Structure and activity of a thermostable thymine-DNA glycosylase: evidence for base twisting to...

doi:10.1006/jmbi.2001.5264 available online at http://www.idealibrary.com on J. Mol. Biol. (2002) 315, 373±384

Structure and Activity of a Thermostable Thymine-DNA Glycosylase: Evidence for Base Twisting toRemove Mismatched Normal DNA Bases

Clifford D. Mol1, Andrew S. Arvai1, Thomas J. Begley2

Richard P. Cunningham2* and John A. Tainer1*

1The Scripps Research InstituteDepartment of MolecularBiology MB4, and, SkaggsInstitute for Chemical Biology10550 North Torrey Pines RdLa Jolla, CA 92037, USA2Department of BiologicalSciences, Center forBiochemistry and Biophysics,State University of New York-Albany, Albany, NY12222, USA

Present address: T. J. Begley, DepBioengineering and EnvironmentalMassachusetts Institute of Technolo16-771, Cambridge, MA 02139-4309

Abbreviations used: HhH, helix-hmismatch glycosylase; m5C, 5-meth3-methyladenine DNA glycosylaseendonuclease III; UDG, uracil-DNAapurinic:apyrimidinic; MBD, methy

E-mail addresses of the [email protected]; [email protected]

0022-2836/02/0030373±12 $35.00/0

The repair of T:G mismatches in DNA is key for maintaining bacterialrestriction/modi®cation systems and gene silencing in higher eukaryotes.T:G mismatch repair can be initiated by a speci®c mismatch glycosylase(MIG) that is homologous to the helix-hairpin-helix (HhH) DNA repairenzymes. Here, we present a 2.0 AÊ resolution crystal structure and comp-lementary mutagenesis results for this thermophilic HhH MIG enzyme.The results suggest that MIG distorts the target thymine nucleotide bytwisting the thymine base �90 � away from its normal anti positionwithin DNA. We propose that functionally signi®cant differences exist inDNA repair enzyme extrahelical nucleotide binding and catalysis that arecharacteristic of whether the target base is damaged or is a normal basewithin a mispair. These results explain why pure HhH DNA glycosylasesand combined glycosylase/AP lyases cannot be interconverted by simplyaltering their functional group chemistry, and how broad-speci®cityDNA glycosylase enzymes may weaken the glycosylic linkage to allow avariety of damaged DNA bases to be excised.

# 2002 Academic Press

Keywords: DNA repair; DNA glycosylase; DNA mismatch; methylation;base twisting
*Corresponding authors
Introduction

Cytosine methylation is the most common meth-od used by cells to control epigenetic processesthat alter the cellular phenotype while maintainingthe genetic integrity of DNA. In bacteria, the meth-ylation of cytosine to 5-methylcytosine (m5C)within target sequences prevents their DNA frombeing cleaved by endogenous restriction endonu-cleases that protect the cell from viral and otherpathogens. In humans and higher eukaryotes, cyto-sine methylation at CpG islands clustered near

artment ofHealth,gy, 77 Mass Ave., USA.airpin-helix; MIG,ylcytosine; AlkA,II; Endo III,glycosylase; AP,

l-binding domain.ding authors:

gene promoters ensures that the expression ofinappropriate genes is repressed in different tissuesand during development and differentiation.1 ± 4

While promoter hypermethylation of DNAdamage response and repair protein genes hasbeen implicated in breast5 and colorectal cancers,6

genome-wide loss of methylation may be an earlystage in carcinogenesis and leads to highermutation rates and chromosomal anomalies.7,8

Both bacterial and human cells face a fundamentalproblem, however, in maintaining the methylationstate of target cytosine bases, in that m5C bases areparticularly susceptible to spontaneous, hydrolyticdeamination.9 Accordingly, single base substi-tutions at m5C nucleotides account for approxi-mately one third of the mutations seen in heritablehuman diseases,10 despite the fact that m5Cnucleotides comprise only about 3 % of the totalcytosine in human DNA.11,12

Unlike general cytosine deamination, whichcreates U:G wobble mispairs that can be ef®cientlyrepaired by uracil-DNA glycosylase (UDG), theT:G mispairs resulting from m5C deamination

# 2002 Academic Press

374 Base Twisting in DNA Mismatch Repair

must be repaired by distinct enzymes that speci®-cally recognize T:G mismatches. In bacteria, theenzymes possessing this activity include the veryshort-patch repair (VSR) endonuclease,13 whichnicks the DNA backbone 50 of a T:G mismatch,and the T/U:G mismatch speci®c glycosylase(MUG). Despite low amino acid sequence simi-larity, MUG is structurally homologous toUDG,14,15 but lacks the base-speci®c, hydrogen-bonding interactions seen in UDG:DNA complex-es.16 ± 18 In humans, enzymes that recognize T:Gmismatches include the MUG homolog thymine-DNA glycosylase termed TDG,19 and also MBD4,20

which contains a small m5CpG.TpG methyl-bind-ing domain (MBD) linked to a DNA repair domainthat belongs to the helix-hairpin-helix (HhH)superfamily of DNA repair enzymes.21,22 T:G mis-match-speci®c HhH DNA glycosylases also includethe mismatch glycosylase (MIG) from thehyperthermophilic archaebacteria Pyrobaculum aero-philium23 and the thermophile Methanobacteriumthermoformicicum.24 This latter MIG enzyme isfound on a cryptic plasmid and functions within aDNA restriction modi®cation system that methyl-ates the internal cytosine in a CCGG sequence25

and acts in DNA base excision repair to counteractspontaneous mutagenic deamination of these m5Cresidues.26 We have previously termed this M. ther-moformicicum T:G mismatch-speci®c HhH enzymethymine-DNA glycosylase II (TDGII),24 but nowadopt theMIG nomenclature to avoid confusion with thestructurally unrelated human TDG enzyme,19

which is not a member of the HhH DNA repairenzyme superfamily.27

The HhH enzymes target distinct base lesionsand function in DNA repair as either pure glycosy-lases or combined glycosylase/apurinic:apyrimidi-nic (AP) lyases. The structure of endonuclease III(Endo III) established the HhH enzyme fold,21,28

consisting of two a-helical domains with the HhHmotif and enzyme active site localized at thedomain interface. Crystal structures of HhH DNArepair enzymes include: Endo III, which cleavesoxidized pyrimidines; the mismatch repair enzymeMutY,29 which cleaves adenine mispaired withguanine and 8-oxoguanine;30 human Ogg1,31

which removes 8-oxoguanine paired withcytosine;27 and 3-methyladenine DNA glycosylaseII (AlkA),32,33 which removes a variety of alkylatedpurine bases.34 Additionally, a MutY:adenine com-plex,29 and co-crystal structures of AlkA35 andhuman Ogg1 with DNA31 have delineated the gen-eral structural features of HhH DNA repairenzymes that govern DNA binding and catalysis.However, only the combined glycosylase/AP lyaseOgg1:DNA structure contains a damaged base inthe recognition pocket, and there is no structure-based model for how an HhH glycosylase speci®-cally recognizes and cleaves a mismatched thyminebase from DNA.

The HhH motif consists of two a-helices withan intervening b-hairpin loop that contains the

conserved sequence (LPGVGX(K/S)), which non-speci®cally binds the DNA backbone via theglycine backbone amide groups. Aside from theDNA glycosylase/AP lyases, the HhH motif isalso found in diverse enzyme families includingDNA polymerases36 and the Holliday junction-binding protein RuvA.37 For the base excisionrepair enzymes, the HhH motif impacts directlyon the excision chemistry at the active site.Combined glycosylase/AP lyase enzymes, suchas Endo III and human Ogg1, contain a lysineresidue in their HhH motifs that forms a Schiffbase at the C10 atom of the target nucleotide.Breakdown of this covalent enzyme:DNA inter-mediate by b-elimination results in cleavage ofthe DNA backbone 30 of the target nucleotide,conferring these enzymes with AP lyase activity.In contrast, the pure HhH DNA repair glycosy-lases, which do not contain a lysine residue intheir HhH motifs, generally do not formcovalent enzyme:DNA intermediates and arethought to cleave the target glycosylic bond viaattack of an activated water nucleophile. TheT:G mismatch-speci®c HhH enzymes, includingMIG and MBD4, are glycosylases without APlyase activity, containing a conserved tyrosinerather than a lysine residue in their HhH motifs.Mutation of this tyrosine residue to lysine con-fers M. thermoformicicum MIG with AP lyaseactivity against pre-exisiting AP sites in DNA,but destroys T:G mismatch-speci®c glycosylaseactivity.24

Here we present a 2.0 AÊ resolution crystal struc-ture and mutational results for the M. thermoformi-cicum T:G mismatch speci®c HhH MIG enzyme.These data provide the ®rst description of keyinteractions for the recognition and cleavage of anextrahelical thymine nucleotide by an HhH glyco-sylase. The results suggest that the MIG HhHmotif tyrosine residue is key to binding an extra-helical thymine base, and suggest a unifyingstructural and mechanistic feature of DNA repairenzymes that are targeted to normal DNA baseswithin mispairs. These enzymes may enhance theircatalytic power by physically distorting the targetextrahelical nucleotide. This model also highlightsa potential structural and functional discriminationbetween the pure HhH glycosylases and the com-bined HhH glycosylase/AP lyases, in which thelatter enzymes do not distort their target nucleo-tides. Moreover, the results also suggest howbroad-speci®city DNA glycosylases may achievecatalysis despite having enzyme active sites thatare only weakly complementary to target baselesions.

Results and Discussion

MIG structure and HhH enzyme homology

To initiate structure-function analyses of HhHglycosylase-initiated repair of T:G mismatches, wehave cloned, expressed, characterized, and deter-

Base Twisting in DNA Mismatch Repair 375

mined the crystal structure of a thermophilic thy-mine-DNA mismatch glycosylase enzyme, MIG(see Materials and Methods). Phylogenetic analysisindicates that the MIG enzymes are more closelyrelated to MutY than to other members of the HhHenzyme superfamily.23 The amino acid sequence ofMIG bears the hallmarks of the HhH DNA glyco-sylases, including the conserved, catalytic asparticacid residue, the DNA-binding HhH motif, andthe cysteine residues that ligate the four iron/foursulfur (Fe4S4) cluster, which is a commonlyobserved structural feature in the HhH DNA repairenzymes. Importantly, MIG and the glycosylasedomain of human MBD4 share distinctivesequences, including the HhH motif tyrosine resi-due, that are not generally found in other HhHenzymes (Figure 1).

The HhH enzyme superfamily is typi®ed by thecore, two-domain architecture with the active sitelocated at the interface between the six-helix barreland four-helix domains. The six-helix barreldomain contributes the HhH motif and minor-groove, nucleotide-¯ipping wedge, and the four-helix domain provides the conserved, catalytic Aspresidue (Figure 2(a)). This modular organization ofthe HhH enzymes allows for additional proteindomains or loops to be appended to the amino or

Figure 1. Structure-based sequence alignments for MIGMBD4, MutY, Endo III, AlkA, and Ogg1. Residues of the coDNA helix at the ¯ipped-out target nucleotide are boxed.domain (a6[a-f]) and four-helix domain (a4[a-d]) are listed abcated by a dot. For MIG and MBD4 the alignment is basedof MIG with MutY, Endo III, AlkA, and Ogg1 is based on aresidues implicated in activity by enzyme kinetics and mutag

carboxyl termini, which can affect enzyme activityas in MutY,38 or contribute to enzyme speci®city,as seen in the human Ogg1:DNA structure.31 Thetwo core domains are joined by ¯exible hingeloops, and variations in the relationship betweenthe two domains suggest that HhH DNA repairenzymes close down onto their bound target extra-helical damaged nucleotides.29,32 This interdomain¯exibility can also complicate crystal structure ana-lyses, and we determined the crystal structure ofMIG by the molecular replacement technique usinga series of overlaid homologous enzyme modelsthat mimic this interdomain variation (seeMaterials and Methods). The MIG amino acidsequence was ®t to bias-reduced omit electron den-sity maps and the structure, consisting of residues3 to 219, re®ned to a crystallographic R value of0.211 (Rfree � 0.248) for all data to 2.0 AÊ resolution.

The two-domain MIG structure (Figure 2(a))exhibits strong homology to other Fe4S4 cluster-containing HhH enzymes, including Endo III andMutY, but also resembles the more divergent non-cluster containing enzymes such as AlkA andOgg1 (Figure 2(b)). This structural similarity is con-centrated in the six-helix barrel domain with mostof the Ca atoms of MIG superimposable with EndoIII and MutY within a root-mean-square (rms)

and the HhH glycosylase/AP lyase family includingnserved HhH motif, and the residue that penetrates theMIG helices and nomenclature for the six-helix barrelove the alignment, and every tenth MIG residue is indi-on amino acid sequence homology, while the alignmentstructural superposition performed with SEQUOIA. MIGenesis (Table 1) are shown in red.

Figure 2 (legend opposite)



deviation of <1.5 AÊ , and nearly three-quarterssuperimposable with AlkA and Ogg1 residues at2-2.5 AÊ rms deviation (Figure 2(b)). These lattertwo enzymes both contain a loop insertionbetween helices a6b and a6c that packs with theunique amino-terminal domains in these enzymes(Figures 1 and 2(b)). The relationship between thetwo MIG domains falls within the range observedin other HhH DNA repair enzyme structures. ForMIG this interdomain angle is similar to thedomain relationships observed in the DNA-boundhuman Ogg131 and adenine-bound MutYstructures,29 and lies between the most open EndoIII structure and most closed AlkA and AlkA:DNAstructures.32,33,35

For all ®ve HhH enzymes with known struc-tures, the more diverse four-helix domains adoptthe same right-handed four-a-helix bundle top-ology, consisting of an N-terminal helix, a4a,and the three contiguous C-terminal a-helices,a4b-a4d that follow the conserved Asp residue(Figures 1 and 2(a) and (b)). A number of thesea-helices, from both the four-helix bundle andsix-helix barrel domains, direct their positivelycharged amino-terminal helix dipoles towardsthe interdomain active site cleft and contributeto forming the electrostatically positive DNA-binding surface (Figure 2(c)) that likely orientsthe enzyme with the target DNA substrate.Importantly, M. thermoformicicum MIG has signi®-cant structural and functional similarity withother HhH DNA repair enzymes, including thelocation of the damaged-base binding pocket,and the DNA minor groove-binding, nucleotide-¯ipping motif.

Minor groove DNA binding andnucleotide flipping

The speci®c homology MIG shares with knownHhH enzyme structures allows for particularenzyme residues to be assigned roles in bindingT:G mismatch-containing DNA. Inspection of theAlkA:DNA complex,35 the Ogg1:DNA complex,31

and the MutY:adenine complex29 structures over-laid with that of MIG, shows how the bent DNAand/or extrahelical nucleotides from these struc-tures may be accommodated by the MIG DNA-

Figure 2. Stereo views of the MIG structure, enzyme homoillustrating the two-domain architecture with the a-helices (pdomain (bottom) numbered sequentially. The a-helices and bsulfur cluster (large spheres), and the positions of key resshown. (b) The MIG structural homology with other HhHknown structures of HhH DNA repair enzymes are supeaccording to conserved structural elements within their sixin the same orientation as shown in (a), and colored by�2.0 kT/e). The positively-charged DNA-binding face of MIsylases and combined glycosylase/AP lyases and assists inlesions into the active site cleft. DNA (red tubes) is shownAlkA:DNA complex structure.

binding surface and active site (Figure 2(c)).Aspects of MIG speci®city and activity may derivefrom the particular amino acid side-chain thatinserts through the minor groove and into theDNA base stack to ¯ip the target thymine nucleo-tide into the active site. Despite strong structuralconservation of the nucleotide-¯ipping wedge and¯anking a-helices (a6a and a6b, Figures 1 and2(b)), the amino acid sequence, and in particularthe identity of the residue that penetrates the DNAminor groove, varies widely among the HhHenzymes. Based on structural homology, this resi-due is Arg47 in MIG, while at the homologousposition in AlkA and Ogg1 a leucine or an aspara-gine residue, respectively, penetrates the DNAminor groove,31,35 and both MutY and Endo IIIcontain a glutamine residue at this position(Figure 1).

In other DNA repair enzyme systems, replace-ment of a DNA-intercalating leucine residue witharginine in human UDG slows enzyme dissociationfrom product DNA,17,18 whereas removal of anintercalating arginine residue in human AP-endo-nuclease-1 results in a faster, more ef®cientenzyme.39 The rate of product dissociation fromMIG is 0.606 minÿ1 at 65 �C, indicating that theenzyme remains bound to DNA for about twominutes after catalysis (T.J.B and R.P.C, unpub-lished results). Thus, by inserting through theDNA minor groove and into the base stack tointeract with DNA phosphate groups, Arg47 mayslow product dissociation at elevated temperaturesto ensure that every mismatched thymine that isbound is in fact cleaved and removed.

This DNA-binding model for MIG is consistentwith known HhH glycosylase/AP lyase DNAcomplex structures, and with most other DNArepair glycosylase structures, in that binding ismediated exclusively via the DNA minor groove.Interestingly, the human enzyme MBD4 contains aC-terminal HhH DNA glycosylase domain, whichis similar to MIG (Figure 1), linked to an N-term-inal methyl-binding domain (MBD), which prefer-entially binds to m5CpG.TpG mismatches.22

Solution structures show that MBD domains con-sist of a compact a/b fold with an extended loopbetween two anti-parallel b strands,40,41 and thatthese b strands insert into the DNA major groove

logy and DNA-interaction surface. (a) The MIG structureurple) of the four-helix domain (top), and six-helix barrel-hairpin loop of the HhH motif (orange), the Fe4S4 iron-

idues lining the active site at the domain interface areglycosylases and combined glycosylase/AP lyases. The

rimposed on the structure of M. thermoformicicum MIGa-helix barrel domains. (c) The MIG molecular surface

electrostatic charge (color bar: red, ÿ2.0 kT/e to blue,G is a conserved feature of the homologous HhH glyco-orienting the DNA for nucleotide-¯ipping of target basesuperimposed on the MIG surface from the homologous

Figure 3. Proposed simultaneous binding of the MBDand MIG domains of MBD4 to duplex DNA with anm5CpG.TpG mismatch. (a) Binding of the MIG domainof MBD4 (dark blue a helices and purple loops) to theDNA minor groove can occur simultaneously with bind-ing of the MBD domain (light blue helices, gold bstrands and green loops) to the DNA major groove atm5CpG.TpG mismatches. The �66 � bend of the DNA(red tubes), overlaid from the AlkA:DNA complex(1DIZ), can still accommodate speci®c recognition of thetarget lesion by the MBD domain via the major grooveas determined by NMR,40 suggesting that both domainscontribute to forming the functionally relevant complex.The model is presented at the level of the Ca trace andwas created by superposition of MIG onto DNA-boundAlkA and manually docking the MBD domain anti-par-allel b-ribbon into the major groove with residues impli-cated in recognizing the m5CpG.TpG mismatch40

positioned near the m5CpG.TpG base-pairs. (b) Modelfor coordination of m5CpG.TpG mismatch repairthrough MBD targetting of thymine-DNA glycosylaseactivity to regions of methylated DNA. Speci®cm5CpG.TpG mismatches (red DNA bases) are recog-nized via the DNA major groove by the MBD domain


where particular protein residues bind methylatedCpG sequences.40 The MBD domain's sequence-speci®c, DNA major-groove binding mode iscomplementary to the DNA minor-groove bindingmode of the MIG domain, suggesting that theMBD and MIG domains of MBD4 can cometogether at m5CpG.TpG mismatches to engulf theDNA (Figure 3(a) and (b)). Although the isolatedglycosylase domain of MBD4 is enzymaticallyactive,42 this dual-binding model provides a mech-anism to restrict MBD4 thymine-DNA glycosylaseactivity in vivo to biologically relevant substrates atthe promoters of silenced genes without interfer-ence from the U:G mispairs that arise continuouslythroughout the genome from general cytosinedeamination.

Thymine-binding pocket

The MIG active site structure, and comparisonwith homologous enzyme DNA complexes,suggests a consistent model for how MIG binds atarget extrahelical thymine nucleotide. The MIGthymine base-binding pocket is similar to theMutY adenine base-binding pocket, and surpris-ingly the substrate speci®city of MIG can beexpanded to include the A:G mismatches targetedby MutY with two simple residue substitutions(L187Q, A50V), which complement the Watson-Crick face of adenine.43 Thus, MIG likely binds athymine base in the same orientation as adenine isobserved to bind in the MutY:Adenine complex,29

forming bidentate hydrogen bonds from thymineN3 and O4 to Glu42 Oe1 and Oe2, respectively(Figure 4(a)). The pKa of Glu42 is likely elevateddue to its sequestration from bulk solvent in aDNA-bound complex, and a similar interaction isdirectly observed between the homologous Glu37residue of MutY and the N7 atom of its targetadenine base (Figure 4(b)), supporting an edge-onorientation of the thymine base to the Glu42 side-chain. Any other orientation for the thymine basein the MIG pocket would defeat the hydrogen-bonding capability of Glu42 in thymine binding,nor would it be consistent with the substrate speci-®city expansion results.

The proposed orientation for a bound thyminebase places the thymine O2 atom in position toaccept a hydrogen bond from the -OH group ofTyr126, while the thymine C5 methyl group can ®tinto a hydrophobic cleft bordered by Met190,Phe27, Leu187 and Arg30 (Figures 2(a) and 4(a)).

(red), allowing binding of the extrahelical thyminenucleotide and bond cleavage by the MIG domain(blue). In this way the glycosylase activity of MBD4 canbe targeted to relevant biological lesions and not be les-sened by binding to and being inhibited by normal U:Gmismatches arising from cytosine deamination in bulkDNA.

Table 1. Enzyme activity and DNA binding for wild-type and site-directed mutants of MIG

Enzyme Kst (10ÿ2 minÿ1)a %Wild-type activity Kd (mM)b Fold difference in binding

Wild-type 68.0 100 2.5-2.6 1E42D 15.0 22 9.5-9.7 3.7E42S 2.6 4 4.0-5.2 1.8Y126F 1.1 1.6 1.3-2.0 0.65Y126S 0.26 <1 7.5-9.0 3.2Y126 K Inactive <1 2.9-3.1 1.2D144A 4.2 6 3.5-4.5 1.9D144N Inactive <1 2.0-2.2 0.83N146E 0.27 <1 5.9-9.4 3.0N146H 0.73 1 9.2-10.1 3.7R47A 4.1 6 2.6-3.0 1F27A 56.3 83 5.8-6.2 2.4W29F 30.3 45 2.7-3.6 1.2T127V 63.0 93 7.3-9.0 3.0L187Q 6.7 10 1.3-1.5 �1.5

a Single turnover kinetic constant for a 19mer with a T:G mismatch. Error for Kst < 10 %.b Dissociation constant for a 19mer tetrahydrofuran AP site mimic opposite G.


Superposition of an attached deoxyribose sugarfrom the homologous Ogg1:DNA complex(Figure 2(b) and (c)) suggests that Asn146 deliversa hydrogen bond from Nd2 to the deoxyribose O40atom of the bound nucleotide. The side-chain con-formation of conserved, catalytic Asp144 isrestricted by a capping interaction with the N ter-minus of the a4b helix, but is appropriately alignedto contact the thymine deoxyribose (Figures 2(a)and 4(a)). Thus, both Asp144 and Asn146 are posi-tioned to interact with the deoxyribose and assistin destabilizing the glycosyl bond, by both shield-ing it from bulk solvent and stabilizing a positivelycharged incipient oxocarbenium ion.

Key residue mutations impairglycosylase activity

We generated site-directed mutants of keyenzyme residues and assayed these mutantenzymes for glycosylase activity against T:G mis-matches, and for general DNA binding activityto oligonucleotides containing a synthetic AP-siteopposite guanine. As expected, substitution ofany of the key catalytic residues identi®ed fromthe structural analysis diminishes enzymeactivity without abolishing general DNA binding(Table 1). Substitution of residues likely to inter-act with the thymine base functional groups,such as the conservative E42D mutation, resultsin ®vefold lower activity, but the more drasticE42S substitution has very low activity. It isnoteworthy that the analogous E37S mutant inthe MIG paralog MutY also greatly reducesMutY activity.29 Even the conservative Y126Fsubstitution, which could still shield the activesite from bulk solvent but lacks the ability toform hydrogen bonds, results in a �60-foldreduction to <2 % of wild-type activity, whiletruncation of Tyr126 to serine essentially inacti-vates the glycosylase activity of the enzyme. TheY126 K mutant, which recreates the Schiff base-

forming HhH motif residue for the AP lyases(Figure 1), is no longer a T:G mismatch glycosy-lase. Interestingly, this MIG lysine variantenzyme acquires AP lyase activity against pre-existing AP sites in double-stranded (ds) DNA.24

The isosteric D144N substitution can still packwith the thymine deoxyribose, but lacks a formalnegative charge that could complement a devel-oping positively charged transition state, therebyseverely inhibiting enzyme activity, whereas thesmaller side-chain of the D144A mutant cannotpack with the bound deoxyribose and is less dis-ruptive. Substitutions at Asn146 also impair cata-lysis. Both the N146E and N146H mutationsseverely impact activity.

The critical role of the minor-groove, nucleotide-¯ipping wedge in achieving catalysis is illustratedby the effect on enzyme activity of truncating theside-chain of Arg47 to alanine (Table 1). The smal-ler alanine side-chain cannot intercalate into theDNA base stack to ¯ip the target thymine nucleo-tide into the active site pocket. Thus, although theR47A mutation has no effect on binding toAP-DNA oligonucleotides (Table 1), the mutantenzyme cannot easily ¯ip a target thymine fromthe dsDNA helix to form a catalytically competentcomplex. This latter mutation highlights the bipar-tite nature of DNA repair enzyme activity. Whilethe active site chemistry necessary to cleave theglycosylic bond may be intact, the enzyme activitymay be signi®cantly reduced by changes thatimpair achieving an extrahelical conformation forthe target nucleotide.

A distinction between pure DNA glycosylasesand DNA glycosylase/AP lyases

A poorly understood aspect of HhH DNA repairenzymes are the structural requirements for anyparticular HhH enzyme acting as a pure glycosy-lase versus a combined glycosylase/AP lyase. Atthe level of excision chemistry, there is the obvious

Figure 4. The active sitedamaged-base binding pockets forthe pure HhH glycosylases MIGand MutY, and the combined gly-cosylase/AP lyases Ogg1 and EndoIII. (a) The MIG thymine-bindingpocket delineated from structureand mutagenesis results. The mod-eled thymine base is shownattached to the extrahelical deoxy-ribose from the overlaidOgg1:DNA structure (1EBM). Forthe thymine to form speci®c con-tacts with Glu42 and Tyr126 (greytubes), as suggested by the muta-genesis results, the thymine basemust be twisted �90 � from its nor-mal anti position in duplex DNA.(b) The MutY adenine-bindingpocket, in the same orientation asMIG:thymine in (a), illustratingadenine-speci®c interactions asobserved in the MutY:adenine com-plex (1MUN.PDB), with the deox-yribose overlaid as for MIG. Forthe observed adenine-speci®c inter-actions, including the similar rolefor Glu37 in MutY and the homolo-

gous Glu42 in MIG, the adenine base must also be twisted �90 � relative to the deoxyribose. (c) The Endo III thymineglycol-binding pocket viewed in a similar orientation to, and overlaid deoxyribose as in, (a) and (b). The insertedloop containing Thr22 and Glu23 (upper left) is not present in MIG or MutY and prevents binding of thymine glycolin a twisted conformation. Lacking an aromatic ring structure to twist, the modelled thymine glycol binds in a nor-mal anti position relative to the attached deoxyribose. (d) The Ogg1 8-oxoguanine-binding pocket as seen in theOgg1:DNA co-crystal structure, viewed similarly to (a), (b) and (c). The inserted Gly42 loop (upper left), from theunique N-terminal Ogg1 domain forms the only speci®c 8-oxoguanine interaction with a hydrogen bond between theGly42 carbonyl group and the protonated 8-oxoguanine N7 position. This inserted loop prevents the 8-oxoguaninefrom binding in a twisted manner and the 8-oxoguanine binds in the anti conformation it would possess in the target8-oxoG:C base-pair in DNA.


distinction that the combined glycosylase/AP lyaseenzymes utilize an HhH motif lysine residue toform a Schiff base intermediate with the C10 atomof the DNA, while the pure glycosylases do notform covalent enzyme:DNA intermediates. DNAbase-excision repair enzymes can also be differen-tiated by whether they excise damaged DNA basesor remove normal DNA bases from mispairs. Com-paring pure HhH gycosylases, including the MIGthymine-binding pocket delineated here, and theadenine-binding pocket of MutY, with thedamaged-base binding pockets of the combinedHhH glycosylase/AP lyases Ogg1 and Endo III,suggests a fundamental difference in the manner inwhich the target, extrahelical nucleotide is bound(Figure 4). In each case, the extrahelical base-bind-ing pockets of the pure HhH glycosylases are in aregion that is occupied by loops that insert into thedomain interface in the combined glycosylase/APlyase enzymes (Figure 4(c) and (d)). These insertedloops shift the base recognition pockets for the APlyases to a different region of the domain interface,but more importantly they also alter the relation-ship between the extrahelical target base and itsattached deoxyribose sugar. The functional signi®-

cance of this interplay between alternate damaged-base recognition pockets is that the pure glycosy-lases (Figure 4(a) and (b)) impart a �90 � twist tothe damaged base upon binding, whereas the com-bined DNA glycosylase/AP lyases bind their tar-get nucleotides in the �anti conformation normallyseen in dsDNA (Figure 4(c) and (d)).

Base twisting

The process of base twisting in binding nucleo-tides is a general phenomenon that has beendirectly observed in unrelated enzyme systems.Furanose sugars, such as ribose and deoxyribose,are prone to oxocarbenium ion formation,44,45 andbase twisting favors the formation of oxocarbe-nium ion transition states.46 The �90 � twisting of atarget, extrahelical base has been directly observedin a high-resolution, uncleaved UDG:DNA sub-strate complex,16 in the structure of the broad-speci®city human enzyme alkyladenine DNAglycosylase (AAG) bound to DNA containing1,N6-ethenoadenine,47 and also in the enzyme pur-ine nucleoside phosphorylase (PNP), which per-forms a chemical reaction very similar to that

Figure 5. Structurally implied active site chemistry forpure HhH glycosylase MIG. MIG interactions of Glu42and Tyr126 with the O4, N3 and O2 positions of thy-mine facilitate glycosylic bond dissociation, while the�90 � clockwise twist and distortion enforced by theMIG thymine-binding pocket allows the three normallyorthogonal O40, N-C10, and p electron orbitals to over-lap. This orbital overlap facilitates catalysis by promot-ing the electron transpositions needed for glycosylicbond cleavage.16


catalyzed by the DNA glycosylases.44 Both UDG48

and PNP46 have had their transition state struc-tures established from kinetic isotope effects, con-®rming that catalysis proceeds via a primarilydissociative oxocarbenium ion transition state.

Based on the results presented here, we proposethat the pure HhH DNA glycosylases, such as MIGand MutY, also twist their target extrahelical bases(Figure 5). It is notable that MIG, MutY and UDGall cleave normal bases with stable glycosylicbonds from dsDNA, suggesting that the structu-rally implicated physical twisting of the target baseis an important component of the active site chem-istry that allows these enzymes to ef®cientlyremove normal bases from promutagenic mispairs.Additionally, base twisting provides a means forthese enzymes to avoid signi®cant inhibition byfree nucleotides that are present within the cell.For enzymes which excise a wide range of DNAbases, and thus do not make speci®c contacts toDNA base functional groups, such as AAG inhumans and its functional homolog in Escherichiacoli, the HhH glycosylase AlkA,35 this physical

twisting of the target base may be the primarymeans for enhancing catalysis. Interestingly, theMIG base-recognition pocket could also recognizecytosine bases via the other tautomer of Glu42(Figure 5). However, unlike thymine and uracil,cytosine does not form mispairs that are suscep-tible to being ¯ipped-out of the DNA helix. Thus,MIG does not remove thymine from A:T base-pairsnor cytosine from any base-pair. This further high-lights the bipartite nature of DNA repair glycosy-lase activity.

The functional signi®cance of HhH AP lyaseactivity may derive from the nature of the targetdamaged base, as neither Ogg1 nor Endo III appar-ently distort the extrahelical nucleotide. For Ogg1,which removes 8-oxoguanine from a normal Wat-son-Crick base-pair with cytosine, the binding ofthe extrahelical base in a normal anti conformation(Figure 4(d)) may provide an extra level of speci-®city to preclude the binding and removal of syn8-oxoguanine bases from mispairs with adenine,which are the target mispairs for MutY. For EndoIII, which removes oxidized pyrimidines such asthymine glycol, the target base lesion no longerhas a rigid, aromatic ring structure, and thus EndoIII cannot induce twisting and strain to facilitatebond cleavage.

In summary, complementary structural andmutagenesis results for a thermostable thymine-DNA glycosylase suggest that glycosylic bondcleavage is enhanced by twisting of the bound,extrahelical target base. This base twisting may bea requirement for the ef®cient removal of normalDNA bases within mispairs, or for the removal ofa wide range of damaged bases by DNA glycoy-lases with broad speci®city. Enzyme loops insertedinto HhH AP lyase enzyme active sites apparentlyblock this base twisting, and these enzymes bindtheir damaged DNA target bases in the �anti con-formation normally seen in duplex DNA. The evi-dence for base twisting to enhance catalysis byMIG, and perhaps also by MBD4 and MutY, iscompelling, and suggests that nature has exploiteddifferent reaction chemistries within similar struc-tural frameworks.

Materials and Methods

Enzyme activity and mutagenesis

The cloning, expression and puri®cation of M. thermo-formicicum MIG was as described previously.24 Site-directed mutants were generated as described forY126 K and employed the megaprimer mutagenesismethod.49 Enzyme activity was measured by the alkali-induced cleavage of AP sites generated by MIG actingon double-stranded oligonucleotides containing a T:Gmismatched base-pair. Wild-type or mutant enzymeswere incubated with mismatched 32P-labeled substrate in50 mM Hepes-KOH (pH 7.5), 100 mM NaCl, at 65 �C.Addition of NaOH to 0.15 M and incubation at 65 �C for15 minutes inactivates the enzyme and cleaves at any APsites that are created. Samples were separated on dena-turing polyacrylamide gels for two hours at 200 V in


50 mM Hepes-NaOH (pH 7.5), and the amount of pro-duct formed quanti®ed on a Storm phosphorimager,with enzyme activity (A) determined according to theformula A � P/(P � S), where P is the amount of pro-duct formed and S the amount of substrate remaining.Single turnover kinetics for thymine removal weremeasured under similar conditions as above except withenzyme (47 mM) in excess of substrate oligonucleotidescontaining a central T:G mismatch (15 mM), and the reac-tion terminated by the addition on NaOH at 0, 0.5, 1, 2,3, 4, 6, and 8 minutes. The products of these reactionswere separated on denaturing polyacrylamide gels andquanti®ed using a Storm phosphorimager, with theamount of product formed at the time-points used tocalculate a single turnover rate constant, kcat-st. The datawere ®tted using KaleidagraphTM according to the equa-tion:50 [Product] � A(1 ÿ exp(ÿkcat-st)(t)), where Ais the normalization factor for the endpoint of productformation and kcat-st is the single turnover rate constant.

Crystallization, data collection, and processing

For crystallization experiments, puri®ed, wild-typeMIG was concentrated to �20 mg/ml in 10 mM Hepes-NaOH (pH 7.5), 100 mM NaCl. Crystals are grown byhanging drop vapor diffusion by mixing 2 ml of proteinsolution with an equal volume of reservoir solution con-taining 60 % saturated ammonium sulfate, 100 mMHepes (pH 8.0), 10 mM zinc acetate, 2 mM DTT, andbelong to the tetragonal space group P41212,a � b � 68.2 AÊ , c � 98.9 AÊ with one molecule in theasymmetric unit. X-ray diffraction data were collectedfrom a single crystal (0.4 mm � 0.2 mm � 0.2 mm)frozen in a stream of liquid nitrogen in mother liquorsupplemented with 20 % (v/v) glycerol, at beamline 5.0.2of the Advanced Light Source (ALS) at UC Berkeley. Dif-fraction data extending to 2.0 AÊ resolution werecollected, with 209,829 observations of 16,125 uniquere¯ections (99.9 % complete), an overall I/sI of 8.7 and amerging R value for symmetry-related re¯ections of0.066.

Structure determination, refinement and analysis

Initial molecular replacement calculations employingseparate HhH glycosylases, or their individual four-helixand six-helix domains, as search models were unable to®nd the correct solution. By utilizing a feature withinAMoRe,51 whereby the overlaid structures of Endo IIIand MutY were used as search models simultaneously,the correct solutions were found with a correlation coef-®cient of 0.23 and an R value of 0.479, with the valuesfor the next highest peak of 0.178 and 50.6, respectively,for data in the resolution range 10.0 to 4.0 AÊ . Bothmodels were re®ned in parallel in CNS,52 and the MIGamino acid sequence ®tted to bias-reduced, simulated-annealed omit maps. The Endo III model was chosen forfurther re®nement, with manual inspection and rebuild-ing of the structure performed with XFIT,53 as necessary.The ®nal re®ned model contains the four Fe and four Satoms of the iron-sulfur cluster; 1782 protein atoms,including alternate conformations for Ile38, Ile103,Met132, and Cys182; 200 ordered solvent molecules; fouracetate and two chloride ions; and one Zn2�. The crystal-lographic R value is 0.211 (Rfree � 0.248) for 16,107F > 0sF re¯ections from 20.0 to 2.0 AÊ resolution, withrms bond length deviations of 0.011 AÊ and bond angledeviations of 1.30 �. Electrostatic surface calculations

were performed with UHBD.54 Structure super-positions and sequence alignments were done withSEQUOIA (http::/www.scripps.edu/ � bruns), andFigures prepared using the Advanced Visualization Sys-tems software (AVS Inc., Waltham, Mass.). Structuresused in the enzyme superpositions and preparation ofFigures are: Ogg1:DNA (PDB entry code 1EBM),AlkA:DNA (1DIZ), AlKA (1MPG), MutY:adenine(1MUD), and Endo III (2ABK).

Coordinates

The coordinates for M. thermoformicicum MIG havebeen deposited with the Protein Data Bank under acces-sion code 1KEA.pdb.

Acknowledgments

We thank B. R. Chapados, D. S. Daniels, M. DiDo-nato, S. S. Parikh, C. D. Putnam, and T. I. Wood forhelpful discussions, and the staff and facilities atBeamline 5.0.2 of the Advanced Light Source (ALS)for data collection. ALS is supported by the Director,Of®ce of Science, Of®ce of Basic Energy Sciences,Materials Sciences Division, of the U.S. Department ofEnergy under Contract no. DE-AC03-76SF00098 atLawrence Berkeley National Laboratory. Work onDNA repair in the authors' laboratory is supported bythe National Institutes of Health (GM46312 J.A.T. andR.P.C.), and the Skaggs Institute for Chemical Biology(C.D.M.).

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Edited by K. Morikawa

(Received 11 July 2001; received in revised form 28 October 2001; accepted 30 October 2001)

Structure and activity of a thermostable thymine-DNA glycosylase: evidence for base twisting to...

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