Influence of DNA structure on hypoxanthine and 1,N6-ethenoadenine removal by murine 3-methyladenine DNA glycosylase

Michael D. Wyatt1 and Leona D. Samson

Department of Cancer Cell Biology, Harvard School of Public Health, Boston, MA 02115, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
3-Methyladenine DNA glycosylases initiate base excision repair by flipping the nucleotide bearing the target base out of double-stranded DNA into an active site pocket for glycosylic bond cleavage and base release. Substrate bases for the murine 3-methyladenine DNA glycosylase (other than 3-methyladenine) include hypoxanthine and 1,N6-ethenoadenine, two mutagenic adducts formed by both endogenous and exogenous agents. Using double-stranded DNA oligonucleotides containing damaged bases at specific sites, we studied the relative removal rates for these two adducts when located in different sequence contexts. One of the sequence contexts was an A:T tract, chosen because DNA secondary structure is known to change along the length of this tract, due to a progressive narrowing of the minor groove. Here we report that removal rates for hypoxanthine, but not for 1,N6-ethenoadenine, are dramatically affected by its location within the A:T tract. In addition, the removal rates of hypoxanthine and 1,N6-ethenoadenine when paired opposite thymine or cytosine were examined, and in each sequence context hypoxanthine removal decreased by at least 20-fold when paired opposite cytosine versus thymine. In contrast, 1,N6-ethenoadenine removal was unaffected by the identity of the opposing pyrimidine. We conclude that the removal of certain bases by the mouse 3-methyladenine DNA glycosylase can be modulated by both adjacent and opposing sequence contexts. The influence of DNA sequence context upon DNA repair rates, such as those described here, may contribute to the creation of mutational hot spots in mammalian cells.

Abbreviations: AAG, human alkyladenine DNA glycosylase; Aag, mouse alkyladenine DNA glycosylase; AP, apurinic/apyrimidinic; BER, base excision repair; {varepsilon}A, 1,N6-ethenoadenine; Hx, hypoxanthine (inosine); 3-MeA, 3-methyladenine; MMS, methyl methanesulfonate.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
Base excision repair (BER) is one of several DNA repair pathways that help to maintain the stability of the genome. Incorrect or damaged bases are removed from double-stranded DNA by DNA glycosylases via cleavage of the N-glycosylic bond between the base and the deoxyribose, thus initiating BER. A 5'-AP (apurinic/apyrimidinic) endonuclease, or an AP lyase, generates a DNA strand break at the resulting abasic site and, after trimming of the ends to leave a 3'-hydroxyl and 5'-phosphate, the nucleotide gap is filled by DNA polymerase and the remaining DNA strand break rejoined by DNA ligase (1,2).

DNA glycosylases that remove replication-blocking 3-methyladenine (3-MeA) DNA adducts have been isolated from bacteria, yeast, plants and mammals (311). Although these DNA glycosylases are extremely efficient at removing 3-MeA lesions, most 3-MeA DNA glycosylases display a broad substrate range that includes numerous structurally diverse base lesions. For example, mouse and human 3-MeA DNA glycosylases have been shown to excise 7-methylguanine, 3-methylguanine, 7-hydro-8-oxoguanine (8-oxoG), hypoxanthine (Hx), 1,N6-ethenoadenine ({varepsilon}A) and 3,N2-ethenoguanine (6,1220). Several of these substrates are mutagenic DNA lesions that can be induced by endogenous cellular metabolites (21). It is therefore possible that BER initiated by mammalian 3-MeA DNA glycosylases could serve to limit spontaneous mutation rates as well as to protect against the toxic effects of methylating agents, such as methylmethane sulfonate (MMS) (22).

In order to study the role of mammalian 3-MeA DNA glycosylases in protecting against alkylation-induced toxicity and in limiting spontaneous mutation, the mouse 3-MeA DNA glycosylase gene (Aag) was cloned, and Aag null mouse embryonic stem cells were generated (6,22). Aag null cells are sensitive to the induction of chromosome aberrations, sister chromatid exchange, cell killing and apoptosis by MMS and MeOSO2(CH2)2-N-methylpyrrole dipeptide (methyl-lexitropsin), an agent that specifically produces 3-MeA DNA lesions (2224). In addition, the Aag null cells are sensitive to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and mitomycin C, two chemotherapeutic alkylating agents that produce more complex alkylation products. The Aag null embryonic stem cells were found to lack detectable 3-MeA, Hx and {varepsilon}A DNA glycosylase activity, suggesting that Aag is the major glycosylase for these lesions, at least in this cell type. Aag null mice were then generated for the purpose of examining Aag's role in the whole animal; the liver, testes and kidney were each found to lack any detectable 3-MeA, Hx and {varepsilon}A DNA glycosylase activity, suggesting that Aag is indeed the major DNA glycosylase in mice for several types of damage (14).

Evidence is accumulating that many different types of DNA-modifying enzymes, including DNA glycosylases, perform their function by flipping the nucleotide bearing the base to be excised out of the helix and into an active site pocket (25,26). Indeed, a nucleotide-flipping mechanism was demonstrated for the human 3-MeA DNA glycosylase (AAG, MPG) 3-MeA DNA glycosylase, co-crystallized with DNA containing a pyrrolidine transition state mimic (27). The crystal structure revealed that the AAG enzyme has a relatively flat DNA-binding surface that contacts both strands of the duplex DNA, primarily through phosphate contacts. The DNA is bent away from AAG at an overall angle of 22°, and the abasic pyrrolidine is rotated out of the DNA helix into an active site lined with amino acids that presumably interact with a target base. A tyrosine residue (Y162) enters the DNA helix via the minor groove and intercalates into the DNA to occupy the site vacated by the flipped out nucleotide. Three amino acids appear to form a rigid structure that press against the base opposing the flipped out abasic site, resulting in destabilization of the stacking interactions of the opposing base with its 5'-adjacent neighbor. Mouse Aag shares 83% sequence identity to human AAG over a 232 amino acid stretch that includes the active site pocket of AAG. Therefore, mouse Aag is also likely to act through a nucleotide-flipping mechanism. Factors affecting catalysis may therefore include interactions of the target base with its neighboring bases, both adjacent and opposing. A target base that interacts strongly with adjacent or opposing neighbors, because of a sequence-dependent local DNA conformation, may be more difficult to flip out of the helix and may thus be excised more slowly than a target base that does not interact strongly with its neighbors. Here we explore some structural determinants predicted to influence base removal by mammalian 3-MeA DNA glycosylases.

The removal of Hx and {varepsilon}A by Aag was determined with these modified bases located in different sequence contexts. These two bases were selected because they are easily incorporated into oligonucleotides using standard synthesis procedures. DNA sequence motifs with distinct sequence dependent structures were chosen and we demonstrate that motifs affecting the dimensions of the minor groove profoundly influence Aag-mediated Hx removal, while having little effect on {varepsilon}A removal. In addition, the base that is paired opposite the target base profoundly influences Hx, but not {varepsilon}A, removal. The implications of these results for understanding why certain DNA sequence motifs are hot spots for mutation are discussed.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
Purification of recombinant Aag
The mouse Aag cDNA was subcloned from pBE 1.1 (6) into the pCALn vector (Stratagene) to express Aag as an N-terminal fusion protein in-frame with the 4 kDa calmodulin-binding protein. The subcloned cDNA fragment contained a 43 amino acid N-terminal deletion designed to aid its purification; such an N-terminal deletion of 48 amino acids retains wild-type activity for 3-MeA removal while facilitating purification (28). Similar results were reported for a 63 amino acid N-terminal deletion of the human AAG enzyme (15). The pCALnAag vector was transfected into the alkylation sensitive MV1932 DE3 Escherichia coli (a kind gift from Dr Anne Britt, University of California, Davis). The pCALnAag-containing E.coli were induced with IPTG (final concentration of 1 mM) for 2 h at 37°C, lysed by sonication and then the cell debris removed by centrifugation. The fusion protein was purified using a calmodulin affinity resin following the protocol specified by Stratagene (La Jolla, CA). The eluent fractions were concentrated using a Centricon C10 filter, analyzed by SDS–PAGE and the protein concentration determined by Bradford analysis (BioRad). Crude extracts and protein isolated under identical conditions as above from the same bacterial strain transfected with the pCALn vector lacking the Aag cDNA provided the negative control. No Hx or {varepsilon}A DNA glycosylase activity was detected in four independent preparations of the negative control.

Oligonucleotides containing Hx and {varepsilon}A substrates
The substrate-containing oligonucleotides used in this study are shown in Figure 1Go. The -CAXGT-, -TTTTTX- and -AAAAX- symbols indicate mixed, thymine rich and adenine rich flanking sequence, respectively. For instance, -AAAAHx- signifies an oligonucleotide containing Hx flanked by an adenine tract on the 5' side and a mixed sequence on the 3' side. The -CAHxGT- oligonucleotide has the sequence 5'-GGATAGTGTCCA(Hx)GTTACTCGAAGC. The {varepsilon}A-containing oligonucleotide -CA{varepsilon}AGT- has the same sequence as -CAHxGT- except that an {varepsilon}A is positioned in place of Hx. The -TTTTTHx- oligonucleotide has the sequence 5'-GGATCATCGTTTTT(Hx)GCTACATCGC. The {varepsilon}A-containing oligonucleotide -TTTTT{varepsilon}A- has the same sequence as -TTTTTHx- except that an {varepsilon}A base is positioned in place of Hx. The -AAAAHx- oligonucleotide has the sequence 5'-GCGATGTAGCTAAAA-(Hx)CGATGATCC. The {varepsilon}A-containing oligonucleotide -AAAA{varepsilon}A- has the same sequence as -AAAAHx- except that an {varepsilon}A base is positioned in place of Hx. The -AAHxAA- oligonucleotide has the sequence 5'-GCGATGTAGCTAA(Hx)AACGATGATCC. The -HxAAAA- oligonucleotide has the sequence 5'-GCGATGTAGCT(Hx)AAAACGATGATCC. The -CAHxGT-, -TTTTTHx- and -AAAAHx- oligonucleotides were purchased from Amitof (Boston, MA). The -TTTTT{varepsilon}A- and -AAAA-{varepsilon}A- oligonucleotides were purchased from Midlands Certified Reagents (Midlands, TX). The -CA{varepsilon}AGT- oligonucleotide was a gift from Drs O.Scharer and G.Verdine, Harvard University. The -HxAAAA-, -AAHxAA- and all the complementary strand oligonucleotides were purchased from Ransom Hill Biosciences (Ramona, CA). Each of the oligonucleotides was polyacrylamide gel purified. Oligonucleotides were annealed to the complementary strand by heating in a 90°C water bath, then cooled to room temperature over 60 min. Annealed oligonucleotides with a thymine opposite the modified base are, for example, referred to as -CAHxGT-, -CA{varepsilon}AGT- (Figure 1Go, oligonucleotides 1–5). When the Hx and {varepsilon}A-containing oligonucleotides were annealed to a complementary strand containing a cytosine opposite the modified base, the oligonucleotides are, for example, referred to as -CAHxGT-:C, -CA{varepsilon}AGT-:C (Figure 1Go, oligonucleotides 6–8).



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Fig. 1. Sequences of the oligonucleotides, numbered from 1 to 8. The -CAXGT-, -TTTTTX- and -AAAAX- symbols indicate mixed, thymine rich and adenine rich flanking sequence, respectively. Oligonucleotide sequence 1, of mixed sequence, is referred to as -CAXGT-. Oligonucleotide sequence 2 contains the modified base immediately 3'- to the T tract and is referred to as -TTTTTX-. Oligonucleotide sequence 3 contains the modified base immediately 3'- to the A tract and is referred to as -AAAAX-. Oligonucleotide sequences 4 and 5 place Hx in the middle or 5'-end of the A tract and are referred to as -AAHxAA- and -HxAAAA-, respectively. Oligonucleotide sequences 6–8 are the same as 1–3 except that a cytosine is paired opposite Hx and {varepsilon}A, and are referred to as -CAXGT-:C, -TTTTTX-:C and -AAAAX-:C.

 
Oligonucleotide-based assay for DNA glycosylase activity
The oligonucleotides, containing site specifically located base lesions, were 5'-end labeled with T4 polynucleotide kinase and [{gamma}-32P]ATP, then annealed to a 2-fold excess of the appropriate complementary strand. Complete annealing was confirmed by electrophoresis on a 20% non-denaturing acrylamide gel. For the time course experiments, 100 fmol (5 nM) labeled and annealed Hx or {varepsilon}A-containing oligonucleotide and 1.45 µg Aag DNA glycosylase from the purified Aag preparation were used. The incubations were carried out in buffer containing 20 mM Tris–HCl pH 7.8, 100 mM potassium chloride, 2 mM EDTA, 1 mM EGTA and 5 mM 2-mercaptoethanol at 37°C for the times indicated. Following incubation, NaOH was added to 0.1 M and the samples were heated to 70°C for 30 min, in order to convert the abasic sites created by DNA glycosylases into DNA strand breaks. The DNA fragments were separated on a 20% denaturing polyacrylamide gel. Gels were exposed to a phosphorimaging screen and quantitation of the gels was carried out on a BioRad GS525 Molecular Imager. Relative initial rates of catalysis are means calculated using three data points within the linear range and corresponding to less than 50% of the total substrate in the reaction; the rates presented are derived from a minimum of two independent experiments and are expressed as fmol/min.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
The mouse Aag DNA glycosylase is highly likely to remove modified bases via nucleotide flipping, as shown for the human AAG (27) and other DNA glycosylases (25,26). We predicted that the interactions of the target base with neighboring and opposing bases could influence the efficiency of Aag DNA glycosylase function. We therefore set out to determine whether sequences known to affect local DNA conformation could influence Aag's efficiency. DNA containing a poly(dA): poly(dT) tract has an unusual conformation distinct from normal B-DNA (2932). Based upon structural studies, the stacking interactions between A:T base pairs within poly(dA):poly(dT) tracts result in a progressive narrowing of the minor groove from 5'- to 3'- on the poly(dA) strand (and thus 3' to 5' on the poly(dT) strand) (2932). Additionally, there is a disruption of A:T base stacking at the junction between a poly(dT) run and a purine immediately 3' to that run (3035). Figure 2AGo summarizes the structural changes known to occur at an A:T tract.



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Fig. 2. (A) Schematic representation of the unique structural features of an A:T tract deduced from structural studies. As a result of the stacking interactions between A:T base pairs, the minor groove width progressively narrows from 5'- to 3'- along the A tract. There is a disruption of the A:T base stacking at the junction between the T tract and a purine (R) immediately 3' to that run. (B–D) Detection of Aag-mediated Hx removal from the -TTTTTHx-, -AAAAHx- and -CAHxGT- oligonucleotides. Representative 20% denaturing polyacrylamide gels showing the conversion of the full length (B) -TTTTTHx-, (C) -AAAAHx- and (D) -CAHxGT- oligonucleotides to truncated 14-, 15- and 12mer fragments, respectively, as a function of time incubated with Aag. Identically prepared protein from E.coli containing the expression vector lacking the Aag cDNA had no detectable Hx or {varepsilon}A DNA glycosylase activity (lanes labeled `–'). The incubation times in minutes for each lane are listed below each gel.

 
Influence of sequence context upon Aag-mediated Hx removal
Hx removal by Aag was studied using oligonucleotides 1–3 shown in Figure 1Go. The Hx in the -CAHxGT- oligonucleotide is placed within a mixed sequence and opposite thymine; note that Hx:T base pairs would be produced upon adenine deamination in double-stranded DNA. The Hx in -TTTTTHx- lies immediately 3' to a (dT)5 tract and thus where the minor groove is the widest. The Hx in -AAAAHx- lies immediately 3' to a (dA)4 tract and thus where the minor groove is the narrowest. Figure 2B–DGo shows representative gels measuring the time course of Aag-mediated Hx removal from the -TTTTTHx-, -CAHxGT- and -AAAAHx- oligonucleotides, as visualized by the production of 14mer, 12mer and 15mer fragments, respectively (see Materials and methods). Identically prepared protein from E.coli containing an empty expression vector had no detectable Hx or {varepsilon}A DNA glycosylase activity (Aag –ve lanes, Figure 2B–DGo). Figure 3AGo and Table IGo show that the rate of Hx removal by Aag differed by as much as ~25-fold depending on the sequence context. The removal rate for Hx located immediately 3' to the (dT)5 (in the -TTTTTHx- oligonucleotide) was 39.8 fmol/min, whereas the removal rate for Hx located immediately 3' to the (dA)4 tract (in the -AAAAHx- oligonucleotide) was 1.53 fmol/min (Table IGo). The removal rate for Hx from the -CAHxGT- oligonucleotide was intermediate at 12.1 fmol/min (Table IGo).



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Fig. 3. (A) Aag-mediated Hx removal from three different oligonucleotide sequence contexts. The oligonucleotides were incubated with Aag for the times listed. Aag mediated Hx removal from the -CAHxGT- ({blacktriangleup}), -TTTTTHx- (•), and -AAAAHx- ({blacksquare}) oligonucleotides was measured by following the conversion of full length oligonucleotide to cleaved fragment. Error values (standard deviations) were calculated for each data point; however, error values less than the symbol size were not displayed. (B) Aag-mediated {varepsilon}A removal from three different oligonucleotide sequence contexts. The oligonucleotides were incubated with Aag for the times listed. Aag mediated {varepsilon}A removal from the -CA{varepsilon}AGT- ({blacktriangleup}), -TTTTT{varepsilon}A- (•) and -AAAA{varepsilon}A- ({blacksquare}) oligonucleotides was measured by following the conversion of full length oligonucleotide to cleaved fragment.

 

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Table I. Relative initial rates of Aag-mediated removal of Hx and {varepsilon}A from different sequence contextsa
 
Influence of sequence context upon Aag-mediated {varepsilon}A removal
Aag-mediated removal of {varepsilon}A was examined in the same three sequence contexts as shown in Figure 3AGo for Hx (Figure 1Go, oligonucleotides 1–3). {varepsilon}A is an alkylation product of adenine that is paired opposite thymine when it is produced in double-stranded DNA. Surprisingly, the rates of {varepsilon}A removal from this set of sequence contexts differed from each other by less than 2-fold (Figure 3BGo, Table IGo). Removal of an {varepsilon}A located immediately 3' to the (dA)4 tract appeared to be slightly better (7.0 fmol/min) than that located immediately 3' to the (dT)5 tract (5.2 fmol/min), while {varepsilon}A removal from -CA{varepsilon}AGT- (4.2 fmol/min) was the least efficient.

Influence of A:T base pair stacking upon Aag-mediated Hx removal
Structural studies demonstrate that the minor groove width decreases progressively from 5' to 3' along oligo(dA) tracts (2932). In order to test whether Aag-mediated Hx removal within the (dA)4 tract is progressively affected by the minor groove narrowing, two additional oligonucleotides were studied (Figure 1Go, oligonucleotides 4 and 5). In the -HxAAAA- oligonucleotide, the Hx is located immediately 5' to the (dA)4 tract and is paired opposite the 3'-T of the (dT)5 run on the complementary strand. The minor groove width within oligo(dA) tracts is least affected at the 5' end of the poly(dA) strand, presumably because, on the complementary strand, the junction of the 3'-T in the poly(dT) run and the neighboring purine disrupts base stacking (3035). The rate of Hx removal in the -HxAAAA- oligonucleotide (24.5 fmol/min) was substantially higher than that seen for Hx located immediately 3' to the (dA)4 tract in the -AAAAHx- oligonucleotide (1.53 fmol/min; Figure 4Go and Table IGo). In the -AAHxAA- oligonucleotide, the Hx lies in the middle of the (dA)4 tract, and its rate of removal (12.3 fmol/min) was intermediate to that seen for the other two sequence contexts. We infer that as the minor groove narrows, due to base stacking between A:T base pairs, the efficiency of Aag-mediated Hx removal decreases.



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Fig. 4. Aag-mediated Hx removal placed at different positions along the A tract. The oligonucleotides containing Hx placed at the 5'-end of the A tract (x), at the 3'-end ({blacksquare}) and in the middle of the A tract ({blacklozenge}) were incubated with Aag for the times listed.

 
Influence of the opposing base upon Aag-mediated Hx removal
Hx can pair with cytosine and induces predominantly A:T to G:C transition mutations in bacterial and mammalian cells (3638). In order to test whether Aag can discriminate between Hx:T base pairs formed in double-stranded DNA and Hx:C base pairs formed upon DNA replication, the -CAHxGT, -TTTTTHx- and -AAAAHx- oligonucleotides were each annealed to complementary strands that placed either thymine or cytosine opposite the Hx (Figure 1Go, oligonucleotides 1–3 and 6–8). For each sequence context, pairing Hx with cytosine dramatically reduced Aag-mediated Hx removal relative to removal of Hx paired with thymine (Figure 5AGo); in each context the reduction was at least 20-fold (Table IGo). It is interesting to note that the fastest repair rate for Hx opposite C (in the -TTTTTHx- context) is roughly the same as the slowest repair rate for Hx opposite T (in the -AAAAHx- context). In other words, although Hxs opposite cytosines are generally removed more slowly than Hxs opposite thymines, this does not mean that all Hxs opposite cytosines are repaired more slowly than all Hxs opposite thymines.



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Fig. 5. (A) Aag-mediated Hx removal opposite thymine or cytosine. The -TTTTTHx- (top panel), -CAHxGT- (middle) or -AAAAHx- (bottom) oligonucleotides were annealed to complementary strands that placed cytosine ({circ}, {square}, {triangleup}) or thymine ( •, {blacksquare}, {blacktriangleup}) opposite Hx. The oligonucleotides were incubated with Aag for the times listed. (B) Aag-mediated {varepsilon}A removal opposite thymine or cytosine. The -TTTTT{varepsilon}A- (top panel), -CA{varepsilon}AGT- (middle) or -AAAA{varepsilon}A- (bottom) oligonucleotides were annealed to complementary strands that placed cytosine ({circ}, {square}, {triangleup}) or thymine (•,{blacksquare},{blacktriangleup}) opposite {varepsilon}A. The oligonucleotides were incubated with Aag for the times listed.

 
Influence of the opposing base upon Aag-mediated {varepsilon}A removal
{varepsilon}A can pair with cytosine and induces predominantly A:T to G:C transition mutations in bacterial and mammalian systems (39,40). The -CA{varepsilon}AGT-, -TTTTT{varepsilon}A- and -AAAA{varepsilon}A- oligonucleotides were annealed to complementary strands that placed either thymine or cytosine opposite {varepsilon}A (Figure 1Go, oligonucleotides 1–3 and 6–8). For each sequence context, {varepsilon}A was removed at the same rate whether paired with cytosine or thymine (Figure 5BGo and Table IGo). Thus, in contrast to Hx, {varepsilon}A removal by Aag is not greatly affected by either the neighboring sequence context or the opposing base.

Effect of substrate concentration
The oligonucleotide substrate concentration used in all the reaction time courses presented in this study was 5 nM. In order to confirm that the initial reaction rate values obtained at this concentration were well within the linear range for each substrate, we measured the initial reaction velocity for Aag over a wide substrate concentration range, namely 12.5–200 nM. Incubation times where less than 25% of the substrate (at 5 nM) was converted to product were chosen; this corresponded to a 30 s time point for the -TTTTTHx- oligonucleotide, a 60 s time point for the -CAHxGT- oligonucleotide, and a 5 min time point for the -AAAAHx- oligonucleotide. Figure 6Go shows that the initial velocity values increased over the entire substrate concentration range, and that the velocity measured with 5 nM substrate is well below the Vmax for each oligonucleotide. Importantly, the effect of sequence context is apparent across the entire range of substrate concentrations examined. The plot shows that the sequence context effect on Aag-mediated removal is not dependent on substrate concentration.



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Fig. 6. Plot of initial velocity versus substrate concentration. The plot shows initial velocity (fmol/min) versus substrate concentration (nM). The time point for the -TTTTTHx- ({blacklozenge}) oligonucleotide was 30 s. The time point for the -CAHxGT- ({blacktriangleup}) oligonucleotide was 60 s. The time point for the -AAAAHx- ({blacksquare}) oligonucleotide was 5 min. Error bars were calculated from four independent experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
Each mutagen produces a characteristic mutational spectrum, and the factors influencing these spectra include the following: the chemical nature and distribution of the induced DNA damage; how the DNA replication machinery copies the damaged template; and the efficiency of DNA repair at the damaged sites. It is now becoming clear that the local sequence context of certain kinds of DNA damage can modulate some of these factors. For instance, the in vivo distribution of induced DNA damage has been shown to be non-random for several mutagenic agents, including UV damage, 1-nitrosopyrene and benzo[a]pyrene (4144). The in vivo repair rates for pyrimidine dimers in the p53 gene were found to be highly variable and sequence dependent, and the sites at which dimer repair is relatively slow correspond to p53 mutational hotspots in skin tumors (45). A recent report noted differences of up to 185-fold for the repair of 7-MeG at different positions (46). These results suggest that the influence of sequence context on the distribution of DNA damage and on the efficiency of repair helps to shape mutational spectra. DNA sequences that are highly susceptible to damage but refractory to repair may contribute to the presence of mutational hotspots in the genome. Here we explore the influence of DNA sequence context upon repair by the mammalian Aag DNA glycosylase, known to be the major DNA glycosylase for the repair of three types of endogenous DNA base damage, namely, 3-MeA, Hx and {varepsilon}A (14,47).

There are relatively few reports documenting DNA sequence context effects on base removal by DNA glycosylases. Uracil DNA glycosylases (UDGs) from calf thymus and E.coli have been shown to vary by up to 15-fold in their ability to remove uracil from different sequence contexts, and at least for E.coli, these differences appear to contribute to the spontaneous mutation spectrum (48,49). In order to identify sequence contexts that might significantly enhance the mutagenicity of 8-oxoG, Hatahet et al. (50) used an in vitro selection process to identify sequence contexts that affected the rate of repair and/or the frequency of misincorporation during DNA synthesis. For the human AAG DNA glycosylase, a correlation between the thermal stability of {varepsilon}A-containing 15mer oligonucleotides and {varepsilon}A base removal has been reported (51). Here we report up to ~25-fold differences in the ability of mouse Aag DNA glycosylase to remove Hx bases embedded within different sequence contexts, where the sequence dependent effect is likely to be the result of changes in the local secondary structure. Surprisingly, the sequence contexts that dramatically affect Hx removal have little effect on {varepsilon}A removal by the same enzyme.

The general applicability of a nucleotide flipping mechanism for removing damaged bases is underscored by X-ray crystal studies of five different DNA glycosylases (27,5255). The structures of AlkA and the human AAG proteins are particularly interesting because, despite the fact that these two enzymes have quite different folds, they excise a similar range of substrates. The structural feature that AlkA and AAG do share is an active site rich in aromatic amino acids and a catalytically essential carboxylate (aspartate in AlkA, glutamate in AAG) that is thought to activate a water for nucleophilic attack to release the base (27,52). The mouse Aag and human AAG cDNAs share a strong identity (83% over 232 amino acids). The dramatic differences in the ability of Aag to remove Hx from each particular sequence context are presumably due to the nature of Aag's interaction with the local DNA conformation in which the target Hx base is located. In the three-dimensional structure of AAG complexed to pyrrolidine-containing DNA, the DNA helix is bent away from the AAG protein at an approximate angle of 22°. The contacts that AAG makes with the DNA helix appear to be limited to phosphate backbone contacts, except for the tyrosine that inserts into the DNA helix, and residues 164 and 165 (27). Methionine 164 and tyrosine 165 appear to press against the base opposing the flipped out abasic site, destabilizing the stacking interactions of the opposing base with its 5' adjacent neighbor. Because tyrosine 162 inserts into the DNA helix via the minor groove, a narrowing of the minor groove width might reduce the efficiency of such interactions. Alternatively, it may be harder for the glycosylase to induce structural perturbations in the DNA at such sequences where the minor groove width narrows. DNase footprinting of AAG bound to an abasic site indicated that AAG covered a span of approximately seven base pairs (56), in agreement with the crystal structure. For DNA repair enzymes that locate damaged bases amongst vast tracts of undamaged bases, it would make sense for the enzyme to contact DNA in a base sequence-independent manner. The present study indicates, however, that the mouse Aag glycosylase might be susceptible to certain structural features of DNA.

{varepsilon}A differs from Hx in several respects. Whereas Hx can form hydrogen bonds with both thymine and cytosine (38), the hydrogen bonding face for {varepsilon}A is eliminated by the alkene bridged between the purine ring N1 and the exocyclic N6 positions. NMR studies indicate that although {varepsilon}A:T base pairs do not disrupt the hydrogen bonding of adjacent base pairs, the {varepsilon}A:T base pair itself is not coplanar; in fact the thymine is displaced in order to accommodate the exocyclic alkene ring of the {varepsilon}A base. Such non-coplanar base pairs would clearly disrupt base stacking adjacent to the {varepsilon}A-containing base pair. Thus, unlike Hx, the `flippability' of {varepsilon}A may be independent of hydrogen bonding with the opposing base, and independent of base stacking interactions with adjacent bases because these interactions are already disrupted. Differences in {varepsilon}A removal were previously noted for AA{varepsilon}AAA or TT{varepsilon}ATT versus GG{varepsilon}AGG or CC{varepsilon}ACC sequence contexts (51), and such differences appeared to correlate with the thermal stability of the 15mer duplexes.

DNA base lesions that are mutagenic because they mispair during replication present a special problem to the cell, a problem not shared by cytotoxic DNA base lesions that simply inhibit replication. This problem centers on the timing of the initiation of BER; if BER is initiated at the base lesion after it has formed a mispair (e.g. after Hx has paired with cytosine), then the completion of BER will rapidly fix a base substitution mutation into the genome using the mispaired base as template. In E.coli, this problem is solved for 8-oxoG lesions that frequently mispair with adenine in two complementary ways: (i) the major 8-oxoG DNA glycosylase (MutM, fpg) efficiently removes 8-oxoG from 8-oxoG:C base pairs and is almost inactive for removing 8-oxoG from 8-oxoG:A mispairs; (ii) another DNA glycosylase (MutY) can efficiently remove adenine from the 8-oxoG:A mispairs that are formed during replication. Together, MutM and MutY reduce the induction of G:C to T:A transversions at 8-oxoG lesions. Here we find that the Aag enzyme strongly discriminates between Hx lesions paired with thymine or cytosine. A Hx DNA glycosylase displaying a similar discrimination was previously reported (57), but whether this activity represents an Aag homolog is not clear. It is currently unclear why Aag excises Hxs paired with T better than those paired with C, but it should be noted that, in crystal structure studies, Hx:T and Hx:C base pairs adopt different conformations. In a crystal structure of a DNA octamer, a Hx:T base pair adopted a wobble conformation (58). In contrast, an Hx:C base pair adopted a very similar conformation to a G:C base pair in a dodecamer crystal structure (59). It should also be noted that the ability of Aag to discriminate between Hx:T and Hx:C base pairs would be most beneficial to the organism if there exists a complementary DNA glycosylase for the removal of cytosine from the Hx:C mispair. Such a repair activity is currently being sought.

DNA sequence motifs that are particularly susceptible to mutation, such as minisatellites, triplet repeats, inverted repeats and homopolymeric runs have been termed `at risk motifs' (ARMs) and as such represent mutational hotspots (60). Indeed, it was recently shown that the inheritance of an ARM in the human APC gene confers a predisposition to colon cancer, even though the APC allele containing the ARM encodes a perfectly functional protein; this particular ARM contains a homopolymeric run of As which creates a local hypermutable region in the APC gene (61). Most of the ARMs thus far characterized appear to influence DNA replication fidelity. DNA sequences that are refractory to DNA repair may represent another important class of ARMs, especially if these sequences are also prone to DNA damage. An understanding of how sequence context influences DNA repair efficiency will contribute to our understanding of the factors that govern why some regions of the genome are mutational hotspots.


    Note added in proof
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
Two recent publications report similar findings for the preference of the human, rat, E.coli AlkA and S.cerevisiae MAG 3-MeA DNA glycosylases for Hx paired opposite T versus Hx paired C. [Saparbaev et al. (2000) Nucleic Acids Res., 28, 1332; Asaeda et al. (2000) Biochemistry, 39, 1959.]


    Acknowledgments
 
This work was supported by NIH grants RO1 CA55042 and PO1 ES03926 to L.S. L.S. was supported by a Burroughs Wellcome Toxicology Scholar Award. M.D.W. was supported by a National Institutes of Health Radiation Biology Training Grant 5T32CA09078 and an NIH Individual Fellowship Award CA73135-01.


    Notes
 
1 To whom correspondence should be addressed at: Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of South Carolina, Columbia, SC 29208, USA Email: wyatt{at}cop.sc.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
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Received November 1, 1999; revised January 12, 2000; accepted January 21, 2000.