doi:10.1006/jmbi.2001.5189 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 314, 735±749

Mechanism and Cleavage Specificity of the H-N-HEndonuclease Colicin E9

Ansgar J. Pommer1, Santiago Cal2, Anthony H. Keeble1, Daniel Walker1

Steven J. Evans2, Ulrike C. KuÈ hlmann4, Alan Cooper3

Bernard A. Connolly2, Andrew M. Hemmings1,4, Geoffrey R. Moore4

Richard James5 and Colin Kleanthous1*

1School of Biological Sciences,University of East AngliaNorwich NR4 7TJ, UK2Department of Biochemistryand Genetics, University ofNewcastle, Newcastle uponTyne, NE2 4HH, UK3Department of ChemistryUniversity of GlasgowGlasgow, G12 8QQ, UK4School of Chemical SciencesUniversity of East AngliaNorwich NR4 7TJ, UK5Division of Microbiology andInfectious Diseases, UniversityHospital, Queen's MedicalCentre, University ofNottingham, NottinghamNG7 2UH, UK

Present addresses: A. J. Pommer:R&D, Zenit Building, Haus 65, LeipMagdeburg, Germany. S. Cal, DepaBioquimica y Biologia Molecular, EGascon. Campus del Cristo, Univer33006, Oviedo, Asturias, Spain.

Abbreviations used: ds, double-stendonuclease domain of colicin E9;triethanolamine; T(npp)2, thymidinenitrophenyl)-phosphate; TpA, thymdeoxyadenosine; ss, single-stranded8-anilinonaphthalene-1-sulfonic acid

E-mail address of the correspondc.kleanthous@uea.ac.uk

0022-2836/01/040735±15 $35.00/0

Colicin endonucleases and the H-N-H family of homing enzymes share acommon active site structural motif that has similarities to the active sitesof a variety of other nucleases such as the non-speci®c endonucleasefrom Serratia and the sequence-speci®c His-Cys box homing enzymeI-PpoI. In contrast to these latter enzymes, however, it remains unclearhow H-N-H enzymes cleave nucleic acid substrates. Here, we show thatthe H-N-H enzyme from colicin E9 (the E9 DNase) shares many of thesame basic enzymological characteristics as sequence-speci®c H-N-Henzymes including a dependence for high concentrations of Mg2� or Ca2�

with double-stranded substrates, a high pH optimum (pH 8-9) and inhi-bition by monovalent cations. We also show that this seemingly non-speci®c enzyme preferentially nicks double-stranded DNA at thyminebases producing 30-hydroxy and 50-phosphate termini, and that theenzyme does not cleave small substrates, such as dinucleotides or nucleo-tide analogues, which has implications for its mode of inhibition in bac-teria by immunity proteins. The E9 DNase will also bind single-strandedDNA above a certain length and in a sequence-independent manner,with transition metals such as Ni2� optimal for cleavage but Mg2� a poorcofactor. Ironically, the H-N-H motif of the E9 DNase although resem-bling the zinc binding site of a metalloenzyme does not support zinc-mediated hydrolysis of any DNA substrate. Finally, we demonstrate thatthe E9 DNase also degrades RNA in the absence of metal ions. In thecontext of current structural information, our data show that the H-N-Hmotif is an adaptable catalytic centre able to hydrolyse nucleic acid bydifferent mechanisms depending on the substrate and metal ion regime.

# 2001 Academic Press

Keywords: homing endonuclease; DNA; metal ions; mechanism; toxin

*Corresponding author

MelTec GmbH,zigerstr. 44, 39120rtamento dedi®cio Santiagosidad de Oviedo,

randed; E9 DNase,TEA,-30,50-di-(p-idylyl (30!5)-20-; ANS,.

ing author:

Introduction

Homing endonucleases are found throughoutbiology. Encoded by group I and group II intronsand inteins they promote the homing of the geneticelements coding for them into intronless/inteinlessallelic sites. Traditionally, these ubiquitousenzymes are classi®ed into families based on activesite amino acid sequence identities with four mainclasses: LAGLIDADG, GIY-YIG, His-Cys box, andH-N-H.1,2 In keeping with their biological import-ance, numerous studies have been reported onthese enzyme families, with the LAGLIDADG,His-Cys box and GIY-YIG families the best studiedbut with few mechanistic reports for any H-N-H

# 2001 Academic Press

736 Nuclease Mechanism and Speci®city of Colicin E9

enzyme. In the present paper we analyse thenucleic-acid cleavage speci®city and metal depen-dence of the H-N-H endonuclease from colicin E9,a bacterial toxin that kills Escherichia coli cellsthrough a non-speci®c DNase activity, and assignputative functionality to many of its amino acidresidues.

A characteristic feature of homing endonucleasesis that their DNA-binding sites are extensive (15-40 bp) but degenerate, with double-strand cleavageoccurring some distance away and generally leav-ing a 30 overhang.2 The enzymology of homingenzymes has been enhanced by structural andmechanistic studies on homologous endonucleasesfound in different biological settings. The non-speci®c, catabolic DNA/RNA nuclease from Serra-tia marcescens, for example, is a homologue of theHis-Cys box enzyme I-PpoI from Physarum polyce-phalum. Although both are dimers there is no struc-tural similarity between these enzymes except attheir active sites, cleaving DNA by the same basicmechanism.3,4 Another example of active site simi-larity between homing enzymes and other endonu-cleases is that of the H-N-H family of enzymes andcolicin endonucleases.5 Colicins are plasmidencoded toxins released by some strains of E. colifollowing SOS-mediated induction. Cell entry bythese bactericidal agents occurs through the parasi-tisation of outer membrane proteins normally usedin nutrient uptake, such as BtuB in the case of Egroup colicins, and periplasmic proteins usedeither in nutrient uptake or for maintaining theintegrity of the cell envelope (reviewed byLazdunski et al.6). Enzymatic E group colicins are60 kDa toxins that translocate their C-terminalcytotoxic domains across the inner membrane inorder to elicit cell death through the hydrolysis ofnucleic acid. Four H-N-H endonuclease colicinshave been identi®ed, E2, E7, E8 and E9, all ofwhich target the bacterial chromosome. Here, thefocus is the 15 kDa, monomeric endonucleasedomain of colicin E9 (E9 DNase) domain from coli-cin E9, an H-N-H enzyme that is the subject ofongoing enzymological studies in our laboratory.7,8

The only crystal structures of H-N-H DNasescurrently available are those of colicins E7 and E9bound to their cognate immunity proteins Im7 andIm9, respectively.9,10 Immunity proteins bind andinhibit colicins in the producing host. Homology tohoming enzymes in colicin DNases is restricted to12 residues of the C-terminal 32 residue H-N-Hmotif itself. The structural work on the E9 DNasesuggested that the classical description of homingenzymes into four families be re-evaluated sincethe H-N-H motif is structurally homologous to theactive sites of both I-PpoI, a His-Cys box enzyme,and Serratia nuclease even though these enzymesshare almost no sequence similarity with E9.11

Rather more unexpectedly, this analysis also high-lighted that the three enzymes position a metal ionin the same location. However, where this is amagnesium ion in Serratia and I-PpoI, the site isoccupied by a transition metal ion in the structures

of the H-N-H enzymes; zinc in the case of the E7DNase and nickel in the E9 DNase, the latter a con-sequence of the puri®cation protocol. KuÈ hlmannet al.11 proposed that these enzymes constitute anew group of metal-dependent endonucleasescalled the ``bba-Me'' family, identifying the struc-tural elements involved in the motif and the metalion. The same motif (referred to as a ``b-®nger'') isalso found in bacteriophage T4 endonuclease VII,which is thought to be mechanistically similar toboth Serratia and I-PpoI (see the Discussion).12

These structural similarities have been extendedyet further with the observation that the basicmotif encompassing the active sites of colicin andhoming endonucleases is found in many enzymefamilies, some of which are not even nucleases, butare related by the property that they bind metalions in a so-called ``treble clef ®nger''.13

Little is known of the mechanism by which H-N-H endonucleases cleave nucleic acid substrates.This can in part be attributed to the absence ofextensive sequence identities between the H-N-Henzymes and its structural homologues, but is alsodue to the enigmatic role of metal ions in colicinDNases, given that they bind transition metals in asite where enzymes such as Serratia and I-PpoIbind a magnesium ion.9,10,14,15 Here, we focus onthe mode of action of the DNase H-N-H enzymefrom colicin E9 and de®ne some of the basic con-ditions for optimal cleavage of DNA. We showthat the enzyme exhibits base speci®city fordouble-stranded (ds) DNA as well as cleaving bothsingle-stranded (ss) DNA and ssRNA. The catalyticactivities of colicin endonucleases are discussed inthe context of the structural information on the H-N-H motif in its different metal-liganded statesand its similarity to the active sites of other endo-nucleases. Finally, we discuss the possibility thatcolicin DNases are capable of hydrolysing DNA bytwo catalytic mechanisms, each with a distinctmetal-cofactor requirement.

Results

pH and cation dependence of E9 DNase activity

We have demonstrated that the E9 DNase showsno activity against double-stranded DNA sub-strates in the presence of zinc ions even thoughzinc binds to the H-N-H motif of the enzyme withnanomolar af®nity.8,16 In addition, we have shownthat the enzyme (at nanomolar protein concen-trations) shows highest activity against supercoiledDNA substrates in the presence of Mg2� or Ca2�,8

but the metal ion concentration dependence of thisactivity was not addressed nor were the optimalcleavage conditions assessed. These are reported inFigure 1. We investigated the Mg2� and Ca2�

dependence of the E9 DNase by two assaymethods, one using calf thymus DNA in the spec-trophotometric Kunitz assay, the other a plasmidnicking assay using radiolabelled pUC18 (see Pom-mer et al.8 for further details). From both assays it

Figure 1. Basic enzymological characterisation of theH-N-H motif of the E9 DNase. (a) Effect of calcium ionson endonuclease activity monitored by the spectropho-tometric Kunitz assay (closed symbols, left hand axis)and a plasmid nicking assay (open symbols, right handaxis) in bis-Tris propane at pH 8.5 and 25 �C asdescribed by Pommer et al.8 The DNase concentrationused in the two assays was 1.5 mg/ml (Kunitz assay)and 15 ng/ml (nicking assay). See Materials andMethods for further details. (b) pH optimum for theenzyme assessed in 50 mM Hepes buffer using theKunitz assay at 25 �C and 4 mg/ml E9 DNase. Substi-tution of Tris-HCl or bis-tris propane buffers for Hepesdid not have any in¯uence on the pH maximum (datanot shown). (c) Effect of NaCl on E9 DNase activity(1.5 mg/ml) determined by the Kunitz assay in 50 mMbis-tris propane (pH 8.5) in the presence of 20 mMMg2�.

Nuclease Mechanism and Speci®city of Colicin E9 737

is clear that the enzyme shows a broad metaldependence with an optimum between 10-20 mM,depending on the substrate used. The data inFigure 1(a) are those for Ca2�; very similar resultswere obtained for Mg2� (not shown). We foundthat the pH optimum for the enzyme is 8-9 usingthe Kunitz assay, and that the inclusion of NaClinhibited the activity of the enzyme, most likelydue to the effect of monovalent cations since chlor-

ide salts of buffers have no effect on the enzyme(data not shown) (Figure 1(b) and (c)). Theseresults are similar to those reported recently forI-CmoeI, a site-speci®c H-N-H endonuclease fromChlamydomonas moewusii.17 Hence, enzymes withan H-N-H active site motif likely cleave nucleicacid substrates by similar mechanisms regardlessof their DNA recognition speci®city, a situationreminiscent of the mechanistic similarities betweenthe non-speci®c endonuclease Serratia and thehighly speci®c His-Cys box enzyme I-PpoI.

The E9 DNase displays dsDNA base specificity

Members of the bba-Me family of endonucleaseshave a wide range of DNA base speci®cities; Serra-tia nuclease shows no sequence preference but pre-fers double-stranded A-form nucleic acids,18,19 theT4 endonuclease VII junction resolvase is structuredependent,12 while I-PpoI is highly speci®c for itshoming site.20 To address whether the H-N-H E9DNase possesses any sequence speci®city towarddsDNA, the Mg2�-dependent hydrolysis ofthe 160 bp tyrT promoter element was analysedalongside corresponding sequencing reactions(Figure 2(a)). The tyrT sequence has often beenused to investigate the cleavage preferences ofnon-speci®c endonucleases (see, for example, Drew& Travers21). The E9 DNase was able to cleave atapproximately one third of the 324 potential siteswithin the tyrT promoter sequence, cleaving tractsof GC poorly and having a preference for cleavingafter thymine (Figure 2(b)); 56 % of sites cleavedwere at T, 20 % at G, 14 % at A and 10 % at C.

Analysis of E9 DNase cleavage products

It has not been reported whether the products ofDNA cleavage by H-N-H endonucleases contain 50or 30 phosphates, a critical element in the under-standing of their cleavage mechanism. Hence, weaddressed this issue using the E9 DNase, lookingat the digestion products of l-DNA in the presenceof Mg2�. Following extended digestion, the samplewas divided into two parts, one was treated withcalf thymus alkaline phosphatase, to remove 50phosphate groups, while the other was leftuntreated. Both samples were then reacted withT4 polynucleotide kinase in the presenceof [g-32P]ATP (Figure 3). The rationale for thisexperiment is that if an endonuclease yields 50phosphate groups its products can only be radio-labelled if treated ®rst with alkaline phosphatase.If an enzyme yields 50 hydroxyl groups, radioac-tivity becomes incorporated by the kinase regard-less of the alkaline phosphatase treatment.Cleavage products of the restriction endonucleasesEcoRI and HindIII are known to produce 50 phos-phate groups and so these were included as con-trols in this experiment (Figure 3). Although thereis a low level of incorporated radioactivity in thealkaline-phosphatase-untreated samples, observedfor both the E9 DNase and the restriction enzymes

Figure 2. Cleavage of the tyrT promoter element by the E9 DNase. (a) [50-32P]-end-labelled tyrT DNA was incu-bated for varying times (one to 30 minutes) and with varying amounts of the E9 DNase in the presence of 0.5 mMMg2� and the products separated by gel-electrophoresis alongside sequencing reactions and visualised on a phos-phorimager (see Materials and Methods for further details). The gel data show the E9 DNase to have a clear prefer-ence for cleaving at T residues, where in addition more intense cut sites are generated than at non-T sites. (b)Summary of the tyrT cleavage data with arrows indicating positions where cleavage by the E9 DNase was detected.The four underlined regions are those that are largely refractory to cleavage by DNase I.21 The sequence shown in (a)corresponds to the ®rst 53 nucleotides of the top DNA strand.

738 Nuclease Mechanism and Speci®city of Colicin E9

and most likely due to exchange of 50 phosphategroups, this experiment strongly suggests that theE9 DNase cleavage products contain 50 phosphateand 30-OH termini. The same cleavage productsare seen in the catalysed reactions of Serratia nucle-ase and I-PpoI.

The E9 DNase does not cleave small nucleicacid substrates

The non-speci®c endonuclease from Serratia candigest DNA down to the level of mononucleotides,a consequence of its biological role in degradingextracytoplasmic nucleic acids for metabolism by

Figure 4. The E9 DNase does not cleave small sub-strates. (a) Spectrophotometric assay at 400 nm showingthat at pH 8.0 and at 25 �C the E9 DNase does notrelease nitrophenolate anion from the synthetic substrateT(npp)2 under conditions where DNase I readily showsactivity. See Materials and Methods for further details.(b) Anion exchange HPLC pro®les, eluted using anammonium acetate gradient, of a palindromic 16 bpDNA sequence before (bottom) and after (top) incu-bation with the E9 DNase in 10 mM Tris-HCl (pH 8.0)containing 10 mM MgCl2 at 37 �C for 20 hours. In orderto establish the level of digestion by the E9 DNase themigration positions of each of the four 50-phosphatemononucleotides were also established (bottom trace).

Figure 3. Colicin E9 DNase cleavage of DNA gener-ates 50 phosphate groups and 30-OH groups. Phagelambda DNA (2.5 mg) was incubated either with the E9DNase (left hand panel) or the restriction enzymesEcoRI & HindIII (right hand panel) for 20 minutes at37 �C in 10 mM Tris-HCl (pH 7.5) containing 10 mMMgCl2 and the digested DNA labelled with [g-32P]ATPusing T4 polynucleotide kinase (PK), with or withoutprior treatment by calf intestinal alkaline phosphatase(AK), and the DNA visualised by autoradiography fol-lowing separation on a 1.2 % agarose gel. Endonucleoly-tic cleavages leaving 50-OH groups would be labelledwith or without prior treatment with alkaline phospha-tase while cleavages generating 50 phosphate groupswill only be labelled following this treatment.

Nuclease Mechanism and Speci®city of Colicin E9 739

the organism.22,23 Consistent with this activity is itsability to also cleave nucleotide nitrophenyl esterssuch as thymidine-30,50-di-(p-nitrophenyl)-phos-phate (T(npp)2),3 which have also proved to beconvenient substrates for kinetic and mechanisticstudies of other non-speci®c endonucleases such asDNase I.24 Cleavage of T(npp)2 by DNase I gener-ates the p-nitrophenolate anion which can befollowed spectrophotometrically at 400 nm. Wetherefore investigated the cleavage of T(npp)2 bythe E9 DNase, using DNase I as a positive control(Figure 4(a)). Nitrophenolate anion was not gener-ated by the E9 DNase, in contrast to the reportedactivities of I-PpoI and Serratia both of which willcleave T(npp)2,3 and which have active sites thatare structurally homologous to the E9 DNase.11

Hence, although there are similarities between H-N-H enzymes such as E9 and other nucleases it isclear that there are also differences between them,particularly in terms of the substrates that they canhydrolyse.

To investigate this further we tested the abilityof the E9 DNase to digest the simple dinucleotidesubstrate thymidylyl (30 ! 50)-20-deoxyadenosine(TpA) in the presence of Mg2� but could detect no

The arrow in each pro®le indicates the injection point.

Figure 5. dsDNA and ssDNA binding to the E9DNase followed by extrinsic ¯uorescence quenching.DNA binding to the E9 DNase was monitored by thequench in ANS ¯uorescence (lex 365 nm/lem 490 nm)following the addition of ANS (40 mM) to the inactivemutant, E9 DNase H127A (0.2 mg/ml; 13.2 mM) in Tris-HCl buffer at pH 7.5 and 25 �C. (a) ANS ¯uorescencewith increasing molar ratio of 12mer dsDNA-to-proteinshowing that approximately two molecules of E9 DNasebind the duplex. (b) ANS ¯uorescence change as a resultof adding randomised sequences of equimolar ssDNAof increasing length (see Materials and Methods) show-ing that the E9 DNase prefers to bind substrates >tennucleotides in length. Under these conditions, ssDNA(10mer and above) binds to the enzyme stoichiometri-cally (data not shown).

740 Nuclease Mechanism and Speci®city of Colicin E9

activity with this substrate (data not shown). Thiswas con®rmed by HPLC analysis of E9 DNasecleavage products following incubation, for 20hours at 37 �C in the presence of Mg2�, with 16 bpduplex DNA (see Materials and Methods fordetails) that did not yield mononucleotide pro-ducts even though the starting material wasdigested to completion (Figure 4(b)). Takentogether these results show that, in the presence ofMg2�, the E9 DNase will not cleave mononucleo-tide phosphoryl esters or dinucleotide substrates,implying that substrate size likely plays an import-ant role in the ability of the enzyme to hydrolyseDNA.

dsDNA and ssDNA binding to the E9 DNasemonitored by 8-anilinonaphthalene-1-sulfonicacid (ANS) fluorescence

The absence of catalytic activity by the E9DNase against small nucleotide substrates could bedue to their inability to bind to the enzyme and soDNA binding to the E9 DNase was investigated,capitalising on work showing that metal bindingto the H-N-H motif causes substantial changes tothe ¯uorescence of the extrinsic ¯uorophoreANS.16 These changes occur as a result of theincrease in stability metal binding causes and theconcomitant expulsion of ANS from hydrophobicsurfaces of the DNase. For these studies, we used acatalytically inactive mutant, E9 DNase H127A,identi®ed through a random mutagenic screen,25

and initially investigated the binding of dsDNA.dsDNA caused a �30 % quench in the ANS-derived ¯uorescence on binding to E9 DNaseH127A, saturating at a stoichiometry of approxi-mately two protein molecules per 12mer duplex(Figure 5(a)). Since E9 DNase is able to bindssDNA (see below) it is possible that the ¯uor-escence changes were due to DNase-inducedmelting of the duplex and the binding of single-strands. However, this is unlikely since the meltingtemperature of the duplex used (40 �C) is signi®-cantly greater than the temperature at which theexperiment was conducted (25 �C), and since theintercalating dye YOPRO-1, which is only ¯uor-escent when intercalated into dsDNA, gave a high¯uorescence when added to the dsDNA-E9 DNaseH127A complex that was lost only when activeendonuclease (DNase I) was added to the mixture(data not shown).

Early studies on the DNase of colicin E2 indi-cated that it was able to cleave single-strandedDNA,26 although this activity, demonstrated usingsingle-stranded phage DNA, was not described indetail nor reported for other DNase colicins.Hence, using ANS ¯uorescence spectroscopy, wesought to determine whether the E9 DNase couldbind single-stranded oligonucleotides. In prelimi-nary experiments we found that, as with dsDNA,ssDNA causes a quench in ANS-derived ¯uor-escence but that this quench was dependent on thelength of DNA and that the binding was stoichio-

metric at micromolar protein concentrations (datanot shown). Hence, we analysed the binding ofequimolar concentrations of ssDNA of varyinglength to the E9 DNase H127A mutant (13.2 mM)and quantitated the ¯uorescence quench(Figure 5(b)). The ssDNA used was randomisedduring synthesis so that each of the four bases wasrepresented in every position in order to excludeany base information. The data demonstrate thatsmall oligonucleotides (<®ve bases) bind poorly ifat all to the E9 DNase, consistent with the lack ofhydrolytic activity toward dinucleotides andT(npp)2 (Figure 4), and that the optimal length forbinding is >ten bases. The stoichiometry and af®-nity of ssDNA binding to the E9 DNase weredetermined by isothermal titration calorimetry

Figure 6. Monitoring ssDNA binding to the E9 DNaseby isothermal titration calorimetry. Figure shows thebinding of 10mer single-stranded DNA of de®nedsequence (insert) to the inactive mutant E9 DNaseH127A. The top panel shows the calorimetric responseof 30, 10 ml injections of E9 DNase (0.53 mM) to ssDNA(50 mM) in 50 mM TEA buffer (pH 7.5) and 25 �C. Thebottom panel shows integrated injection heats for theabove data ®tted to a single, non-cooperative bindingmodel and the continuous line represents the theoreticalisotherm. Thermodynamic parameters from this ®t areshown in Table 1.

Nuclease Mechanism and Speci®city of Colicin E9 741

(Figure 6 and Table 1). Here, we compared thebinding of single-stranded DNA of randomsequence (12mer) with DNA of de®ned sequence(10mer) and found similar af®nities (Kd � mM) and1:1 binding for both, consistent with the ANS datashowing ssDNA >10 bases is optimal for binding.

Table 1. Thermodynamic parameters for ssDNA binding totitration calorimetry at pH 7.5 and 25 �C

ssDNA sequence n

GAC-GTA-AGA-G 0.89, 0.96Randomised 12mer 1.06

The thermodynamic parameters for a randomised 12mer are verydent measurements on two protein preparations are shown). For fur

ssDNA cleavage requires a transition metal ion

The question of whether E9 DNase cleavesssDNA and with what metal dependence wasaddressed using a 23 bp 32P-labelled ssDNA frag-ment (Figure 7). Very limited cleavage of the oligo-nucleotide was observed in the presence of 10 mMMg2� in a 15 minute incubation. Addition of Zn2�

did not support catalysis of ssDNA and did notenhance the ability of Mg2� to catalyse its hydroly-sis, whereas Ni2� (Kd, 0.7 mM16) yielded the highestactivity toward ssDNA. These experiments showthat E9 DNase will cleave ssDNA in the presenceof metal ions, with the highest activity associatedwith Ni2� and only weak activity associated withMg2�. This order of reactivity is the same as thatobserved using calf thymus DNA in the spectro-photometric Kunitz assay but the reverse of metalion reactivity in the nicking of supercoiled plas-mids, where Mg2� (or Ca2�) is the preferred metalover Ni2�.8 Interestingly, the nanomolar proteinconcentration required to observe an appreciableactivity in the plasmid nicking assay is signi®-cantly lower than that required for either theKunitz assay or the cleavage of ssDNA (100 and10,000-fold, respectively) suggesting that theselater activities are ``star'' activities and may not becentral to the cytotoxicity of colicins which areactive against bacteria at nanomolar concen-trations.

E9 DNase will cleave ssRNA

Non-speci®c endonucleases such as Serratianuclease,18 NucA,27 and Mitogenic Factor secretedby Streptococcus pyogenes.28 which cleave ss anddsDNA will also cleave ssRNA in the presence ofMg2�. Therefore, potential RNase activity for theE9 DNase was investigated by incubating theenzyme with a synthetic, ¯uorescently-labelledssRNA 10mer under a variety of metal regimes.We found that the enzyme does indeed cleaveRNA and that this activity could be ascribed to theE9 DNase and its H-N-H motif since it was inhib-ited by Im9 binding and by the H-N-H mutationHis127Ala (Figure 8(a)). Surprisingly, however, wefound that the enzyme cleaved RNA in the absenceof divalent cations and that this activity was notstimulated either by Mg2� or Zn2�, but was stimu-lated by Ni2� (Figure 8(b)). This is in contrast tothe work of Schaller & Nomura26 on colicin E2

the E9 DNase H127A mutant determined by isothermal

Kd (M) �H (kcal/mol)

0.4, 0.7 � 10ÿ6 ÿ12.0, ÿ10.22.3 � 10ÿ6 ÿ13.0

similar to those of a 10mer of de®ned sequence (two indepen-ther details see the legend to Figure 6. (1) cal � 4.184 J.

Figure 8. The E9 DNase cleaves RNA in the absenceof metal ions. Carboxy-¯uoresceine-labelled 10merssRNA (GAC GUA AGA G) was incubated with the E9DNase (0.15 mg/ml) in 50 mM TEA buffer (pH 7.5) for30 minutes at 30 �C and the products analysed by aphosphorimager following separation by acrylamide gelelectrophoresis. Three time points are indicated (15seconds, 15 minutes and 30 minutes), with ``c'' identify-ing the migration of substrate RNA in the gel. (a) TheRNase activity of the enzyme occurs in the absence ofmetal ions and is abolished by Im9 binding and by ahistidine-to-alanine mutation at residue 127, the latterresult indicating that the H-N-H motif of the enzyme isresponsible for this activity. (b) The effect of metal ionson the RNase activity of the E9 DNase. The concen-trations of the Ni2� and Zn2� used (10 mM) were abovetheir respective Kd values16 while that of Mg2� (10 mM)is optimal for DNA cleavage (Figure 1).

Figure 7. Cleavage of ssDNA by the E9 DNaserequires transition metal ions. 23mer ssDNA was end-labelled with 32P and incubated with the E9 DNase(0.2 mg/ml) over a 15 minute time course under a var-iety of metal regimes and in TEA buffer (pH 7.5) at37 �C (see legend to Figure 8). The products were separ-ated using a 20 % polyacrylamide gel containing 8 Murea and visualised using a phosphorimager. The datashow that Zn2� does not support ssDNA cleavagewhereas Ni2� yields rapid digestion, while Mg2� is apoor cofactor for this substrate.

742 Nuclease Mechanism and Speci®city of Colicin E9

where no RNase activity could be detected againstphage RNA. However, these early studies usedlower concentrations of the toxin (20 mg/ml) thanused here with the E9 DNase domain (150 mg/ml),emphasizing that this is a weak RNase activity.

Discussion

Enzymological similarities and differencesbetween the E9 DNase and other nucleases

The non-speci®c endonuclease from colicin E9shares many of the same basic enzymologicalcharacteristics as sequence-speci®c H-N-H homingendonucleases such as I-CmoeI as well as with theHis-Cys box enzyme I-PpoI, including a require-ment for high concentrations of Mg2�, a pH opti-mum �8.5 and inhibition by monovalent cations.We show, for the ®rst time, that an H-N-H endo-nuclease releases 50 phosphate and 30-OH termini,which is also the case for Serratia nuclease andI-PpoI. Moreover, the E9 DNase will cleave ssDNA,ssRNA and dsDNA, demonstrating a lack of speci-®city for the sugar moiety of nucleic acid, which isalso the case for Serratia nuclease. These similaritiesto other nucleases suggests that, in the presence ofMg2�, the E9 DNase may cleave nucleic acid by asimilar mechanism to that used by I-PpoI and Ser-ratia nuclease, two enzymes that have been verywell characterised in terms of their catalyticactivity, and this is addressed below.

The similarities and differences between the E9DNase and other nucleases, revolving around sub-strate preferences and base speci®city, may pointto their evolutionary ancestry. Not only does theE9 DNase act on a wide range of polynucleotides

but it also shows a very strong preference for cut-ting at T bases in dsDNA. This speci®city is pre-sumably a re¯ection of the biological role of theenzyme, digestion of the relatively A/T rich E. coligenome. Pronounced selectivity for a single base israre with deoxyribonucleases since any preferenceseen with low-speci®city DNasess is usuallyascribed to gross structural features of the nucleicacid. For example, both the Serratia nuclease19 andDNase I21 cut d(A).(T) tracts poorly, probably dueto the rigidity of such sequences.29 Staphylococcalnuclease is an example of a low speci®city nucleasethat has a preference for T bases, in exposedsingle-stranded regions of DNA,30 but produces 30phosphate groups and 50 hydroxyl termini which isnot the case with the E9 DNase. Base-speci®c clea-vage is more common with ribonucleases (e.gRNase T1 at G; RNase U2 at A; RNase A at C/U)suggesting that staphylococcal nuclease and E9DNase, both of which are active on RNA, mayhave evolved from base-selective RNases. Inrelation to this, it is interesting to note that cation-independent cleavage of RNA but cation-depen-dent cleavage of DNA by a single nuclease activesite is reminiscent of the RNase a-sarcin from

Nuclease Mechanism and Speci®city of Colicin E9 743

Aspergillus which cleaves RNA in the absence ofmetal ions but requires Mg2� to cleave DNA.31

Finally, since colicin E9 expression is induced byDNA damage through the SOS response, the E9DNase may have evolved from a DNA repairendonuclease, a possibility given credence by theobservation that H-N-H endonuclease domainshave been found in mismatch repair enzymes.32

E9 DNase nucleic acid binding characteristics

Unlike enzymes such as Serratia nuclease, the E9DNase does not cleave dinucleotides or simplenucleotide derivatives since these bind poorly if atall to the enzyme (Figure 5). The colicin E9 DNasecan bind ssDNA in a sequence- independent man-ner preferring DNA above a certain length (>tenbases). Gel-shift experiments using plasmids haveshown that the E9 DNase H127A mutant can binddsDNA.10,25 This is con®rmed by the ANS ¯uor-escence binding data presented here but whichalso show that at least two molecules of theE9 DNase can bind a 12mer duplex. Given thedimensions of the major and minor grooves ofB-form DNA, the dimensions of the E9 DNase(40 AÊ � 25 AÊ � 25 AÊ ) and its preference for bind-ing relatively large substrates, it seems unlikelythat two molecules of the enzyme could be accom-modated simultaneously in the same DNA grooveof a 12mer. More likely, enzyme monomers bindto the phosphate backbone of each DNA strandthus explaining the binding stoichiometry and theability of the enzyme to bind ssDNA with 1:1stoichiometry.

The fact that the E9 DNase prefers to bind (andhydrolyse) DNA above a certain length has twoimportant consequences for our understanding ofcolicin action. First, it implies that the free energyof binding is derived largely from phosphate rec-ognition and that this may play a role in thehydrolysis of DNA, observations consistent withthe natural substrate for colicin DNases being thebacterial chromosome. Second, this has rami®ca-tions for the way in which DNase colicins areinhibited in bacteria. Suicide is prevented in coli-cin-producing cells through the action of an immu-nity protein which binds with very high af®nity(the Kd for Im9 binding the E9 DNase is 10ÿ16 M atpH 7 and 25 �C33) to an Immunity Protein Exosite(IPE) that lies adjacent to the H-N-H motif, withthe immunity protein making no direct interactionswith catalytic residues.10,34,35 The immunity proteinis thought to inhibit the enzyme primarily throughsteric hindrance and electrostatic repulsion of sub-strate binding and this is supported by modellingstudies,10 and by the absence of conformationalchanges in the active site on immunity binding asreported by NMR spectroscopy.36 Hence, it wouldbe a disadvantage to a colicin-producing organismfor the enzyme to retain endonuclease activityagainst small, single-stranded oligonucleotide sub-strates since the immunity protein would not inhi-bit their binding. The results presented here show

that the E9 DNase shows no activity against dinu-cleotides and binds ssDNA less than ten basespoorly, observations consistent with the exositeinhibitory mechanism of immunity proteins.

Since there are no structures of the E9 DNasebound to nucleic acid, an important questionremains as to whether the H-N-H motif representsthe site of DNA binding. Three lines of evidencestrongly suggest the motif as the site of DNA bind-ing in the E9 DNase: (1) The motif forms the majorpart of a positively charged cleft in the proteinwhere phosphate readily binds, and acidicmutations within this cleft abolish DNA binding toE9 (T. Georgiou and C.K., unpublished results); (2)DNA is known to bind to the equivalent site in I-PpoI;37 (3) The intrinsic tryptophan ¯uorescence ofthe E9 DNase is sensitive to ligand binding, withtransition metal binding to the H-N-H motifquenching the ¯uorescence but immunity proteinbinding to the IPE yielding a ¯uorescentenhancement.16,38 ssDNA and dsDNA both quenchthe intrinsic tryptophan ¯uorescence on binding tothe E9 DNase which is consistent with the H-N-Hmotif being the site of binding (A.H.K. & C.K.,unpublished results). It should also be noted thatDNA binding to the H-N-H motif is metal inde-pendent, as is that to I-PpoI,20 since the E9 DNaseH127A variant used in these studies does bindtransition metal ions.16 This is in contrast to thereports of Drouin et al.17 on the H-N-H homingendonuclease I-CmoeI where EDTA treated proteinfailed to gel-shift target DNA. However, cautionhas to be taken when treating H-N-H enzymeswith EDTA since NMR experiments in our labora-tory on the E9 DNase have shown that it readilybinds EDTA39 and that this is suf®cient to inhibitDNA binding (data not shown).

Structures of the H-N-H motif in differentliganded states identify a catalytic tetrad

Three structures of colicin DNases are currentlyavailable, two for the E9 DNase (at 2.05 and 1.7 AÊ

resolution), shown in Figure 9, and one for the E7DNase at 2.4 AÊ resolution, each bound to theirimmunity protein, Im9 and Im7, respectively.9,10,40

The active sites of all three complexes, which arenear identical in terms of their amino acidsequence, differ in terms of their metal ligation,with the E9 structures showing the apo- and Ni2�-liganded forms and the E7 structure that of theZn2�-bound form. Superposition of the two tran-sition metal complexes (not shown) reveals that thestructures are very similar, with the metal ions inboth adopting distorted tetrahedral geometries.However, there are subtle differences between thetwo complexes that are revealing: (1) The Ni2� ionhas only two clearly identi®able protein ligands(His102 and His127) with the third (His131) posi-tioned poorly to bind the metal (Figure 9(b)),although solution NMR experiments identify threehistidine ligands to the Ni2� ion.39 The E7 DNasecrystal structure, on the other hand, shows clearly

Figure 9. Stereo representations of the active site hydrogen bonding network within the H-N-H motif of the E9DNase with and without bound Ni2�. In each case the structure of the E9 DNase was solved in complex with theimmunity protein Im9, which does not bind in the active site. (a) Apo-E9 DNase (at 1.7 AÊ resolution; accession code,1emv) from KuÈ hlmann et al.40 The red sphere is a bound water molecule. (b) Ni2�-bound structure (at 2.05 AÊ resol-ution; accession code, 1bxi) from Kleanthous et al.10 The green sphere is the nickel ion. A bound phosphate molecule(red and purple) is present in both structures.

744 Nuclease Mechanism and Speci®city of Colicin E9

that the zinc ion is coordinated by all three histi-dine residues. These differences in coordinationchemistry are re¯ected in the af®nities of themetals for the E9 DNase since the Kd for Zn2� isnM while that for Ni2� (and Co2�) is mM;16 (2)Ni2�-liganded E9 DNase has a bound phosphatemolecule that occupies one of the coordinationsites to the metal ion but this phosphate is replacedby water in the Zn2�-loaded enzyme; (3) a conse-quence of these differences is that the Ni2�-boundE9 DNase has one free coordination site (taken byHis131 in Zn2�-bound E7 DNase) and this mayexplain why nickel (and cobalt) is active as a metalcofactor while zinc, a catalytic metal ion in othernucleases,41 does not support DNase activity in theH-N-H motif of colicins (see below).

The presence of phosphate in the active site ofthe E9 DNase likely denotes the position either of

the scissile bond or, more likely, the product 50phosphate generated by the enzyme, and thisphosphate molecule is bound even in the absenceof metal ions (Figure 9(a)). The hydrogen bondingpattern in the active site of the apo-E9 DNase issubstantially different, although the overall struc-ture of the active site changes little.40 Particularlystriking are changes to the imidazole groups ofHis102, which rotates by 180 �, and His127, both ofwhich form new hydrogen bond interactions.His102 hydrogen bonds to Glu100 while His127(previously hydrogen bonded to Glu100) interactswith Arg96 which in turn forms a salt bridge withGlu100, and so together comprise a catalytic tetrad.Three of these residues are known to be essentialfor catalysis25 and so the entire tetrad is likely tobe central to the catalytic mechanism of colicinDNases.

Figure 10. Putative mechanisms for the hydrolysis ofDNA catalysed by the H-N-H motif in the presence of,(a) Mg2� and (b) Ni2� based on the structures of the E9DNase in the apo and Ni2�-liganded forms.10,40 In (a)the catalytic tetrad at the centre of the motif serves todepress the pKa of His127, which is postulated to act asa ligand for Mg2�, and raise the pKa of His102, pre-dicted to be a general acid involved in protonating the30 oxygen leaving group. In (b) the binding of Ni2�

causes the reorganisation of the tetrad into a triadwhere now His127 is hydrogen bonded to Glu100 andHis102 coordinates the nickel ion. It is proposed that ametal-activated water molecule bound to the metal iontakes on the role of protonating the 30 oxygen, thefourth coordination site used to activate the scissilephosphodiester bond.

Nuclease Mechanism and Speci®city of Colicin E9 745

The H-N-H motif of the E9 DNase is anadaptable catalytic centre

In the following sections we attempt to reconcilethe different activities of the E9 DNase with thestructural information available, and suggest thatthe H-N-H motif adopts different catalytic strat-egies depending on the metal ion regime. We pos-tulate that the apo-form of the E9 DNaserepresents that which undergoes Mg2�-dependenthydrolysis of dsDNA, most likely by a mechanismsimilar to that described for Serratia nuclease and I-PpoI.14,15,42 This mechanism is unusual for Mg2�-dependent endonucleases in that a histidine resi-due, rather than a magnesium ion, activates thehydrolytic water molecule through general basecatalysis (Figure 10(a)). This residue is His103 inthe E9 DNase, which superimposes closely withthe equivalent histidine residues in Serratia (His89)and I-PpoI (His98).11 Stabilisation of the pentacoor-dinate transition state in these enzymes is accom-plished by a magnesium ion liganded to theprotein through the oxygen atom of a single aspar-agine residue (Asn119 in both Serratia and I-PpoI).The equivalent position in colicin DNases is His127(Figure 9). Considering that histidine co-ordinationof magnesium has never to our knowledge beenobserved in an enzyme, this raises doubts as towhether His127 could ful®l such a role. However,we note that there is precedent for His coordi-nation of a calcium ion in squiddiisopropyl¯uorophosphatase43 and that Ca2� is aseffective a cofactor in E9 DNase dsDNA hydrolysisas Mg2� and likely to bind in the same site. Indeed,the equivalent position is occupied by a calciumion in T4 endonuclease VII, albeit bound by anasparagine residue as in Serratia and I-PpoI.12 Thecase for His127 as the Mg2�/Ca2� ligand is madestronger by the fact that there are few potentialoxygen ligands for magnesium within the E9DNase H-N-H motif, especially since the aspara-gine identi®ed in the motif serves a structuralrole.10 Lastly, Arg5 contacts the bound phosphatein the E9 DNase and is equivalent to Arg61 in I-PpoI (and Arg57 of Serratia) where its role is in sta-bilising the product 50 phosphate.

One of the striking differences between theactive sites of I-PpoI and Serratia and that of the E9DNase is the preponderance for histidine residuesin E9, four compared to one, with the two thatdenote the H-N-H motif (His103 and His127,described above) highly conserved and the remain-ing two (His 102 and His131) conserved in mostcases.5 We now speculate on the potential roles ofthese latter residues. The role of His131 is the mostenigmatic but may be involved in binding to thebackbone of substrate DNA. The hydrogen bondof His102 to Glu100 in the apo-form of the enzymeis likely to raise its pKa which, considering the pHoptimum for the enzyme is pH 8-9, argues for arole as a general acid, protonating the 30-oxygenleaving group (Figure 10(a)). This would be in con-trast to I-PpoI, where a magnesium-bound water

molecule protonates the leaving group,14 but isconsistent with T4 endonuclease VII where a histi-dine residue is postulated to take this role.12 Invok-ing the participation of a general acid and ageneral base both of which are histidine residues,and strategically placed above and below thebound phosphate in the E9 DNase apo-structure(Figure 9(a)), may provide an explanation for themetal-independent RNase activity of the E9DNase. However, it remains to be establishedwhether the hydrolytic products of RNA cleavageby the E9 DNase are the same as those of DNAhydrolysis or indeed if a cyclic phosphate inter-mediate is formed, a characteristic feature of manyRNases.

746 Nuclease Mechanism and Speci®city of Colicin E9

The binding of Ni2� to the E9 DNase H-N-Hmotif results in the catalytic tetrad of the apo-enzyme converting to a triad. His102 moves >6 AÊ

to be co-opted as a ligand for the transition metalalong with His127 which moves 3 AÊ (Figures 9(b)and 10(b)). Assuming that the activation of thehydrolytic water remains the role of His103 (itsposition in the Ni2�-bound and apo- structuresdoes not change), then the problem arises as tohow the 30 oxygen becomes protonated sinceHis102 (the putative general acid) is no longeravailable. We postulate that a Ni2�-coordinatedwater molecule ful®ls this role, taking one of fourcoordination sites to the metal ion, the remainingsite used to bind and activate the scissile phospho-diester bond (Figure 10(b)). This mechanism wouldexplain why zinc does not support catalysis in coli-cin DNases since the bound transition metal wouldneed to retain two free metal coordination sites forcatalysis to proceed whereas only one is availablein the tightly bound zinc complex.9 Another conse-quence of which metal ion is used by colicinDNases is substrate preference since magniesumions are optimal for double-stranded substrateswhile transition metals such Ni2� and Co2� areoptimal for ssDNA, although the basis for thesepreferences remains obscure at present.

In summary, the mechanism of DNA hydrolysisby colicin DNases in the presence of transitionmetals is almost certainly different to that in thepresence of Mg2� because of the side-chain move-ments that accompany transition metal ion bind-ing. We postulate that the main difference betweenthese mechanisms is how the 30 oxygen is proto-nated, by a histidine residue in Mg2�-dependenthydrolysis but a metal-activated water moleculewhen transition metals such as Ni2� and Co2� arebound to the enzyme; the absence of two freecoordination sites in the Zn2�-bound complexlikely explaining why it does not support thehydrolysis of nucleic acid. The structuralrearrangements observed in colicin active sitesbegin to explain how a single protein scaffold canrecruit either a magnesium or transition metal ionfor the hydrolysis of phosphodiester bonds andemphasize that the histidine-rich H-N-H motif ofhoming enzymes and colicins is a remarkablyadaptable catalytic centre.

Materials and Methods

Bacterial strains and media

Plasmid pRJ353 (encoding the E9 DNase domain andIm9 with a C-terminal histidine tag) was transformedinto E. coli BL21 (DE3) pLysS and cells grown on Luria-Bertani broth, as described.25

Protein purification and protein andDNA quantitation

The E9 DNase was puri®ed as described by Garinot-Schneider et al.25 and adapted by Pommer et al.16 SinceNi2�-af®nity chromatography is used in the puri®cation

of the E9 DNase and Ni2� binds to the enzyme, all pro-tein preparations were treated with 10 mM EDTA inTris-HCl buffer (pH 7.5) to remove bound metal ionsand then dialysed against the same buffer but containing200 mM NaCl. Protein was ®nally lyophilized fromwater and stored at ÿ20 �C. The absence of transitionmetal contamination in all E9 DNase preparations wasveri®ed as described16 using ANS ¯uorescence. Proteinwas quantitated by absorbance spectrophotometry at280 nm as described,7 while DNA concentrations werequantitated by absorbance at 260 nm.

Kunitz and 3H-plasmid nicking assays of E9DNase activity

Colicin E9 endonuclease activity was routinelymeasured using the Kunitz assay,44 where the change inhyperchromicity of calf thymus DNA was measuredspectrophotometrically at 260 nm, or a plasmid nickingassay where the rate of nicking was quantitated by sep-arating the cleavage products of supercoiled [3H]pUC18by agarose gel-electrophoresis and determining theamount of supercoil remaining by scintillationcounting.8,45 The E9 DNase shows non-linear kinetics inthe Kunitz assay when hydrolysing calf thymus DNA inthe presence of Mg2� or Ca2�,9 and so the hyperchromi-city data were analysed as the change in absorbance at260 nm after a ten minute incubation. Where indicatedthe pH, salt and metal ions in these assays was alteredto determine the effect on E9 DNase activity.

Identification of the colicin E9 DNase cleavage sites

Radiolabelled tyrT promoter DNA was used to deter-mine the positions of cleavage by the E9 DNase.21 tyrTwas PCR ampli®ed using primers with EcoRI andHindIII adapters and cloned using these sites in bothM13mp18 and M13mp19, which makes it possible toidentify positions of cleavage in both strands of theDNA.46 Once the phage single-strand was obtained,double-strand was made using 1 pmol of M13 universalprimer which was 50-end labelled with T4 polynucleotidekinase and [g-32P]ATP. After annealing to the template,the primer was extended by treatment with Klenowenzyme and all four dNTPs. The double strand DNAwas then incubated with the E9 DNase in 5 mM Tris-HCl (pH 8.0) in the presence of 0.5 mM MgCl2 and0.5 mM MnCl2 at 37 �C for one to 30 minutes and theproducts of digestion run in parallel with four sequen-cing lanes obtained using the same primer and accordingto the manufacturers method (Amersham-Pharmacia).Reactions were stopped with stop solution (95 % (v/v)formamide, 20 mM EDTA, 0.05 % (w/v) bromophenolblue and 0.05 % (w/v) xylene cylanol FF) and resultsvisualised with autoradiography for 24-36 hours atÿ70 �C with intensifying screens and bands scannedusing a phosphoroimager (Molecular Dynamics).

Analysis of the terminal nucleotide ofendonuclease-generated fragments

l-DNA (2.5 mg) was incubated for 20 minutes in10 mM Tris-HCl (pH 7.5) containing 10 mM MgCl2 in a®nal volume of 20 ml with either the E9 DNase (40 mg) orEcoRI/HindIII at 37 �C. The reactions were stopped byphenol-extraction (phenol/chloroform/isoamyl alcohol,25:24:1 (v/v)). After splitting each digest into two, 1 mgwas incubated with one unit of calf intestinal alkaline

Nuclease Mechanism and Speci®city of Colicin E9 747

phosphatase (CIAP) (Boehringer Mannheim) with themanufacturers recommended buffer for one hour at37 �C. Three phenol extractions (phenol/chloroform/iso-amyl alcohol (25:24:1 (v/v)) were carried out in order tostop CIAP activity. The dephosphorylated DNA wasthen precipitated with 0.1 volume of sodium acetate(3 M) and three volumes of ethanol and washed with70 % (v/v) ethanol. Both CIAP treated and control frag-ments were labelled at the 50-termini using T4 polynu-cleotide kinase and 1 ml [g-32P]ATP for 40 minutes at37 �C. After two ethanol precipitations (0.1 volumessodium acetate (3 M) and three volumes ethanol) theprecipitates were washed twice with 70 % ethanol. Theproduct was resuspended in 20 ml of TE-buffer (10 mMTris-HCl (pH 8.0), 1 mM EDTA) and samples separatedon a 1.2 % (w/v) agarose gel and analysed by auto-radiography, carried out for three hours at ÿ70 �C withan intensifying screen.

Digestion of T(npp)2 and TpA

The hydrolysis of T(npp)2 was carried out as describedby Liao24 in a volume of 0.5 ml containing 25 mM Tris-HCl (pH 8), 1 mM CaCl2, 10 mM MnCl2 and T(npp)2 ata concentration of 15 mM. The reaction was initiated bythe addition of E9 DNase or DNase I. The rate of clea-vage was determined from the increase in absorbance at400 nm typical for the cleavage product, p-nitrophenol inits deprotonated form. Assays were performed at 25 �Cover ten minutes. The hydrolysis of TpA(e260 � 2.34 � 104 Mÿ1 cmÿ1) was carried out in a volumeof 25 ml containing 5 mM TpA, 10 mg of E9 DNasedomain, 25 mM Tris-HCl (pH 7) and 10 mM MgCl2 at37 �C. The progress of the digestion was followed byHPLC, using a C18 reverse phase column at 55 �C,where 2 ml of the digest was loaded onto the column.Elution was achieved with a gradient formed from bufferA (0.1 M triethylammonium acetate (pH 6.5), 3 % (v/v)CH3CN) and up to 80 % buffer B (as buffer A but with65 % CH3CN) in 40 minutes with a ¯ow rate of 1 ml/min. Elution was monitored by measuring the absor-bance at 254 nm.

Hydrolysis of a 16 bp palindromic oligonucleotide

A 16 bp palindromic sequence (GAA TTC GAT CGAATT C; MWG Biotech) was used to investigate the digestlimit of E9 DNase with dsDNA. The 20 hour digest wascarried out at 37 �C in 30 ml of 10 mM Tris-HCl (pH 8.0)containing 10 mM MgCl2 and DNA. E9 DNase (10 mg)was added to start the digestion and 2 ml of the reactionmixture was analysed using an anion exchange column,PA-100 column (Dionex), coupled to a Gilson HPLCapparatus at 25 �C. The fragments were eluted using agradient formed from buffer A (10 % CH3CN) and bufferB (10 % CH3CN � 1.5 M ammonium acetate (pH 7.5));up to 80 % B in 40 minutes, at a ¯ow rate of 1 ml/min.Elution was monitored by measuring the absorbance at254 nm and the pro®le compared to the retention timeof the four 50 phosphate mononucleotides on the samecolumn under identical conditions.

ANS fluorescence spectroscopy

Fluorescence experiments using the dye ANS wereperformed with an excitation wavelength of 365 nm andmonitoring the emission in the range, 390-650 nm.Quartz cuvettes (3 ml) were used and the reactions con-

tained 1.5 ml TEA buffer (pH 7.5), 13.2 mM E9 DNaseH127A and 40 mM ANS. For all experiments the DNA(supplied by MWG Biotech) was made up in AnalaRwater and added in 1-2 ml aliquots. A 12mer duplex ofde®ned sequence was used (CCA GGT AGC CAG) aswell as single-stranded sequences which were random-ised for each base at every position during synthesis(two to 14 bases in length).

Isothermal titration calorimetry

Isothermal titration calorimetry was carried out essen-tially as described by Pommer et al.16

Digestion of ssDNA and ssRNA

32P-labelled 23-mer (GTT TTC CCA GTC ACG ACGTTG TA; MWG Biotech) was labelled with T4 kinase and[g-32P]ATP and used as a substrate. For the detection ofRNA cleavage, 50-carboxy¯uorescein labelled 10merRNA (GAC GUA AGA G; Cruachem Ltd) was used as asubstrate. In both cases, digests were carried out in50 mM triethanolamine buffer (pH 7.5), in 10 ml and at37 �C for ssDNA, and in 30 ml and at 30 �C for ssRNAcontaining E9 DNase (2 and 4.5 mg, respectively) under avariety of metal regimes, and resolved by acrylamidegel-electrophoresis and visualised using a phosphorima-ger.

Acknowledgements

We thank Ann Reilly and Christine Moore (Norwich)and Pauline Heslop (Newcastle) for excellent technicalassistance. This work was supported by The WellcomeTrust. The Glasgow calorimetry service is funded by theBiotechnology and Biological Science Research Councilof the UK.

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Edited by J. Karn

(Received 13 August 2001; received in revised form 18 October 2001; accepted 19 October 2001)