilFU5-fluorouracilHPLChigh pressure liquid chromatographyhTDGhuman thymine DNA glycosylasekmaxrate constant determined from single turnover kineticsMBD4methyl binding domain IVSMUG1single-strand selective monofunctional uracil DNA glycosylaseUNG2uracil DNA glycosylase. site in the DNA. The repair process is continued by follow-on base excision repair enzymes. apurinic/apyrimidinic 5-bromouracil 2′-deoxyuridine bromodeoxyuridine 5-chlorouracil 5-fluorouracil high pressure liquid chromatography human thymine DNA glycosylase rate constant determined from single turnover kinetics methyl binding domain IV single-strand selective monofunctional uracil DNA glycosylase uracil DNA glycosylase. apurinic/apyrimidinic 5-bromouracil 2′-deoxyuridine bromodeoxyuridine 5-chlorouracil 5-fluorouracil high pressure liquid chromatography human thymine DNA glycosylase rate constant determined from single turnover kinetics methyl binding domain IV single-strand selective monofunctional uracil DNA glycosylase uracil DNA glycosylase. Human thymine DNA glycosylase (hTDG) was discovered as an enzyme that removes thymine from G·T and uracil from G·U mispairs in DNA (4Wiebauer K. Jiricny J. Nature. 1989; 339: 234-236Crossref PubMed Scopus (152) Google Scholar, 5Neddermann P. Jiricny J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1642-1646Crossref PubMed Scopus (138) Google Scholar). In vertebrates, G·T mispairs arise from replication errors, which are handled by the mismatch repair pathway or from the deamination of 5-methylcytosine to T (6Coulondre C. Miller J.H. Farabaugh P.J. Gilbert W. Nature. 1978; 274: 775-780Crossref PubMed Scopus (873) Google Scholar, 7Rideout W.M. II I Coetzee G.A. Olumi A.F. Jones P.A. Science. 1990; 249: 1288-1290Crossref PubMed Scopus (581) Google Scholar). Because cytosine methylation occurs at CpG dinucleotides (8Jones P.A. Takai D. Science. 2001; 293: 1068-1070Crossref PubMed Scopus (1542) Google Scholar, 9Feinberg A.P. Tycko B. Nat. Rev. Cancer. 2004; 4: 143-153Crossref PubMed Scopus (1787) Google Scholar), G·T mispairs caused by 5-methylcytosine deamination are found at CpG sites. It has been shown that hTDG is most active for G·T mispairs with a 5′ C·G pair, suggesting that a predominant biological role of the enzyme is to initiate the repair of CpG·T lesions (10Waters T.R. Swann P.F. J. Biol. Chem. 1998; 273: 20007-20014Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 11Sibghat U. Gallinari P. Xu Y.Z. Goodman M.F. Bloom L.B. Jiricny J. Day R.S. II I Biochemistry. 1996; 35: 12926-12932Crossref PubMed Scopus (82) Google Scholar). DNA methylation at CpG plays a fundamental role in many cellular processes, including transcriptional regulation and the silencing of repetitive genetic elements (8Jones P.A. Takai D. Science. 2001; 293: 1068-1070Crossref PubMed Scopus (1542) Google Scholar, 9Feinberg A.P. Tycko B. Nat. Rev. Cancer. 2004; 4: 143-153Crossref PubMed Scopus (1787) Google Scholar). Suggesting a biological imperative to maintain the integrity of CpG sites, another human DNA glycosylase exhibits specificity for G·T mispairs at CpG sites; methyl binding domain IV (MBD4) (12Hendrich B. Hardeland U. Ng H.H. Jiricny J. Bird A. Nature. 1999; 401: 301-304Crossref PubMed Scopus (526) Google Scholar, 13Bellacosa A. Cicchillitti L. Schepis F. Riccio A. Yeung A.T. Matsumoto Y. Golemis E.A. Genuardi M. Neri G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3969-3974Crossref PubMed Scopus (221) Google Scholar, 14Petronzelli F. Riccio A. Markham G.D. Seeholzer S.H. Stoerker J. Genuardi M. Yeung A.T. Matsumoto Y. Bellacosa A. J. Biol. Chem. 2000; 275: 32422-32429Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 15Millar C.B. Guy J. Sansom O.J. Selfridge J. MacDougall E. Hendrich B. Keightley P.D. Bishop S.M. Clarke A.R. Bird A. Science. 2002; 297: 403-405Crossref PubMed Scopus (259) Google Scholar). In addition to its CpG·T activity, hTDG has been shown to remove a variety of damaged bases (5Neddermann P. Jiricny J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1642-1646Crossref PubMed Scopus (138) Google Scholar, 16Hardeland U. Bentele M. Jiricny J. Schar P. Nucleic Acids Res. 2003; 31: 2261-2271Crossref PubMed Scopus (115) Google Scholar, 17Liu P. Burdzy A. Sowers L.C. DNA Repair (Amst). 2003; 2: 199-210Crossref PubMed Scopus (47) Google Scholar, 18Saparbaev M. Laval J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8508-8513Crossref PubMed Scopus (164) Google Scholar, 19Yoon J.H. Iwai S. O'Connor T.R. Pfeifer G.P. Nucleic Acids Res. 2003; 31: 5399-5404Crossref PubMed Scopus (66) Google Scholar), most of which are shown in Fig. 1. We recently identified several new hTDG substrates (20Bennett M.T. Rodgers M.T. Hebert A.S. Ruslander L.E. Eisele L. Drohat A.C. J. Am. Chem. Soc. 2006; 128: 12510-12519Crossref PubMed Scopus (137) Google Scholar), including 5-chlorouracil (ClU), 5-iodouracil (IU), 5-flourocytosine (FC), and 5-bromocytosine (BrC) (the activity is weak for IU, FC, and BrC and is probably not biologically relevant). The ability of hTDG to remove a broad range of damaged nucleobases is consistent in its relatively large and nonspecific active site (21Barrett T.E. Scharer O.D. Savva R. Brown T. Jiricny J. Verdine G.L. Pearl L.H. EMBO J. 1999; 18: 6599-6609Crossref PubMed Scopus (122) Google Scholar, 22Baba D. Maita N. Jee J Nature. 2005; 435: 979-982Crossref PubMed Scopus (185) Google Scholar). Yet despite its substrate promiscuity, hTDG exhibits exceedingly weak activity for the excision of cytosine and 5-methylcytosine (11Sibghat U. Gallinari P. Xu Y.Z. Goodman M.F. Bloom L.B. Jiricny J. Day R.S. II I Biochemistry. 1996; 35: 12926-12932Crossref PubMed Scopus (82) Google Scholar, 16Hardeland U. Bentele M. Jiricny J. Schar P. Nucleic Acids Res. 2003; 31: 2261-2271Crossref PubMed Scopus (115) Google Scholar, 20Bennett M.T. Rodgers M.T. Hebert A.S. Ruslander L.E. Eisele L. Drohat A.C. J. Am. Chem. Soc. 2006; 128: 12510-12519Crossref PubMed Scopus (137) Google Scholar). We recently showed that for a broad range of C5-substituted uracil and cytosine bases, hTDG specificity depends on substrate reactivity (i.e. the stability of the scissile C-N bond) rather than the selective recognition of substrates in the active site (20Bennett M.T. Rodgers M.T. Hebert A.S. Ruslander L.E. Eisele L. Drohat A.C. J. Am. Chem. Soc. 2006; 128: 12510-12519Crossref PubMed Scopus (137) Google Scholar). Moreover, we showed that specificity against the excision of cytosine from the huge excess of normal G·C pairs in DNA is largely explained by the very low reactivity of dC rather than the inability of hTDG to flip cytosine into its active site (20Bennett M.T. Rodgers M.T. Hebert A.S. Ruslander L.E. Eisele L. Drohat A.C. J. Am. Chem. Soc. 2006; 128: 12510-12519Crossref PubMed Scopus (137) Google Scholar). Consistent with this catalytic mechanism and the enhanced reactivity of 5-halogenated dU substrates, we found that hTDG rapidly excises FU, ClU, and BrU from CpG sites (20Bennett M.T. Rodgers M.T. Hebert A.S. Ruslander L.E. Eisele L. Drohat A.C. J. Am. Chem. Soc. 2006; 128: 12510-12519Crossref PubMed Scopus (137) Google Scholar). Indeed, compared with CpG·T, the activity is 920-fold greater for CpG·FU, 550-fold greater for CpG·ClU, and 53-fold greater for CpG·BrU (20Bennett M.T. Rodgers M.T. Hebert A.S. Ruslander L.E. Eisele L. Drohat A.C. J. Am. Chem. Soc. 2006; 128: 12510-12519Crossref PubMed Scopus (137) Google Scholar). The robust activity observed for CpG·ClU and CpG·BrU suggests that hTDG may also have significant activity for removing ClU and BrU from DNA contexts other than CpG, raising the possibility that hTDG could play a role in the mutagenic and cytotoxic effects associated with ClU and BrU incorporation into DNA (23Morris S.M. Mutat. Res. 1991; 258: 161-188Crossref PubMed Scopus (115) Google Scholar, 24Morris S.M. Mutat. Res. 1993; 297: 39-51Crossref PubMed Scopus (84) Google Scholar). These lesions can arise in DNA when the 5-chloro-dUTP or 5-bromo-dUTP pools become elevated, which can be promoted by the activity of peroxidases during inflammation (25Jiang Q. Blount B.C. Ames B.N. J. Biol. Chem. 2003; 278: 32834-32840Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 26Henderson J.P. Byun J. Takeshita J. Heinecke J.W. J. Biol. Chem. 2003; 278: 23522-23528Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The very strong hTDG activity for CpG·FU substrates is also of interest because FU has been used for decades to treat many types of cancer (27Longley D.B. Harkin D.P. Johnston P.G. Nat. Rev. Cancer. 2003; 3: 330-338Crossref PubMed Scopus (3512) Google Scholar). The mechanism of FU cytotoxicity is thought to involve multiple pathways, including a repetitive cycle of U and FU incorporation into DNA followed by the excision of these bases by a DNA glycosylase, increasing the burden abasic sites and leading to DNA strand breaks (27Longley D.B. Harkin D.P. Johnston P.G. Nat. Rev. Cancer. 2003; 3: 330-338Crossref PubMed Scopus (3512) Google Scholar). Thus, hTDG could potentially be involved in the cytotoxicity of FU, as suggested by a report that inactivation of TDG in fission yeast and in mouse embryonic fibroblasts diminishes the sensitivity of these cells to FU treatment (28Cortazar D. Kunz C. Saito Y. Steinacher R. Schar P. DNA Repair (Amst). 2007; 6: 489-504Crossref PubMed Scopus (160) Google Scholar). To further examine these possibilities, it is important to determine the activity of hTDG for removing FU, ClU, and BrU from DNA contexts other than CpG, because the incorporation of these bases into DNA or their presence in the template strand can be expected to give predominantly A·X pairs but also some G·X pairs and with no significant preference for a CpG sequence context. Although previous studies have examined the effect of altering the 5′-flanking pair on hTDG activity for G·T and G·ϵC (10Waters T.R. Swann P.F. J. Biol. Chem. 1998; 273: 20007-20014Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 29Abu M. Waters T.R. J. Biol. Chem. 2003; 278: 8739-8744Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), quantitative studies have not been reported for the many other hTDG substrates. Moreover, previous studies (and our findings here) indicate that the effect of the 5′-flanking pair depends strongly on the nature of the target base (29Abu M. Waters T.R. J. Biol. Chem. 2003; 278: 8739-8744Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), so the results for G·T substrates do not necessarily predict the 5′-neighbor effects for other substrates. In addition, the effect of pairing the target base with adenine rather than guanine (i.e. A·X versus G·X) has not been rigorously examined for substrates other than U (10Waters T.R. Swann P.F. J. Biol. Chem. 1998; 273: 20007-20014Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 30Wibley J.E. Waters T.R. Haushalter K. Verdine G.L. Pearl L.H. Mol. Cell. 2003; 11: 1647-1659Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), and the effect of altering the 5′-flanking pair for A·X substrates is completely unexplored. Here, we use single turnover kinetics experiments to compare the activity of hTDG (kmax) for substrates that contain a G·X lesion with various 5′-flanking base pairs, i.e. CpG·X, TpG·X, GpG·X, and ApG·X, where X represents FU, ClU, BrU, U, or T. We also examine the effect of pairing the target base with adenine rather than guanine (i.e. CpA·X versus CpG·X). Finally, we examine the combined effect of pairing the target base with adenine and altering the 5′-flanking base pair using CpA·X, TpA·X, GpA·X, and ApA·X substrates. These studies provide the relative activity of hTDG for the excision of U, FU, ClU, and BrU from DNA contexts other than CpG, i.e. those in which they might be expected to arise in vivo. In addition, by systematically altering the CpG context for a series of target bases, our findings illuminate the catalytic role of the putative interactions that hTDG forms with the opposing guanine and with the 5′ C·G base pair. DNA Synthesis and Purification-Duplex DNA substrates were hybridized in 10 mm Tris, pH 8.0, 0.1 m NaCl, and 0.1 mm EDTA by rapid heating to 80 °C and slow cooling to room temperature. Single-strand DNA oligonucleotides were synthesized at the Biopolymer Genomics Core Facility, University of Maryland, Baltimore and at the Keck Foundation Biotechnology Resource Laboratory of Yale University. The 5-chlorodeoxyuridine phosphoramidite was obtained from ChemGenes Corp. (Wilmington, MA). Oligonucleotides were purified by anion exchange HPLC using a Zorbax Oligo column (Agilent Technologies), desalted by gel filtration using pre-packed Sephadex G25 columns (GE Healthcare), and stored at –20 °C. Oligonucleotide purity was verified by analytical anion-exchange HPLC under denaturing (pH 12) conditions using a DNAPac PA200 column (Dionex Corp.), as described previously (20Bennett M.T. Rodgers M.T. Hebert A.S. Ruslander L.E. Eisele L. Drohat A.C. J. Am. Chem. Soc. 2006; 128: 12510-12519Crossref PubMed Scopus (137) Google Scholar). Oligonucleotides were quantified by absorbance (260 nm) using the pairwise extinction coefficients, calculated as described (31Fasman G. CRC Handbook of Biochemistry and Molecular Biology. 1975; Google Scholar). Expression and Purification of hTDG-Escherichia coli BL21(DE3) cells (Stratagene) were transformed with a pET-28-based expression plasmid for human TDG, 410-amino acids (32Hardeland U. Steinacher R. Jiricny J. Schar P. EMBO J. 2002; 21: 1456-1464Crossref PubMed Scopus (259) Google Scholar). Expression cells were grown in Luria broth (typically 2 liter) at 37 °C until A600 = 0.8, the temperature was reduced to 15 °C, and hTDG expression was induced with 0.25 mm isopropyl β-d-thiogalactoside and continued for about 15 h. Cells were harvested by centrifugation and suspended in ∼25 ml of lysis buffer (0.05 m sodium phosphate, pH 8.0, 0.3 m NaCl, 0.02 m imidazole, 0.01 m β-mercaptoethanol) with 1 mg/ml lysozyme and a protease inhibitor mixture (Roche Applied Science). The cell suspension was frozen on dry ice, thawed, and incubated on ice for 30 min with stirring followed by an additional 30 min with DNase (Novagen). The lysate was cleared by centrifugation and incubated with 4 ml of nickel-nitrilotriacetic acid metal affinity resin (Qiagen) for 1 h at 4 °C. The lysate-resin mix was placed in a gravity-flow column, washed with 30 ml of lysis buffer containing 1 m NaCl and 20 mm imidazole followed by 30 ml of lysis buffer containing 20 mm imidazole, and hTDG was eluted with lysis buffer containing 150 mm imidazole. hTDG was purified further using an SP-Sepharose HP column (GE Healthcare) with buffers IE-A (25 mm Tris, pH 7.5, 75 mm NaCl, 1 mm dithiothreitol, 0.2 mm EDTA, 1% glycerol) and IE-B (IE-A with 1 m NaCl) and a gradient of 5–20% IE-B over 60 min at 2.5 ml/min. hTDG was further purified using a Q-Sepharose HP column (GE Healthcare) with the same IE-A and IE-B buffers and a gradient of 0–100% IE-B over 60 min at 2.5 ml/min. The purity of hTDG was >99% as judged by SDS-PAGE stained with Coomassie. Purified hTDG was dialyzed overnight versus storage buffer (20 mm HEPES 7.5, 0.1 m NaCl, 1 mm dithiothreitol, 0.5 mm EDTA, 1% glycerol), concentrated to about 0.1 mm, flash-frozen in small aliquots, and stored at –80 °C. The concentration of hTDG was determined by absorbance using ϵ280 = 31.5 mm–1cm–1 (33Gill S.C. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5048) Google Scholar). Single Turnover Kinetics-Because hTDG is strongly inhibited by its abasic DNA product (10Waters T.R. Swann P.F. J. Biol. Chem. 1998; 273: 20007-20014Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), the rate constant obtained from steady-state kinetics experiments (kcat) is dominated by product release and is not useful for comparing hTDG activity for various substrates. Here, we use single turnover kinetics under saturating enzyme conditions to obtain rate constants (kmax) that are not impacted by product release or the association of enzyme and substrate and thereby reflect the maximal enzymatic activity for a given substrate. To ensure that the observed rate constants represent the maximal value (i.e. kobs ≈ kmax), single turnover experiments were collected using a large excess of enzyme over substrate and with an enzyme concentration that is more than 100-fold higher the KD = 41 nm reported as the apparent binding affinity of hTDG for DNA containing a CpG·T mispair (29Abu M. Waters T.R. J. Biol. Chem. 2003; 278: 8739-8744Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). To confirm saturating enzyme conditions, experiments were in some cases conducted with two or more hTDG concentrations, typically 5 and 10 μm, providing rate constants that were equivalent within experimental uncertainty. Substrate DNA concentrations were 500 nm unless noted otherwise. Single turnover reactions were performed either manually or using a rapid chemical quenched-flow instrument (RQF-3, Kintek Corp.). The reactions were conducted at 22 °C in HEMN.1 buffer (20 mm HEPES, pH 7.50, 0.2 mm EDTA, 2.5 mm MgCl2, 0.1 m NaCl) with 0.1 mg/ml bovine serum albumin, quenched with 50% (v:v) 0.3 m NaOH, 0.03 m EDTA, and heated for 15 min at 85 °C to induce cleavage of the DNA backbone at AP sites. The extent of product formation was analyzed by HPLC, as described below. Rate constants were determined by fitting the single turnover data to a single exponential equation using nonlinear regression with Grafit 5 (34Leatherbarrow R.J. Grafit 5. 1998; Google Scholar). In most cases, the reactions proceeded to full completion, except those that are very slow (i.e. kmax <∼1 × 10–4 min–1). HPLC Assay for Monitoring hTDG Activity-We recently developed a HPLC assay for monitoring hTDG activity (20Bennett M.T. Rodgers M.T. Hebert A.S. Ruslander L.E. Eisele L. Drohat A.C. J. Am. Chem. Soc. 2006; 128: 12510-12519Crossref PubMed Scopus (137) Google Scholar). Samples taken during a kinetics experiment contain a mixture of substrate and products that is comprised of four oligonucleotides; that is, the full-length target strand and its complement and two shorter strands resulting from alkaline cleavage of the nascent abasic strand. These strands are resolved by anion exchange HPLC using denaturing (pH 12.0) conditions with a DNAPac PA200 column (Dionex Corp.). The alkaline conditions serve to suppress hybridization of ssDNA during chromatography and have the added benefit of resolving strands that are of the same length but differ in the number of thymine and guanine bases, which are negatively charged at pH 12. The elution buffer is 0.02 m sodium phosphate pH 12.0 containing either 0.03 m NaClO4 (buffer A) or 0.50 m NaClO4 (buffer B). The oligonucleotides are detected by absorbance (260 nm), and the fraction product (F) is determined from the integrated peak areas for the target strand (AS) and product strands (AP1 and AP2) using the equation F = (AP1 + AP2)/(AS + AP1 + AP2). The determination of fraction product using this assay is reproducible to within 1%, as determined from multiple analyses of identical samples. In a recent study we determined the activity of hTDG (kmax) for a series of 5-substituted uracil and cytosine substrates in which the target base was placed in a CpG context (20Bennett M.T. Rodgers M.T. Hebert A.S. Ruslander L.E. Eisele L. Drohat A.C. J. Am. Chem. Soc. 2006; 128: 12510-12519Crossref PubMed Scopus (137) Google Scholar). We found that kmax is much higher for CpG·FU, CpG·ClU, and CpG·BrU than for CpG·T, suggesting that hTDG may have significant activity for FU, ClU, and BrU in DNA contexts other than CpG. Here, we examine the effect of altering the CpG context on the activity of hTDG for the excision of five different target bases (X = T, U, FU, ClU, and BrU) using a series of substrates as shown in Fig. 2. Thus, one set of substrates examines the effect of altering the 5′-flanking pair on G·X activity (i.e. YpG·X). Another set examines the effect of pairing the target base with adenine rather than guanine while preserving the 5′ C·G pair (CpA·X versus CpG·X). A final set examines the effect of both, pairing the target base with adenine and altering the 5′-flanking pair (YpA·X). Single Turnover Kinetics-Like many DNA glycosylases, hTDG is strongly inhibited by one of its reaction products, abasic DNA (10Waters T.R. Swann P.F. J. Biol. Chem. 1998; 273: 20007-20014Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 14Petronzelli F. Riccio A. Markham G.D. Seeholzer S.H. Stoerker J. Genuardi M. Yeung A.T. Matsumoto Y. Bellacosa A. J. Biol. Chem. 2000; 275: 32422-32429Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 35O'Neill R.J. Vorob'eva O.V. Shahbakhti H. Zmuda E. Bhagwat A.S. Baldwin G.S. J. Biol. Chem. 2003; 278: 20526-20532Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 36Porello S.L. Leyes A.E. David S.S. Biochemistry. 1998; 37: 14756-14764Crossref PubMed Scopus (175) Google Scholar, 37McCann J.A. Berti P.J. J. Biol. Chem. 2003; 278: 29587-29592Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Indeed, previous studies show that under limiting enzyme conditions, the turnover of hTDG is exceedingly slow after it converts one molar equivalent of G·T (or G·U) substrate to G·AP product (10Waters T.R. Swann P.F. J. Biol. Chem. 1998; 273: 20007-20014Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 38Waters T.R. Gallinari P. Jiricny J. Swann P.F. J. Biol. Chem. 1999; 274: 67-74Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 39Steinacher R. Schar P. Curr. Biol. 2005; 15: 616-623Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Thus, the rate constant obtained from steady-state kinetics, kcat, is dominated by product release and cannot provide a meaningful comparison of activity for different substrates (Fig. 3). In contrast, single turnover kinetics conducted under saturating enzyme conditions provide a rate constant (kmax) that is not impacted by product release or the association of enzyme and substrate and, therefore, reflects the maximal activity for a given substrate (Fig. 3). For the hTDG reaction, kmax reflects the rate constant for the chemical step (kchem) and is also influenced by the equilibrium constant for base flipping (Kflip). In the base-flipping step, the target nucleotide flips out of the DNA duplex and into the active site, a process that likely involves a conformational change in hTDG, as observed for uracil DNA glycosylase (40Jiang Y.L. Stivers J.T. Biochemistry. 2002; 41: 11236-11247Crossref PubMed Scopus (57) Google Scholar). Thus, differences in kmax that result from alterations to the CpG context reflect a change in kchem and/or Kflip. Effect of the 5′ Base Pair on G·X Activity-We determined the effect of varying the 5′ neighboring base pair on hTDG activity (kmax) for G·FU, G·ClU, G·BrU, G·U, and G·T using the YpG·X series of substrates (Fig. 2). The results are given in Table 1 and Fig. 4. Previous studies showed that hTDG activity for G·T substrates depends strongly on the 5′-flanking pair, with relative activity of CpG·T » TpG·T > GpG·T > ApG·T (10Waters T.R. Swann P.F. J. Biol. Chem. 1998; 273: 20007-20014Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 11Sibghat U. Gallinari P. Xu Y.Z. Goodman M.F. Bloom L.B. Jiricny J. Day R.S. II I Biochemistry. 1996; 35: 12926-12932Crossref PubMed Scopus (82) Google Scholar). We find a similar trend here; compared with CpG·T, kmax is reduced by 37-, 96-, and 582-fold for TpG·T, GpG·T, and ApG·T, respectively. The influence of the 5′ neighbor is much smaller for G·U activity; compared with CpG·U, kmax is decreased by 3.3-, 2.9-, and 22-fold for TpG·U, GpG·U, and ApG·U, respectively. The 5′-neighbor effect is also small for G·FU activity; compared with CpG·FU, kmax decreases by merely 1.8-, 1.6-, and 11-fold for TpG·FU, GpG·FU, and ApG·FU, respectively (Fig. 4B). The 5′-neighbor effects are much larger for G·ClU activity; compared with CpG·ClU, kmax is decreased by 6-, 11-, and 82-fold for TpG·ClU, GpG·ClU, and ApG·ClU, respectively (Fig. 4C). The results are similar for G·BrU; compared with CpG·BrU, kmax is decreased by 9-, 26-, and 75-fold for TpG·BrU, GpG·BrU, and ApG·BrU, respectively.TABLE 1Kinetic Parameters for hTDGSubstratekmaxaThe rate constants (kmax) reflect the maximal enzymatic activity of hTDG for a given substrate, as determined using single turnover kinetics experiments with saturating enzyme conditions.-Fold change relative to CpG·X-Fold change relative to CpG·T-Fold change relative to CpA·X-Fold change relative to YpG·Xb-Fold change relative to YpG·X gives the effect of pairing the target base (X) with adenine rather than guanine for a given 5′ base pair (Y), i.e. the rate of TpA·U relative to TpG·U.Min-1G·X CpG·T0.22 ± 0.0411 TpG·T0.0060 ± 0.00010.0270.027 GpG·T0.0023 ± 0.00020.0100.010 ApG·T0.00038 ± 0.000050.00170.0017 CpG·U2.6 ± 0.3112 TpG·U0.79 ± 0.040.3033.6 GpG·U0.88 ± 0.110.3404.0 ApG·U0.117 ± 0.0030.0450.5 CpG·FU202 ± 161918 TpG·FU113 ± 10.558513 GpG·FU125 ± 110.618568 ApG·FU18 ± 10.08975 CpG·ClU120 ± 61546 TpG·ClU20.9 ± 0.50.17495 GpG·ClU11.1 ± 0.30.09351 ApG·ClU1.46 ± 0.150.0126.7 CpG·BrU11.6 ± 1.0153 TpG·BrU1.2 ± 0.10.1065.6 GpG·BrU0.44 ± 0.060.0382." @default.
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• W2061458403 title "Excision of 5-Halogenated Uracils by Human Thymine DNA Glycosylase" @default.
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