Characterization of the Substrate Specificity of a Human 5

Loma Linda, California 92350, and Divisions of Pediatrics and Molecular Medicine,. City of Hope National Medical Center, 1500 East Duarte Road, Duarte...
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Chem. Res. Toxicol. 2002, 15, 33-39

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Characterization of the Substrate Specificity of a Human 5-Hydroxymethyluracil Glycosylase Activity David Baker, Pingfang Liu, Artur Burdzy, and Lawrence C. Sowers* Department of Biochemistry and Microbiology, Loma Linda University School of Medicine, Loma Linda, California 92350, and Divisions of Pediatrics and Molecular Medicine, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, California 91010 Received July 10, 2001

The oxidation of pyrimidine 5-methyl groups, derived from either thymine or 5-methylcytosine, can generate 5-hydroxymethyluracil (HmU) in DNA. An activity from HeLa cells that removes 5-hydroxymethyluracil (HmU) from DNA has been partially purified and characterized using a battery of oligonucleotides containing modified bases. This partially purified activity preferentially removes HmU mispaired with guanine. The HmU repair activity also acts on uracil and fluorouracil but not 5-substituted uracil derivatives with halogens larger than fluorine. However, neither mispaired thymine nor ethenocytosine are substrates. HmU is readily removed when paired with guanine, hypoxanthine (deoxyinosine), and purine (deoxynebularine), but not from single-stranded substrates. Upon the basis of these substrate preferences, we conclude that (1) the mispaired HmU repair activity is distinct from previously reported glycosylases including UDG, TDG, MUG, and SMUG1 activities, (2) the binding pocket is highly selective for the 5-hydroxymethyl group, and (3) the preference for mispaired HmU derives from reduced thermal stability of the mispair, as opposed to selective recognition of the mispaired guanine residue in the opposing DNA strand.

Introduction Endogenously occurring alkylating agents, water, and reactive oxygen species constantly attack the DNA of all living organisms. The rate constants for the formation of individual damage products are relatively small. However, because of the large number of bases present in the human genome, the consequences of endogenous DNA damage are considered to be biologically significant (1-3). To maintain the integrity of the genome in the face of substantial endogenous and exogenous DNA damage, all living organisms contain complex repair pathways that recognize and remove DNA lesions. Many DNA glycosylases have been identified from numerous organisms that remove damaged and/or mispaired pyrimidines. Interestingly, glycosylases with high degrees of sequence homology very often have quite different substrate specificities, whereas some glycosylases with little homology share common substrates (4-39). Efforts are currently underway in many laboratories to identify and characterize such glycosylase activities. Previously, glycosylase activities have been reported (36, 37) which remove 5-hydroxymethyluracil (HmU),1 an oxidation damage product derived from thymine or 5-methylcytosine (5mC). Although HmU-glycosylase activity has been known for some time, the gene encoding this activity has not yet been identified. The HmU derived from thymine would be found paired with ad* To whom correspondence should be addressed. Phone: (909) 5584527. E-mail: [email protected]. 1 Abbreviations: HmU, 5-hydroxymethyluracil; 5mC, 5-methylcytosine; TDG, thymine-DNA glycosylase from methanobacterium thermoautotrophicum; UDG, uracil-DNA glycosylase; MUG, E. coli mismatch uracil DNA glycosylase; UGI, uracil glycosylase inhibitor protein.

enine. Recently, we reported that human cells had an unexpectedly abundant activity for HmU mispaired with guanine (38). The higher activity against HmU:G, compared with HmU:A, was surprising because the HmU:G base pair would be derived from the 5mC:G base pair by two sequential and relatively slow reactions. However, the conversion of 5mC to HmU could result in a 5mC to T transition mutation. Such mutations are frequently found in human cancer (40-42). The potential involvement of the HmU-glycosylase activity in protecting against such transition mutations prompted us to further characterize the activity in human cells. We report here the partial purification of the major HmU glycosylase activity from HeLa cell extracts. This partially purified activity was subsequently characterized using a series of oligonucleotide substrates containing a panel of purine and pyrimidine analogues, and the results compared with a group of known, cloned glycosylases including uracil-DNA glycosylase (UDG), Escherichia coli mispaired uracil DNA glycosylase (MUG) and methanobacterium thermoautotrophicum thymine-DNA glycosylase (TDG). The subset of base analogues that are substrates for the partially purified HmU-glycosylase activity are distinct from those recognized by any other as yet reported glycosylase.

Materials and Methods Oligonucleotide Synthesis and Characterization. The oligonucleotide substrates for the glycosylase assay were prepared by solid-phase synthesis methods. The phosphoramidites of the normal bases as well as those for uracil, fluorouracil, bromouracil, iodouracil, purine, hypoxanthine, and 2,6-diaminopurine were obtained from Glen Research (Sterling, VA). Special deprotection conditions were used when recommended by the supplier. The phosphoramidites for HmU (43), 2-ami-

10.1021/tx010113b CCC: $22.00 © 2002 American Chemical Society Published on Web 12/13/2001

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Figure 1. Sequences of the duplex oligonucleotides used in this study, where X ) U, T, 5-fluorouracil, 5-bromouracil, 5-iodouracil, HmU, ethenocytosine (C), and P ) A, G, 2-aminopurine, purine (deoxynebularine), hypoxanthine (deoxyinosine) or 2,6diaminopurine. nopurine (44), and 5-chlorouracil (45) were prepared as previously described. The oligonucleotides containing ethenocytosine were obtained from Trevigen, Inc. (Gaithersburg, MD). The sequences of the oligonucleotides used in the cleavage assays, designated duplex 1, 2, or 3, are shown in Figure 1. Following synthesis and deprotection, oligonucleotides were purified using C-18 sep-pak cartridges from Waters Associates (Milford, MA). Oligonucleotides were purified to homogeneity by gel electrophoresis when needed. The composition of the oligonucleotides was confirmed by GC/MS following acid hydrolysis (46-48). In all cases, chromatographic peaks were detected with mass spectra corresponding to both normal and modified bases, when expected. Cloned Glycosylases. The E. coli mismatch uracil-DNA glycosylase (MUG protein) and thymine DNA glycosylase from methanobacterium thermoautotrophicum (TDG) were obtained from Trevigen, Inc. Uracil-DNA glycosylase (UDG) was obtained from USB Corp. (Cleveland, OH). The uracil glycosylase inhibitor protein (UGI) was obtained from New England Biolabs (Beverley, MA). Glycosylase Cleavage Assay Conditions. The HmU:G glycosylase assay used to screen fractions obtained from the HeLa cells contained 3 pmol of 32P-end labeled duplex oligonucleotide (duplex 1 plus duplex 2, sequences shown in Figure 1) containing 10 µM ZnCl2. The most active fraction contained 2.5 µg of total protein. The final volume was adjusted to 50 µL with HE buffer (25 mM Hepes/NaOH, pH 7.8, 1 mM EDTA, 1 mM DTT, 10% glycerol) and allowed to react for 2 h at 37 °C. Samples were then passed over Micro Bio-Spin 6 chromatography columns from Bio-Rad (Hercules, CA) that had been equilibrated with water, and dried. Samples were resuspended in 10 µL of water, 5 µL of 0.1 M NaOH, and 15 µL of 98% formamide dye solution, heated at 95 °C for 30 min and cooled to -70 °C for at least 30 min. To ensure that the oligonucleotide strand containing the modified base remained single-stranded during electrophoresis, an excess (400 pmol) of unlabeled competitor oligonucleotide of the same sequence, containing normal bases was added, the solution was heated to 95 °C for 15 min, and rapidly cooled. The reaction products were then analyzed by polyacrylamide gel electrophoresis (PAGE) on an 18% denaturing gel. The glycosylase assays for the partially purifed fraction described above, and cloned glycosylases, were performed in a final volume of 15 µL with 1.25 pmol of 32P-end labeled duplex oligonucleotide, 2.5 µL of buffer (50 mM piperazineethanesulfonic acid, pH 6.7, 10 µM ZnCl2, 0.5 mM EDTA, and 2 mM DTT). Following glycosylase cleavage, samples were purified by spin column chromatography. Samples were denatured and prepared for electrophoresis as described above. The assays with the partially purified HmU-glycosylase fraction described above, and the cloned glycosylases, were performed with 1.25 pmol of duplex oligonucleotide (duplex 3, Figure 1) at 37 °C. The assays with the partially purified HmU-glycosylase contained 0.5 µg of total protein in the isolation buffer, in a final volume of 15 µL, and were incubated for 2 h. The assays with MUG (Trevigen)

Baker et al. contained 1 unit of enzyme and buffer supplied by the manufacturer, in a final volume of 10 µL, for 45 min at 37 °C. The TDG assays were performed with 20 units of TDG (Trevigen) and manufacturers buffer in a final volume of 10 µL, for 2.25 h. The assays with UDG (USB) contained 1 unit of enzyme and manufacturer’s buffer in a final volume of 10 µL and were incubated for 1 h. Following the glycosylase reaction, oligonucleotides were denatured and prepared for electrophoresis as described above. The labeled gel bands were visualized with a Molecular Dynamics PhosphorImager and software. Purification of the HmU Glycosylase Activity. The DEAE-sepharose fast-flow resin was obtained from Amersham/ Pharmacia (Uppsala, Sweden). The P-11 cellulose phosphate resin was obtained from Whatman (Fairfield, NJ). The HighQ resin is a strong anion exchanger on a macroporous hydrophobic support. Columns containing the HighQ resin were obtained from BioRad. HeLa-S3 cells were purchased from the National Cell Culture Center (Minneapolis, MN) in 5 g aliquots. Cell lysis, lysate clearing, and DEAE chromatography were performed as previously described (37). Cells (5 g) were quick thawed at 37 °C and suspended in 3 cell volumes, 15 mL, hypotonic swelling buffer (25 mM Hepes/NaOH, pH 7.8, 1 mM EDTA, 2 mM DTT, 1 mM phenylmethanesulfonyl fluoride, 0.5 mM spermidine, and 0.2 mM spermine) for 20 min on ice. The lysate was then homogenized on ice in a 15 mL glass/glass homogenizer with 20-25 strokes. Glycerol (100%) was added to a final concentration of 20%, followed by the addition of saturated and neutralized (NH4)2 SO4 at a rate of 11 mL/100 mL of extract. The mixture was then stirred gently for 30 min at 4 °C and cleared by centrifugation in a Beckman XL 100K ultracentrifuge, using a Type 70Ti rotor with Beckman optiseal tubes at 60,000 rpm for 90 min at 4 °C. The cleared extract was diluted 1:4 with HE buffer. Dilute extract (92.5 mL) containing approximately 200 mg of protein, was incubated with 30 mL of DEAE-Sepharose fast flow resin which had been equilibrated with HE buffer containing 0.1 M NaCl at 4 °C. The slurry was filtered and the cake was resuspended and washed in two matrix volumes with HE buffer containing 0.1 M NaCl. This was allowed to stir for 20 min at 4 °C and then refiltered. The flow through volume and first wash fractions were combined to give a final volume of 137 mL, 88 mg total protein, and stored at -70 oC in 10 13.7 mL aliquots Whatman P-11 cellulose phosphate (3 g), was equilibrated with 0.1 M NaCl in HE buffer and poured into a 1 × 30 cm glass column. The HE buffer containing 0.1 M NaCl was passed through the column using a peristaltic pump at a flow rate of about 1 mL/min. The column was cooled to 4 °C and 14 mL (9 mg of total protein) of the material which was not retained on the DEAE column was passed through the cellulose phosphate column at a flow rate of 1 mL/min. The cellulose phosphate column was washed with HE buffer containing 0.1 M NaCl. A total volume of 23.5 mL containing the HmU-glycosylase activity was obtained containing 4.2 mg of protein. Following the cellulose phosphate purification step, the fraction containing the HmU-glycosylase activity was diluted to 110 mL with HE buffer and applied to a 1 mL HighQ column, equilibrated with HE buffer containing 0.02 M NaCl. The column was eluted at a flow rate of 1 mL/min at 4 °C. The column was washed with 40 mL of HE buffer and then eluted with 16 mL of a linear NaCl gradient from 0 to 0.6 M. Twenty 1.0 mL fractions were collected and assayed for activity. Uracil Glycosylase Inhibition Assays. The assays to examine the effect of uracil glycosylase inhibitor protein (UGI) on glycosylase activity were performed essentially as described above. Sample contained either no inhibitor protein or 2 units UGI (NEB). Molecular Weight Determination. The material obtained following cellulose phosphate chromatography (16 mL) was concentrated to 3 mL with Millipore Centricon YM-3 concentrators (Bedford, MA) in a Beckman JA-20 rotor spun at 6500g for 2.25 h at 4 °C in a Beckman J-25 centrifuge. A volume (200 µL) was layered onto a 4.4 mL, 5-20%, ultrapure sucrose gradient

Mispaired HmU-Glycosylase Activity

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Figure 2. Bioassay-guided fractionation of the HmU-glycosylase activity. Fractions obtained from sequential purification of the HmU-glycosylase activity were assayed simultaneously for U:G activity (duplex 1), and HmU:G activity (duplex 2, X ) HmU, P ) guanine). Following incubation of the end labeled oligonucleotide with a given fraction, oligonucleotides were separated by polyacrylamide gel electrophoresis. Gels were scanned with a PhosphorImager. Lanes marked 1-4 correspond to (1) the negative control, (2) the assay performed with the whole cell extract, (3) the fraction obtained following DEAE chromatography, and (4) the fraction obtained following cellulose phosphate chromatography. The lanes marked F1-F10 correspond to the sequential fractions obtained from chromatography with the HighQ resin. Numbers to the left correspond to the length of the oligonucleotide. The uncleaved uracil-containing oligonucleotide (duplex 1) is 30 bases in length, and when cleaved, 18 bases in length. The uncleaved HmU-containing oligonucleotide is 24 bases in length (duplex 2) and 12 bases in length when cleaved. Fraction F5 contained both HmU- and uracil-glycosylase activities. in HE buffer. Centrifugation was performed using a Beckman SW55Ti rotor and a Beckman XL-100K ultracentrifuge spinning at 44 000 rpm for 22 h at 4 °C. One tube contained internal standards including 40 µg each of bovine milk lactalbumin, 14.4 kDa, carbonic anhydrase, 29.0 kDa, and BSA, 66.0 kDa (Sigma, St. Louis, MO). Fractions obtained from the centrifuge tubes (15 fractions, 0.29 mL each) were collected, dialyzed against HE buffer, and assayed for glycosylase activity. Active fractions (fraction numbers 3-8) were concentrated with YM-3 concentrators and again assayed for activity. The fraction with the highest activity corresponded to a molecular weight of 41 kDa. Glycosylase activities against base pairs containing both uracil and HmU, paired with A or mispaired with G, were all found in this fraction.

Results A standard strategy was employed for the partial purification of an HmU-glycosylase activity as described below. At each stage of the isolation procedure, fractions were assayed for the capacity to remove base analogues from a pair of oligonucleotides. A longer, 30-base duplex oligonucleotide contained a U:G mispair (duplex 1), and a shorter, 24-base oligonucleotide duplex (duplex 2) contained an HmU:G mispair (Figure 1). The cleavage of the uracil-containing 30-mer generated an 18-mer, and cleavage of the HmU-containing 24-mer generated a 12mer. The use of this set of oligonucleotide substrates allowed simultaneous assay of both uracil and HmU glycosylase activities in each fraction (Figure 2). The HmU:G glycosylase activity was isolated by chromatography using DEAE, cellulose phosphate, and HighQ solid-phase supports. In the whole cell extracts and the DEAE wash fractions, the uracil glycosylase activity far exceeded that of the HmU-glycosylase activity. Following cellulose phosphate chromatography, the majority of the uracil glycosylase activity was lost; however, the HmU-

Figure 3. Comparison of the activity of the partially purfied HmU-glycosylase activity with other pyrimidine glycosylases. (A) The partially purified HmU-glycosylase activity, (B) MUG, (C) TDG, and (D) UDG. The oligonucleotide substrates used in this assay correspond to duplex 3 (Figure 1) and contain the central X:P base pair (X is the pyrimidine, P is the purine) indicated at the top of the figure. Ethenocytosine is indicated as C. All four glycosylases cleave the U:G mispair. All four glycosylases can be distinguished based upon their substrate recognition.

glycosylase activity was retained, resulting in a substantial increase in the ratio of the HmU to uracil glycosylase activities. Following chromatography on the HighQ resin, a fraction (F5, Figure 2) was found to contain substantial HmU activity. The apparent molecular weight of the HmU activity was determined to be 41K by sucrose gradient centrifugation. The fraction containing the highest HmU activity was subsequently used for characterization against the panel of oligonucleotides containing base analogues (duplex 3, Figure 1). As shown in Figure 3, the HmU-glycosylasecontaining fraction was active against uracil and HmU base pairs with A, but greater activity was observed in both cases when the pyrimidine was paired with G. No activity was observed against thymine-containing base pairs, and only background activity was observed against ethenocytosine base pairs. We note that the ethenocytosine-containing oligonucleotides can cleave spontaneously under the experimental conditions used here. The weak cleavage bands for the ethenocytosine-containing oligonucleotides seen in Figure 3A represent background hydrolysis rather than enzyme-mediated cleavage. The mispaired uracil glycosylase, MUG, was active against U:A and U:G base pairs, HmU:G and ethenocytosine base pairs (Figure 3B). The thymine-glycosylase TDG was active against all mispaired pyrimidines (Figure 3C),

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Figure 4. Effect of the uracil-glycosylase inhibitor protein, UGI, on UDG and the partially purified HmU-glycosylase. Uracil glycosylase (UDG) and the partially purified HmUglycosylase were incubated with uracil or HmU-containing oligonucleotides as described in the text. The reactions were conducted with (+) or without (-) the UGI protein. UDG is inhibited whereas neither U:G nor HmU:G activities of the partially purified HmU-glycosylase are inhibited.

Baker et al.

Figure 6. Cleavage of oligonucleotides containing HmU paired with a series of purine analogues. The sequence used in the assay corresponds to duplex 2 (Figure 1) where X ) HmU. The identity of the central X:P base pair is shown at the top of the figure. The purine analogues included 2-aminopurine (2AP), purine (P), and 2,6-diaminopurine, also known as 2-aminoadenine (2AA). The presumed structures of the analogue base pairs are shown in Figure 7.

HmU was paired opposite adenine, 2-aminopurine, or 2,6diaminopurine.

Discussion

Figure 5. Cleavage of oligonucleotides containing 5-substituted pyrimidines with increasing substituent size by the partially purified HmU-glycosylase. The assay conditions are described in the text. The sequence used in this assay corresponds to duplex 2 (Figure 1). The identity of the central X:P base pair is shown at the top of the figure. The analogues tested are 5-hydroxymethyluracil (H), 5-fluorouracil (FU), 5-chlorouracil (ClU), 5-bromouracil (BrU), and 5-iodouracil (IU). Analogues carrying 5-substituents larger than fluorine are not cleaved.

whereas uracil glycosylase UDG was active against uracil base pairs only (Figure 3D). We examined the ability of the uracil-glycosylase inhibitor protein to inhibit the uracil- and HmU-glycosylase activities. As shown in Figure 4, UGI-inhibitor strongly inhibits the activity of UDG against uracilcontaining substrates; however, the activities of the partially purified HmU-glycosylase toward uracil and HmU substrates is unaffected by UGI protein. To examine the apparent size of the pyrimidine-binding pocket of the HmU-glycosylase, we measured the activity against a series of 5-substituted pyrimidines with increasing van der Waals radii. As shown in Figure 5, uracil and HmU are good substrates, 5-fluorouracil is a weak substrate, and no activity is observed against thymine, 5-chloro, 5-bromo, or 5-iodouracil. No activity is observed against uracil or HmU in single-stranded substrates. Finally, we examined the activity of the partially purified HmU-glycosylase against a series of purines paired with HmU. As shown in Figure 6, HmU-glycosylase activity was observed when HmU was paired with guanine, hypoxanthine (deoxyinosine), and purine (deoxynebularine), but minimal activity was observed when

The pyrimidine oxidation damage product, HmU, may arise in DNA by oxidation of thymine or by oxidation and deamination of 5-methylcytosine generating HmU:A and HmU:G base pairs, respectively. Repair activities that remove HmU from HmU:A base pairs in DNA have been reported previously; however, the genes encoding these activities have not yet been identified (36, 37, 49). Recently, we utilized synthetic oligonucleotides containing HmU to demonstrate that human cells have abundant activity toward mispaired HmU (38). Here, we report the partial purification of the mispaired HmUglycosylase activity and describe the substrate preferences of this activity. In whole cell extracts, uracil-glycosylase activity is substantially greater than the HmU-glycosylase activity. Following chromatography on cellulose phosphate, the HmU-glycosylase activity is enriched by a factor of approximately 50 and is similar in magnitude to the uracil-glycosylase activity. The apparent molecular weight of the HmU activity was determined to be approximately 41 kDa, similar in size to several previously reported glycosylases. Analysis of the fraction containing the HmU-glycosylase activity by electrophoresis showed several bands in the region of the gel around the expected molecular weight. None of the bands were successfully identified using standard microsequencing and database searching techniques. The partially purifed HmU-glycosylase was active against HmU and uracil base pairs, with a strong preference for mispairs with guanine (Figure 3A). We therefore sought to characterize the partially purified extract by probing against a panel of base analogues as shown in Figure 3. We considered the possibility that our partially purified HmU-glycosylase could be the mispaired uracil-glycosylase MUG. MUG is one of several enzymes identified to date that can remove mispaired uracil residues from DNA. We observed in Figure 3B that MUG does cleave mispaired HmU, but not HmU paired with A. A distinguishing characteristic of MUG is its capacity to remove ethenocytosine residues from DNA. Our enzyme does not cleave ethenocytosine, and

Mispaired HmU-Glycosylase Activity

therefore, we conclude that our enzyme is not MUG. Further, our enzyme has no activity against singlestranded oligonucleotides containing either U or HmU, and therefore, our enzyme is not SMUG1 (30, 31). Another candidate glycosylase, mispaired thymineDNA glycosylase, TDG, removes mispaired thymine and mispaired uracil from DNA. The TDG from methanobacterium thermoautotrophicum recognizes the pyrimidines uracil, HmU, thymine, and ethenocytosine, but only when mispaired with G. Our HmU-glycosylase activity does not recognize mispaired thymine. A series of other glycosylases active against T:G have recently been reported, including MBD4 and the human TDG which shares essentially no homology with the thermophile TDG. Upon the basis of the inability of our HmU-glycosylase activity to recognize mispaired thymine, we conclude that our activity is distinct from the TDG family of glycosylases. We observed that UDG has no activity against HmUcontaining base pairs. Therefore, our HmU-glycosylase is not UDG. However, we also observed that our partially purified fraction contains substantial uracil-glycosylase activity. Uracil glycosylase is known to be inhibited by the uracil-glycosylase inhibitor protein, UGI. As shown in Figure 4, under conditions in which UGI protein results in complete inhibition of cloned UDG, no inhibition of uracil or HmU cleavage is observed with the partially purified HmU glycosylase activity. These data indicate that the HmU-glycosylase activity in HeLa cells is distinct from the predominant uracil glycosylase and that the uracil glycosylase activity that copurifies with the HmU-glycosylase is not UDG. We report here for the first time that MUG and TDG have activity against mispaired HmU. However, the partially overlapping but distinct substrate activities for MUG, TDG, UDG, and HmU-glycosylase described here suggest a potential problem with current strategies for the identification of DNA repair activities. Fractions of proteins could be searched for one that has HmUglycosylase activity. The protein could be sequenced and identification made based upon sequence homology with known proteins. As multiple glycosylases have overlapping substrate specificity, the potential for incorrect identification must be considered. We wished to further probe the HmU-glycosylase activity to determine how it recognizes damaged bases, and we therefore examined the activity of the partially purified fraction against a further panel of substrates. Two sets of substituted oligonucleotide substrates were used. In the first set, a series of 5-substituted uracils with increasing size were placed opposite G. In the second set, HmU was paired with a series of purines to generate the base pairs shown in Figure 7. Uracil glycosylase is known to discriminate between mispaired uracil and mispaired thymine. The basis of this recognition is believed to be the size of the 5-substituent. Structural studies have revealed the presence of a tyrosine residue that presents a steric block to substituents larger than hydrogen, including the thymine methyl group (13). Upon the basis of steric considerations, a glycosylase that had a binding pocket sufficiently large to recognize HmU would also be expected to allow smaller substrates, such as uracil and thymine, as does TDG. It was surprising to find that the HmUglycosylase activity failed to cleave mispaired thymine as described above. We therefore prepared a series of 5-substitued uracil derivatives, including the 5-fluoro,

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Figure 7. Presumed conformations of the analogue base pairs with HmU. The abbreviations used are HmU, 5-hydroxymethyluracil; A, adenine; G, guanine; 2AP, 2-aminopurine; I, hypoxanthine (deoxyinosine); 2AA, 2-aminoadenine (2,6-diaminopurine); P, purine (deoxynebularine).

chloro, bromo, and iodo analogues. As shown in Figure 5, the HmU-glycosylase activity cleaves the 5-fluoro analogue slightly and does not cleave the analogues with larger substituents. This result is quite surprising, suggesting that the pyrimidine-binding pocket of the HmU-glycosylase has been very tightly designed and that the selectivity for the 5-hydroxymethyl substituent is not based primarily on substituent size. Unlike UDG, which has only slight preference for mispaired uracil, both TDG and MUG have strong preferences for the mispaired pyrimidine. Structural studies with MUG indicate that this preference derives from the formation of specific contacts between the “widowed” guanine in the helix and amino acid residues of the glycosylase following exclusion of the mispaired pyrimidine (28). To determine if the observed preference of HmU glycosylase for the HmU:G mispair results from similar recognition of the G in the opposing strand, we constructed a series of base pairs in which HmU was paired with a series of purine derivatives. Surprisingly, the highest activity for the partially purified HmU-glycosylase was observed here with the HmU:hypoxanthine (deoxyinosine) pair which lacks the 2-amino group of G, followed by HmU:purine (deoxynebularine) and HmU: G. The HmU:A and the other analogue base pairs are cleaved at substantially lower rates. The structures of the HmU:A and HmU:G base pairs have been determined in solution by high field NMR methods to be pseudo Watson-Crick and wobble, respectively (50). The HmU:G and, presumably, the HmU base pair with hypoxanthine (51) both would be wobble structures, suggesting a preference of the HmU-glycosy-

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lase for a non-Watson-Crick geometry. However, purine is known to form one hydrogen bond with thymine in a pseudo Watson-Crick geometry (52), and yet the HmUnebularine base pair is a very good substrate for the HmU-glycosylase suggesting that the wobble configuration of the HmU base pair is not the primary determinant of substrate selectivity. Nebularine also lacks the N1imino proton and 2-amino group recognition elements used by MUG to identify the G residue of U:G mispair (28). Therefore, the selectivity of HmU-glycosylase for H:G over H:A base pairs is not based upon specific recognition of the widowed guanine. Singer and co-workers have previously emphasized the role of thermal and thermodynamic stability in determining the rates of glycosylase-mediated reaction rates (24). The melting temperatures of the HmU base pairs have not yet been determined; however, the relative thermal stability of the duplexes falls into two categories. The pseudo-Watson-Crick HmU:A base pair would be among the most stable. Transfer of the 6-amino group of adenine to the 2-position (2-aminopurine) would maintain pseudo Watson-Crick geometry and have only a minor effect upon the thermal stability (53, 56). Similarly, adding an amino group (2,6-diaminopurine) would maintain geometry and slightly elevate or depress the melting temperature, depending upon sequence (54, 59). None of these base pairs with HmU are good substrates for the HmUglycosylase. In contrast, replacement of adenine by guanine changes the geometry from pseudo Watson-Crick to wobble, and would likely result in a substantial decrease in melting temperature, as is observed when comparing the T:A and T:G base pairs (55). Similarly, replacement of adenine by hypoxanthine would change the geometry to wobble, and result in a substantial reduction in melting temperature (57). The replacement of adenine by purine would maintain pseudo-Watson-Crick geometry, but would result in the loss of one of the two hydrogen bonds. Consequently, the purine substitution would decrease the melting temperature (58). The three base pairs with HmU that are good substrates for the HmU-glycosylase would be less thermally stable than the three base pairs which are not good substrates. We conclude that the preference of the HmU-glycosylase for the HmU:G base pair results from the reduced thermal stability of the base pair, as opposed to specific recognition of the guanine residue. Upon the basis of the results obtained with the panel of analogues described here, we conclude that the predominant HmU-glycosylase in human cells is distinct from previously purified glycosylase activities including TDG, MBD4, MUG, and SMUG1. Data with the partially purified HmU-glycosylase and the 5-substituted uracil derivatives indicates that the pyrimidine recognition pocket of the HmU glycosylase is tightly configured and does not discriminate based upon the size of the 5-substituent alone. The preference for mispaired HmU appears related more to the thermal stability of the base pair rather than the presence of functional groups present on the mispaired purine. Efforts are currently in progress to purify the HmU-glycosylase reported here to homogeneity.

Acknowledgment. This work was partially supported by the National Institutes of Health (GM50351, CA84487).

Baker et al.

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