Crystal structures of λ exonuclease in complexwith DNA suggest an electrostatic ratchetmechanism for processivityJinjin Zhanga,b,1, Kimberly A. McCabeb, and Charles E. Bella,b,2

aOhio State Biochemistry Program and bDepartment of Molecular and Cellular Biochemistry, The Ohio State University, 1645 Neil Avenue,Columbus, OH 43210

Edited by Brian W. Matthews, University of Oregon, Eugene, OR, and approved May 31, 2011 (received for review March 2, 2011)

The λ exonuclease is an ATP-independent enzyme that binds todsDNA ends and processively digests the 5′-ended strand to form5′mononucleotides and a long 3′ overhang. The crystal structure ofλ exonuclease revealed a toroidal homotrimer with a central fun-nel-shaped channel for tracking along the DNA, and a mechanismfor processivity based on topological linkage of the trimer to theDNAwas proposed. Here, we have determined the crystal structureof λ exonuclease in complex with DNA at 1.88-Å resolution. Thestructure reveals that the enzyme unwinds the DNA prior to clea-vage, such that two nucleotides of the 5′-ended strand insert intothe active site of one subunit of the trimer, while the 3′-endedstrand passes through the central channel to emerge out the backof the trimer. Unwinding of the DNA is facilitated by several apolarresidues, including Leu78, that wedge into the base pairs at thesingle/double-strand junction to form favorable hydrophobic inter-actions. The terminal 5′ phosphate of the DNA binds to a positivelycharged pocket buried at the end of the active site, while the scis-sile phosphate bridges two active site Mg2þ ions. Our data suggesta mechanism for processivity in which wedging of Leu78 and otherapolar residues into the base pairs of the DNA restricts backwardmovement, whereas attraction of the 5′ phosphate to the posi-tively charged pocket drives forward movement of the enzymealong the DNA at each cycle of the reaction. Thus, processivity ofλ exonuclease operates not only at the level of the trimer, but alsoat the level of the monomer.

DNA repair ∣ enzyme mechanism ∣ homologous recombination ∣recombineering ∣ single-strand annealing

The resection of DNA ends to form a long 3′ overhang is a fun-damental step in the repair of dsDNA breaks by homologous

recombination. Whereas in eukaryotes DNA end-resectioninvolves a complex network of proteins (1), bacteriophage andother DNA viruses encode a simple two-component “SynExo”recombination system that consists of a processive 5′-3′ exonu-clease to resect DNA ends, and a single-strand annealing (SSA)protein to bind the resulting 3′ overhang and promote annealingof complementary strands (2–4). The two proteins interact withone another to form a complex known as a “synaptasome” (4–6),which may serve to physically load the SSA protein onto the 3′overhang as it is generated by the exonuclease. The Redαβ systemof bacteriophage λ, which consists of λ exonuclease and β-protein,is particularly well studied and is currently being employed inpowerful methods for genetic engineering and nanopore DNAsequencing (7–9).

The λ exonuclease is a Mg2þ-dependent enzyme that binds todsDNA ends and processively digests the 5′-ended strand to form5′ mononucleotides and a long 3′-ended ssDNA tail (6, 10). Thecrystal structure of λ exonuclease, determined in the absence ofDNA, revealed a ring-shaped homotrimer with a central channelthat is wide enough to bind dsDNA at one end, but only allowpassage of ssDNA at the other (11). Because the DNA couldremain topologically linked to the trimer via threading of the 3′overhang through the central channel, the structure provided a

compelling basis for the highly processive nature of the reaction.The core fold of λ exonuclease is conserved in type II restrictionendonucleases (12), and in several nucleases involved in DNArepair (13–15), viral replication (16–18), and RNA processing(19, 20). These enzymes all exhibit a conserved PD-(D/E)XKmotif that binds one or more metal ions, usually Mg2þ, to formthe active site for cleavage.

The crystal structure of λ exonuclease provided a compellingmodel for its processive mode of action, but in the absence of astructure with DNA substrate, several questions remain. If theDNA binds within the central channel of the trimer as predicted,how does the 5′ end of the DNA access one of the three activesites, which lie approximately 15 Å from the central channel?Does the enzyme use all three of its active sites in a sequential,cyclical manner, or does the DNA engage with a single active siteon the trimer for multiple rounds of cleavage? Is processivitybased solely on topological linkage of the trimer to the DNA,or does the enzyme possess a “motor” activity that couples energyreleased from cleavage of the phosphodiester bond to forwardmovement along the DNA? Here, we present crystal structuresof λ exonuclease in complex with DNA that show the detailedinteractions between the terminal nucleotides of the DNA andthe active site residues of the protein. Based on these structures,and on accompanying biochemical data, we propose an “electro-static ratchet” mechanism for processivity in which forwardmovement of the enzyme is driven by attraction of the terminal5′ phosphate of the DNA to a positively charged pocket at theend of the active site cleft.

ResultsCrystal Structures of λ Exonuclease Bound to DNA. We have deter-mined two crystal structures of λ exonuclease in complex withDNA. The first is a complex with a symmetric 12 bp duplex with5′-OH ends in the presence of Ca2þ, which replaces Mg2þ butinhibits nuclease activity (10). In this structure, the DNA is boundwithin the central channel of the trimer with the 5′ end of onestrand projecting toward one of the three active sites (Fig. S1A).However, the scissile phosphate of the terminal nucleotide is still11 Å from the active site Ca2þ, and thus the DNA is not fullyinserted into the active site. This structure likely represents anonproductive complex that has been detected in enzymatic

Author contributions: J.Z. and C.E.B. designed research; J.Z., K.A.M., and C.E.B. performedresearch; J.Z., K.A.M., and C.E.B. analyzed data; and C.E.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates and structure factors have been deposited inthe Protein Data Bank, www.pdb.org [PDB ID codes 3SLP (Form 1, WT-12/12-Ca2+) and3SM4 (Form 2, K131A-P14/13-Mg2+)].1Present address: National Institutes of Health, National Institute of Child Health andHuman Development, 35 Lincoln Drive, Bethesda, MD 20892.

2To whom correspondence should be addressed. E-mail: bell.489@osu.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1103467108/-/DCSupplemental.

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studies on DNA substrates with 5′-OH ends (21, 22). Based onthis structure, we designed a DNA with a 12-bp duplex and a5′-dinucleotide overhang, which was intended to extend fully intothe active site. Because previous data indicated the importance ofa 5′ phosphate on the DNA for maximal activity (10, 21, 22),we also added a 5′ phosphate to the 14-mer strand of the duplex.Finally, we used the inactive K131A variant of the enzyme, so thatthe complex could be crystallized with Mg2þ instead of Ca2þ. Theresulting structure, refined at 1.88-Å resolution (Table S1), re-veals a catalytically relevant complex that will now be described.Aside from the noted differences in the active site cleft, the twostructures are very similar to one another.

The DNA binds within the central channel of the trimer as ex-pected, but is asymmetrically tilted so that the 5′-dinucleotideoverhang binds to the active site of subunit B, the green subunitas shown in Fig. 1. The duplex portion of the DNA is essentially Bform (Fig. S2), and there are no large conformational changesthat occur in the protein upon DNA binding: The bound and un-bound trimers superimpose to an rmsd of 1.2 Å for all Cα atoms.The loop connecting helices C and D of each subunit (CD loop,residues 42–50) extends out from rim of the central channel tocontact the downstream portion of the DNA in three differentways (Fig. 1C). As expected for a non-sequence-specific DNA-binding protein, most of the interactions involve hydrogen bondsor ion pairs to the sugar-phosphate backbone, and there are fewcontacts with the bases (Fig. S3). A notable interaction is the in-sertion of Arg45 of subunit C deep into the minor groove of thedownstream portion of the DNA, with its positively charged gua-nidino group lying midway between the two sugar-phosphatebackbones (Fig. 1 C and D). This type of interaction, seen pro-minently in the nucleosome, favors A-tract sequences in which anarrowing of the minor groove leads to an increased negativeelectrostatic potential (23). Although the sequence is not anA tract, the minor groove at this region of the DNA narrowsby about 1–2 Å to close in on the side chain of Arg45. Becausethis interaction does not involve sequence-specific hydrogen

bonds, Arg45 could act as a rudder to help keep the enzymeon track as it moves along the DNA.

The structure reveals that the enzyme unwinds exactly two basepairs from the end of the DNA prior to cleavage. The two nucleo-tides of the 5′ overhang twist away from the duplex to insert intothe active site of subunit B, while the last nucleotide of the 3′-ended strand pokes through the central channel to emerge outthe back of the trimer (Fig. 1E). The loop between helices Dand E (DE loop, residues 69–75) extends across the central chan-nel at the back of the trimer, with the 3′ end of the DNA on oneside and the 5′ end on the other. Four apolar residues of the DEloop, Val73, Ala75, Ala77, and Leu78, pack against the base pairexposed at the end of the duplex to form a hydrophobic wedgethat is positioned to split apart the base pairs as the enzymemoves along the DNA (Fig. 2C). In particular, the side chainof Leu78 inserts between the second and third bases of the 5′-ended strand to separate the two terminal nucleotides in theactive site from the duplex portion of the DNA in the centralchannel. The bases of the two nucleotides in the active site stackon top of one another and are sandwiched by Leu78 on one sideand Trp24 on the other. Trp24 does not form stacking interac-tions, as might be expected, but instead packs against the faceof the terminal base with the edge of its indole ring (Fig. 2A).

In the active site to which the DNA is bound, there are nowtwo Mg2þ ions, both of which exhibit octahedral coordination(Fig. 2). Thus, the structure reveals that λ exonuclease uses a clas-sic two-metal mechanism (24) seen for the proofreading domainof Escherichia coli DNA polymerase 1 (25), RNaseH (26), MutHprotein (13), and several type II restriction endonucleases (27).The two Mg2þ ions, MgA and MgB, are spaced by 4.0 Å andbridged by Asp119 on one side and the scissile phosphate ofthe DNA on the other, which coordinates MgB twice. MgA bindsto the site occupied by Mn2þ or Ca2þ in the absence of DNA (11)(Fig. S1A), and is also coordinated by Glu129, the backbone car-bonyl of Leu130, and two water molecules, one of which is posi-tioned for in-line attack on the scissile phosphate (Fig. 2A). MgB

Fig. 1. Crystal structure of λ exonuclease in complex with DNA. (A) The DNA sequence used for cocrystallization. (B) Stereo ribbon viewwith subunit A, cyan; B,green; and C, wheat. The 5′-dinucleotide overhang of the DNA binds to the active site of subunit B. The two Mg2þ ions are shown as magenta spheres.Phosphate ions of subunits A and C are shown as orange and red spheres, and the two chloride ions of subunit A are shown as gray spheres. (C) Side viewshowing the binding of the CD loops to the downstream portion of the DNA. The side chain of Arg45 of subunit C inserts into the minor groove. The 1.88-ÅFo − Fc electron density map is contoured at 2.0σ around the DNA. (D) Electrostatic surface view generated in PyMOL with negative red and positive blue. Thethree Arg45 residues, which contact the DNA in the central channel, are labeled. (E) Surface view from the back of B, showing how the DE loop splits the twostrands of the duplex. A rear portal through which cleaved mononucleotides could be released is also indicated.

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does not bind in the absence of DNA and is also coordinated bythree water molecules. Glu85, a residue that is conserved as Gluor His in related enzymes (Fig. S4), does not contact either Mg2þion directly, but forms an outer sphere ligand interaction to eachof them, through bound water molecules. Two other highly con-served residues, Gln157 and Tyr154, form hydrogen bonds withthe scissile phosphate and the neighboring 3′ phosphate, respec-tively. By analogy with MutH and other two-metal nucleases(13, 24), the structure supports a mechanism in which MgA,together with the neighboring 3′ phosphate and Lys131, activatesa water molecule for nucleophilic attack, whereas MgB stabilizesthe trigonal bipyramidal geometry of the transition state throughits bidentate coordination to the scissile phosphate.

The 5′ phosphate of the DNA binds to a positively chargedpocket buried at the end of the active site, where it contactsthe guanidino group of Arg28, three backbone amides, two ofwhich are at the N terminus of helix C, three Ser/Thr hydroxyls,and the edge of the indole ring of Trp24 (Fig. 2D and E). Bindingof the 5′ phosphate to this site was predicted from the binding offree phosphate in the uncomplexed structure (11), and the R28Aprotein has substantially reduced activity and processivity (22).Based on these observations and on the fact that the duplex with5′-OH ends binds in a nonproductive manner, attraction of the 5′phosphate to this positively charged pocket appears to be essen-tial for pulling the DNA substrate fully into the active site. All ofthe residues that contact the 5′ phosphate are conserved in alka-line nucleases of Kaposi’s sarcoma-associated herpesvirus(KSHV; ref. 17) and EBV (18), suggesting that 5′-phosphate re-cognition is an important feature of this group of 5′-3′ exonu-cleases (Fig. S4).

The occupancy of the active sites of subunits A and C on thetrimer, which are not bound by DNA, is intriguing (Fig. S5).Neither active site contains Mg2þ, which is present at 5 mM inthe crystal, suggesting that both MgA and MgB require DNAfor tight binding. The active site of subunit C contains a phos-phate ion at the same site as the 5′ phosphate of the DNA, aswas seen in the uncomplexed structure (11). The active site ofthe third subunit, subunit A, also contains a phosphate, but at

a position that is shifted by 8.5 Å toward the back of the trimer,near a small rear portal through which cleaved mononucleotidescould be released (Fig. 1E and Fig. S5C). Movement of the phos-phate is accompanied by a reorientation of the side chain ofArg28, which remains bound to it. At the expected phosphate-binding site on subunit A, near the N terminus of helix C, is apair of chloride ions, spaced by 2.6 Å from one another. The pre-sence of these chloride ions highlights the positively charged nat-ure of this pocket on the enzyme, even when the guanidino groupof Arg28 is displaced. Although it is not clear from the structurewhy the liganded states of subunits A and C are different, it isinteresting to speculate that movement of Arg28 and the phos-phate of subunit A could reflect conformational changes, possiblycoupled to the catalytic cycle, that are involved in ejection ofcleaved mononucleotides out of the active site during processivedigestion.

Structure-Activity Analysis. To probe the roles of key residues seenin the structure, the effects of alanine substitutions at 11 differentresidues on exonuclease activity were determined (Fig. 3). Thetargeted sites include residues within the central channel thatcontact the downstream portion of the DNA (R45, K49, M53,K76, R137), the hydrophobic wedge (L78), the active site(E85, D119, K131), and the 5′-phosphate-binding site (W24,R28). To measure exonuclease activity, digestion of a linear2.7 kB pUC19 DNA by a limiting amount of enzyme was mon-itored by agarose gel electrophoresis with SYBR gold staining.This method allows for simultaneous visualization of the dsDNAsubstrate and ssDNA product of the reaction. At a concentrationof 0.05 nM trimer, wild-type λ exonuclease digests one strand of1.2 nM of duplex within 40 min to release the ssDNA product.Based on this result, we estimate a rate of digestion of approxi-mately 30 nucleotides per second for wild-type enzyme underthese conditions, which is comparable to previous measurements(21, 22, 28, 29). The reaction appears to be completely processivebecause there are no intermediate bands, and the full amount ofssDNA product is released.

Fig. 2. Active site interactions. (A) Close-up view of the active site cleft. The two Mg2þ ions are shown as magenta spheres with coordinating interactions indashed lines. The arrow indicates the proposed attack of the hydrolytic water molecule on the scissile phosphate. (B) Stereo view of electron density around theMg2þ ions. The interactions of three additional conserved residues, Glu85, Gln157, and Tyr154, can be seen in this view. (C) Hydrophobic interactions betweenresidues of the DE loop and the single/double-strand junction of the DNA. The side chains of Val73, Ala75, Ala77, and Leu78 bury 55, 26, 36, and 80 Å2 ofsolvent accessible surface area in the complex, respectively. (D) Interactions of the 5′ phosphate with the positively charged pocket. (E). Electrostatic surfaceview of the 5′-phosphate-binding pocket. The view is from the back of A, looking into the active site through the rear portal. The surface of the protein istransparent so that the terminal nucleotides of the DNA can be seen. The positive charge of the 5′-phosphate-binding pocket, which is deeply buried, can beseen behind the 5′ phosphate. The positive charge in front of the bases is from Lys82, a residue that lines the rear portal.

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The E85A, D119A, and K131A proteins had no detectable ac-tivity (or barely detectable for E85A), consistent with the promi-nent roles of these highly conserved active site residues in thetwo-metal cleavage mechanism. Mutation of two residues thatcontact the 5′ phosphate of the DNA resulted in no activity(R28A) or very little activity (W24A), indicating that these inter-actions are also important for catalysis. As for the residues con-tacting the downstream portion of the DNA, the R45A proteinhad no detectable activity, consistent with the prominent role ofthis residue in binding to the minor groove as seen in the struc-ture. By contrast, the M53A, K49A, and K76A substitutions,which occur at residues that make more subtle contacts withthe DNA, have only modest effects on exonuclease activity.Curiously, the R137A protein had no detectable activity. Theimportance of Arg137, a nonconserved residue that contactsthe fifth phosphate from the 5′ end of the DNA (ca. 16 Å fromMgA), is not apparent from the structure. Finally, the L78A pro-tein exhibited a significantly diminished level of activity, consis-tent with the prominent role of this residue in forming thehydrophobic wedge of the DE loop. Reactions of L78A alsoresulted in the appearance of intermediate bands, evident asreproducible streaking, apparently due to the production ofpartially processed DNA molecules. Such streaking was also seenfor K76A, but not for WT or any of the other mutants.

We also examined the exonuclease activities of the 11 proteinvariants using a continuous assay in which excess enzyme (10 nMtrimer) was added to 0.05 nM of linear pUC19 DNA, and diges-tion was monitored by the decrease in fluorescence of PicoGreen(Fig. S6). An apparent rate of the reaction can be calculatedfrom the initial slope of the decrease in fluorescence. Althoughthe rates are lower in this assay due to the lower pH used forPicoGreen (7.5 vs. 9.4), the relative rates of the protein variantswere similar to what was observed in the gel-based assay.

DiscussionWe report here two crystal structures of λ exonuclease in complexwith DNA. A question from the previous structure of the uncom-plexed enzyme was, if the DNA binds within the central channelas proposed, how does the 5′ end of the DNA access one of thethree active sites, which lie approximately 15 Å from the centralchannel? The structure of the complex shows that binding ofthe 5' end to the active site is accomplished by tilting of theDNA relative to the central axis of the trimer, combined withunwinding to thread two nucleotides of the 5′-ended strand into

the active site cleft. Although the DNA in the crystal contains a5′-dinucleotide overhang and was not actually unwound to formthe complex, because the 5′ and 3′ termini are on opposite sidesof the DE loop, it is clear from the structure that the enzyme mustunwind exactly two base pairs prior to cleavage. Due to the DNAused for crystallization, the structure does not show interactionsthat might occur with the extruded 3′-ended strand. However, asthe rear surface of the trimer is rather flat, such interactions arenot likely to be extensive. Unwinding of the DNA is facilitatedby four apolar residues of the DE loop that wedge into the basepairs to form favorable hydrophobic interactions. A similar topo-logical feature, which is often flexible or disordered, is found inrelated enzymes that resect dsDNA, including RecE protein andthe alkaline nucleases of KSHV and EBV, but not in RecB pro-tein, which acts on ssDNA. In addition, the key Leu78 residueof the DE loop that wedges into the bases appears to be con-served as leucine or valine in the other three proteins (Fig. S4).

The previous structure of λ exonuclease suggested a mechan-ism for processivity based on topological linkage of the trimer tothe DNA, but how or if the enzyme couples energy from phos-phodiester bond cleavage to forward movement along the DNAremained an open question. Myers and coworkers noted that aphosphate bound to Arg28 at a pocket near the active site couldmark the binding site for the terminal 5′ phosphate of the DNA,and observed that the R28A substitution disrupted processivity ofthe enzyme (22). Because a 5′ phosphate is required for normallevels of activity, these observations suggested that binding of the5′ phosphate to the pocket formed by Arg28 is required for cor-rect positioning of the DNA within the active site. The structurewith the 5′-phosphorylated duplex confirms that the 5′ phosphatebinds to this pocket on the enzyme precisely as predicted. More-over, the structure with the 5′-OH terminated duplex reveals thatwithout a 5′ phosphate, a nonproductive complex is formed inwhich the DNA is bound within the central channel, but not un-wound and inserted into the active site cleft. All together, thesedata suggest that attraction of the 5′ phosphate to the positivelycharged pocket is required to drive unwinding of the DNA to pullthe 5′-ended strand into the active site to form the productivecomplex. Interestingly, from a recent structure of Xrn1, an unre-lated enzyme that digests messenger RNA in the 5′ to 3′ direc-tion, it was proposed that attraction of the 5′ phosphate to asimilar positively charged pocket is critical for its mechanism ofprocessivity (30).

Fig. 3. Structure-activity analysis. (A) Structure of the trimer showing the mutated residues, which are colored red for DNA binding, blue for hydrophobicwedge, orange for active site, and black for 5′ phosphate. (B) Exonuclease assays. The indicated concentration of WT or mutant enzyme was incubated with1.2 nM of linear pUC19 DNA at 37 °C. Aliquots removed at the indicated times were analyzed by agarose gel electrophoresis and SYBR gold staining. Controllanes labeled “ds” and “ss” mark the substrate and product of the reaction, respectively. The reactions are sorted by decreasing activity.

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Based on these data, we propose an electrostatic ratchetmechanism for processivity of λ exonuclease (Fig. 4). In step 1,initial capture of the DNA end results in formation of the com-plex seen in the structure with the 5′-OH terminated duplex, inwhich the DNA is bound within the central channel but not fullyinserted into the active site. In step 2, attraction of the 5′ phos-phate to the positively charged pocket, along with insertion of thehydrophobic wedge into the base pairs, drives unwinding of theDNA to form the productive complex seen in the structure withthe 5′-phosphorylated duplex. Although our data do not establishthat steps 1 and 2 are truly distinct from one another, the avail-ability of structures for each provides a framework to test thispossibility. Upon binding of the Mg2þ ions and correct alignmentof the hydrolytic water molecule, the scissile bond is cleaved(step 3), and the resulting 5′ mononucleotide, together withone or both Mg2þ ions, released through the small rear portal(step 4). Release of the cleaved mononucleotide would resultin an intermediate in which the first nucleotide-binding site isempty, and the second site is occupied by the next nucleotideto be cleaved, which now has a newly exposed 5′ phosphate.At this point, the enzyme could in principle move forward orbackward along the DNA. The observation that it is two nucleo-tides of the DNA that are unwound by the enzyme, instead of justthe one that is reacted on, is suggestive, because after cleavage ofthe terminal nucleotide, the DNA would still be unwound by onebase pair, such that interactions between the hydrophobic wedgeof the DE loop and the bases of the DNA would be maintained.These interactions, in particular the insertion of Leu78 betweenthe second and third bases of the 5′-ended strand, could help tokeep a tight grip on the DNA, restricting backward movement.Although attraction of the 5′ phosphate of the next nucleotideto be cleaved to the positively charged pocket at the end ofthe active site could provide a significant force to pull theDNA forward, there is no such force apparent for backwardmovement. After translocation of the enzyme forward alongthe DNA to position the next nucleotide to be cleaved withinthe active site (step 5), repeated cycles of steps 3–5 would leadto processive digestion.

The structure-activity analysis largely supports this model. Thekey roles of Arg45 and Arg28 in binding to the minor groove andthe 5′ phosphate, respectively, are confirmed, because the R45Aand R28A substitutions essentially abolish exonuclease activity.The L78A protein also has significantly diminished activity, con-sistent with the key role proposed for this residue in forming thehydrophobic wedge that unwinds the DNA and restricts back-ward movement. Although the L78A substitution does not totallyabolish activity, alanine at this position does retain some apolarcharacter, and there are three other apolar residues of the DEloop that also help to form the hydrophobic wedge (Val73,Ala75, and Ala77). The presence of intermediate bands for theL78A protein suggests a possible defect in processivity, althoughfurther studies will be necessary to establish this definitively. ThatLeu78 appears to be conserved in RecE and alkaline nucleases ofKSHVand EBV further suggests a key role for this residue in the

reaction. Although Arg28 is also conserved in the latter two en-zymes (but not in RecE), Arg45, and the extended CD loop onwhich it resides, is apparently unique to λ exonuclease (Fig. S4).

According to this model, forward movement of the enzymealong the DNA is driven, at least in part, by electrostatic attrac-tion of the 5′ phosphate to a positively charged pocket at the endof the active site cleft. How substantial is the electrostatic forcebetween the positively charged pocket and the 5′ phosphate likelyto be? In the “intermediate” complex depicted in Fig. 4, the dis-tance between the positively charged pocket and the 5′ phosphateof the next nucleotide to be cleaved is approximately 7 Å, whichmay seem too large to be significant. However, as the attractioninvolves at least two units of opposite charge, and the active sitepocket is largely buried, the electrostatic binding energy is likelyto be significant. Although electrostatic fields in proteins are dif-ficult to compute, the presence of two adjacent chloride ions andone phosphate in the active site of subunit A suggests that thispocket on the enzyme could carry as much as four units of po-sitive charge. If one or both of the Mg2þ ions dissociate fromthe active site after cleavage, as suggested by their absence in sub-units A and C, electrostatic repulsion of the 5′ phosphate with thethree active site carboxylates could destabilize the intermediateto further propel the DNA forward. Binding of the hydrophobicwedge to the single/double-strand junction, or interactions of theterminal bases with one another or with Trp24, could also play arole in forward movement.

It is conceivable that the enzyme could also possess a motoractivity in which energy released from cleavage of the high-energyphosphodiester bond, estimated to be −5.3 kcal∕mol (31), drivesconformational changes that are coupled to movement of the en-zyme along the DNA. However, there are no large-scale confor-mational changes in the trimer, such as subunit reorientations,that occur upon DNA-binding. Thus, the available data do notsupport a motor activity for λ exonuclease that would be analo-gous to other ring-shaped enzymes, such as hexameric helicases,in which binding and hydrolysis of ATP induces large reorienta-tions of the subunits (32). There are, however, some small localchanges in the subunit to which the DNA is bound, most notablyin the DE loop that wedges into the DNA (Fig. S5D), and inLys51, which flips up to contact the fourth phosphate on the5′-ended strand (Fig. S5A). We also observe a significant reorien-tation of the side chain of Arg28 of subunit A, together withmovement of its bound phosphate ion. It is interesting to spec-ulate that these changes could be coupled to the catalytic cycle,to eject cleaved mononucleotides out of the active site throughthe rear portal. We also note that the present structure does notexplain the complete inactivity of the R137A protein, as Arg137makes only a loose contact (4.3 Å) to the fifth phosphate of the5′-ended strand. Thus, there may be as yet unobserved states ofthe enzyme that could be involved in the catalytic cycle.

Single molecule studies of λ exonuclease measured similaraverage rates of digestion, but led to different conclusions regard-ing the sequence dependence of the digestion rate (28, 29). Onestudy observed that the local rate of digestion correlated with theenergy required to unwind the duplex and concluded that meltingof the terminal nucleotide to position it within the active site isthe rate-limiting step of the catalytic cycle (28). Our structuresupports this result and reveals that it is in fact two base pairsthat are melted from the duplex prior to cleavage. A second studyobserved pausing of the enzyme at particular GGCGA sequenceson the DNA, and it was suggested that this sequence could berecognized as ssDNA, possibly through stacking interactions ofthe guanine bases with aromatic residues of the active site cleft(29). Such a mechanism for pause-site recognition is not ex-plained by the current structure, because only two nucleotides ofthe DNA are unwound and there are no interactions with theterminal nucleotides that would appear to favor the GGCGA se-quence. However, the insertion of Arg45 into the minor groove of

Fig. 4. Electrostatic ratchet mechanism for processivity of λ exonuclease. SeeDiscussion for a description of the different steps. The 5′-phosphate group onthe DNA is represented by an asterisk (*). The positively charged pocket atthe end of the active site cleft is represented by the two plus (+) signs.

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the downstream portion of the DNA could provide a means forsequence-dependent pausing, as this interaction favors A-tract se-quences in which the minor groove is narrowed (23). The AT-richsequence of the right half of the most prominent pause site(GGCGATTCT), coupled with the higher energetic barrier tounwinding the GC-rich sequence on the left half, could providea structural basis for pausing. It is conceivable, however, thatpause-site recognition occurs through an off-pathway structurethat is substantially different from the one reported here. Alongthese lines, a structure of the enzyme in complex with DNA con-taining the pause sequence would be illuminating.

Given that λ exonuclease and the related RecE exonuclease(15) both form toroidal oligomers with multiple active sites sym-metrically disposed about a central channel, an interesting ques-tion emerges: Do these enzymes use a sequential mechanism inwhich the DNA moves cyclically from one active site to the nextfor each round of cleavage, or does the DNA engage with a singleactive site on the oligomer for multiple rounds of cleavage? Ourelectrostatic ratchet mechanism posits that processivity operatesnot only at the level of the trimer, but also at the level of themonomer. Thus, we predict that despite its symmetric architec-ture, the enzyme does not use a sequential type of mechanismsuch as that proposed for hexameric helicases (32, 33), F1-ATPase(34), and other toroidal oligomers.

Materials and MethodsThe λ exonuclease proteins were expressed in E. coli as N-terminal 6His fu-sions and purified by nickel affinity and anion exchange chromatography.Site directed mutagenesis was performed by the QuikChange procedure(Stratagene) and mutations confirmed by DNA sequencing and mass spectro-metry of the purified proteins. Oligonucleotides used for crystallization werepurchased HPLC-purified from Integrated DNA Technologies. For crystalliza-tion, 10 mg∕mL λ exonuclease (wild-type or K131A variant) was mixed with a1.1 M excess of DNA duplex in the presence of either 5 mM CaCl2 (Form 1) or5mMMgCl2 (Form 2), and crystallized by hanging-drop vapor diffusion. X-raydiffraction data were collected on flash-frozen crystals at the AdvancedPhoton Source. Structures were determined by molecular replacement. Exo-nuclease activities were determined by incubating a limiting amount ofenzyme at 37 °C with linear pUC19 dsDNA and analyzing the reaction pro-ducts by agarose gel electrophoresis with SYBR gold staining, or by incubat-ing excess enzyme with linear pUC19 and PicoGreen and monitoring thedecrease in fluorescence.

Additional methodological details are reported as SI Text.

ACKNOWLEDGMENTS. This work was funded by National Science FoundationGrant MCB-1021966 (to C.E.B.) and an American Heart Association Predoctor-al Fellowship (J.Z.). Use of the Advanced Photon Source at Argonne NationalLaboratory was supported by the US Department of Energy, Office of Science,Office of Basic Energy Sciences, under Contract DE-AC02-06CH11357. Useof the Lilly Research Laboratories Collaborative Access Team beamline atSector 31 of the Advanced Photon Source was provided by Eli Lilly Company,which operates the facility.

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