φ29 DNA Polymerase Residue Phe128 of the Highly Conserved (S/T)Lx2h Motif is Required for a Stable and Functional Interaction with the Terminal Protein

f29 DNA Polymerase Residue Phe128 of the HighlyConserved (S/T)Lx2h Motif is Required for a Stableand Functional Interaction with the Terminal Protein

Irene Rodrıguez, Jose M. Lazaro, Margarita Salas* and Miguel de Vega

Centro de Biologıa Molecular“Severo Ochoa” (CSIC-UAM)Universidad AutonomaCantoblanco, E-28049 MadridSpain

Bacteriophage f29 encodes a DNA-dependent DNA polymerase belong-ing to the eukaryotic-type (family B) subgroup of DNA polymerases thatuse a protein as primer for initiation of DNA replication. By multiplesequence alignments of DNA polymerases from such a family, we havebeen able to identify two amino acid residues specifically conserved inthe protein-priming subgroup of DNA polymerases, a phenylalanine con-tained in the (S/T)Lx2h motif, and a glutamate belonging to the Exo IIImotif. Here, we have studied the functional role of these residues inreactions that are specific for DNA polymerases that use a protein-primedDNA replication mechanism, by site-directed mutagenesis in thecorresponding amino acid residues, Phe128 and Glu161 of f29 DNA poly-merase. Mutations introduced at residue Phe128 severely impaired theprotein-primed replication capacity of the polymerase, being the inter-action with the terminal protein (TP) moderately (mutant F128A) orseverely (mutant F128Y) diminished. As a consequence, very fewinitiation products were obtained, and essentially no transition productswere detected. Interestingly, f29 DNA polymerase mutant F128Y showeda decreased binding affinity for short template DNA molecules. Theseresults, together with the high degree of conservation of Phe128 residueamong protein-primed DNA polymerases, suggest a functional role forthis amino acid residue in making contacts with the TP during the firststeps of genome replication and with DNA in the further replication steps.

q 2002 Elsevier Science Ltd. All rights reserved

Keywords: DNA polymerase; site-directed mutagenesis; linear DNAreplication; protein-priming; terminal protein*Corresponding author

Introduction

One important issue of DNA synthesis is thereplication of the ends of linear DNA. DNA poly-merases are unable to initiate de novo DNAsynthesis on a DNA template, but require theexistence of a primer containing a free hydroxylgroup to start DNA elongation.1 Generally, RNAprimers provide the 30-hydroxyl (30-OH) groupneeded by the DNA polymerase to elongate theDNA chain. However, replication of lineargenomes cannot be driven using the standardmechanism of RNA priming, since once the primeris removed, no DNA polymerase could fill the

resulting gap. Different mechanisms have evolvedto solve the problem, mainly through the presenceof repetitive terminal sequences that allow DNA-priming through the formation of circular or con-catemeric molecules, as in the case of T4 andlambda phages, the creation of a hairpin structureat the DNA terminus,1 or the use of a protein asprimer, as in the case of bacteriophage f29 andadenovirus (reviewed by Salas et al.2,3).

Bacteriophage f29 genome is a double-strandedDNA (dsDNA) of 19285 bp, with a terminal protein(TP) covalently linked to each 50-end. f29 DNAreplication requires the formation of a heterodimerbetween the f29 DNA polymerase and a free TPmolecule, which is able to recognise and interactwith the replication origin at both ends of thegenome. A histone-like viral protein (p6) forms anucleoprotein complex at the replication originsthat probably contributes to the unwinding of thedouble helix at the DNA ends.4 During initiation,

0022-2836/03/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

E-mail address of the corresponding author:[email protected]

Abbreviations used: Pol I, E. coli DNA polymerase I;Pol Ik, Klenow fragment of Pol I; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA.

doi:10.1016/S0022-2836(02)01130-0 J. Mol. Biol. (2003) 325, 85–97

f29 DNA polymerase catalyses, at both DNA ends,the addition of dAMP to the OH group of Ser232 inthe TP, using as director base the second dTMP ofthe template strand.5,6 Subsequently, the complexslides back one nucleotide to recover the infor-mation of the 30-terminal dTMP, being sequencereiteration a requisite for the sliding-back mechan-ism. This feature is also conserved in other lineargenomes that contain a TP covalently linked totheir DNA ends, such as the Bacillus subtilis, E. coliand S. pneumoniae phages GA-1,7 PRD18 and Cp-1,9 respectively, linear plasmids,2 and the eukary-otic adenovirus.10 Indeed, the replication initiationsite in GA-1, PRD1, Cp-1, and adenovirus DNAcorresponds to an internal nucleotide close to the30-terminal end, and a sliding-back type of mech-anism has been shown to occur in these cases torecover the information of the terminal nucleo-tide(s). For f29 DNA replication, it has beendemonstrated that the same DNA polymerase thataccomplishes initiation, catalyses the synthesis ofa short elongation product (6–9 nt long), whilestill maintaining the heterodimer complex withthe TP (transition step).11 When the tenth nucleo-tide is inserted into the nascent DNA chain, theDNA polymerase dissociates from the primer TP,11

and elongation proceeds processively from bothDNA ends coupled to strand displacement withoutthe need for either processivity factors or helicase-like proteins. Once the two replication forks meet,the two partially replicated parental strands separ-ate and full-length DNA synthesis is completed.12

Bacteriophage f29 DNA polymerase is a singlepolypeptide of 66 kDa, which belongs to theB-type superfamily of DNA-dependent DNA poly-merases (also referred to as eukaryotic or a-likepolymerases) that includes a vast group of DNApolymerases from eukaryotic and prokaryoticorigins.3,13,14 The f29 DNA polymerase has servedas model for the study of the biochemical featuresand of the structure–function relationships ofDNA polymerases by means of an exhaustivemutational analysis of the enzyme, carried outover more than one decade.15 The identification ofmutant DNA polymerases defective in their inter-action with the TP has also allowed the study ofthe DNA polymerase/TP contacts necessary tostart replication of the f29 genome.

On the other hand, taking into account thebimodular structure of DNA polymerases (twomain structural and functional independentdomains, the N-terminal one containing the 30-50

exonuclease activity and the C-terminal domain inwhich the synthetic activities reside) and the factthat those which make use of a protein-primingmechanism have to accommodate a protein(31 kDa in the case of bacteriophage f29) in thesame cleft occupied by the DNA during the furtherreplication steps, it is expected that residues fromboth domains will contribute to the stabilisation ofsuch DNA polymerase/TP interaction. Thus, inthe C-terminal polymerisation domain, residuesThr434 and Arg438 in motif Tx2GR,16 Tyr226 and

Gly229 in the YxGG/A motif17 and Asp332 of theconserved TPR-1 region,18 were identified asinvolved in the interaction with TP. On the otherhand, expression of the C-terminal domain led toa dramatically reduced interaction of TP and DNApolymerase,19 indicating the involvement of theN-terminal domain in such an interaction. In fact,it has been possible to identify residues in thisdomain implicated in making contacts with theTP, as Ser122 of the (S/T)Lx2h motif, and Tyr59,His61 and Phe65 in the Exo II motif.20,21 Theseresults confirm that the interaction with the primerTP involves many contacts with different regionsof the DNA polymerase.

Here, we have focused on residues Phe128 andGlu161 of f29 DNA polymerase, belonging to twowell-characterised regions of the N-terminaldomain of protein-priming DNA polymerases, the“(S/T) Lx2h” and Exo III motifs, respectively. Thebiochemical analysis of f29 DNA polymerasemutants at residue Phe128 allows us to proposethe contribution of this residue in establishing con-tacts with the TP during the first steps of f29 DNAreplication.

Results

Conservation of amino acid residues at the30-50 exonuclease domain of protein-primedDNA polymerases

Figure 1 shows a multiple alignment of tworelevant amino acid segments corresponding tothe 30-50 exonucleolytic domain of proofreadingDNA polymerases belonging to family B (eukary-otic-type), restricted here to the subgroup of thoseDNA polymerases that use a protein-primingmechanism. The first block of amino acid similaritycorresponds to the motif defined by the consensussequence “(S/T)Lx2h”, located between the Exo IIand Exo III motifs.22 Biochemical analysis of site-directed mutants at the consecutive residuesSer122 and Leu123 of f29 DNA polymerasedemonstrated their functional importance assingle-stranded DNA (ssDNA) ligands, being theformer also involved in making contacts with theprimer TP.20 In this sequence segment, an aromaticresidue conserved in 16 out 19 DNA polymerasesis located five residues after Leu123. In 12 ofthem, this aromatic residue is a Phe, whereas inthe other four the aromatic group corresponds toa Tyr residue. The second amino acid similaritysegment shown in Figure 1 is the Exo III motif. Itsimportance resides in the fact that it contains oneof the four catalytic aspartate residues involved inmetal binding and catalysis at the 30-50 exonucleaseactive site in proofreading DNA polymerases,23

and a Tyr residue involved in orientating theattacking water molecule. In the fourth positionpreceding the invariable Tyr residue of the Exo IIImotif there is a Glu residue specifically conservedin 14 out 19 DNA polymerases.

86 f29 DNA Polymerase Phe128 Interaction with TP

The high degree of conservation of the tworesidues described above in the protein-primedDNA polymerases led us to study their involve-ment in the specific reactions catalysed by thiskind of DNA polymerases.

Site-directed mutagenesis at f29 DNApolymerase residues Phe128 and Glu161

To study the physiological role of the residuesdescribed above we made site-directed mutants atresidues Phe128 and Glu161 of f29 DNA polymer-ase. Residue Phe128 was changed either to Tyr(F128Y) to maintain the aromatic group, or to Ala(F128A) to remove it. Residue Glu161 was changedeither to Gln (E161Q) to introduce a positivelycharged amino acid residue or into Ala (E161A) toremove the negative charge. Taking into accountsecondary structure predictions24,25 and generalsuggestions for conservative substitutions,26 all themutations introduced were predicted to maintainthe overall structure in that region of the poly-peptide. These mutant f29 DNA polymerases,overexpressed and purified as describedelsewhere,27 were analysed using a variety of invitro assays corresponding to the different stagesof the TP-primed f29 DNA replication mechanism.

Mutations at residues Phe128 and Glu161 off29 DNA polymerase do not affect thepolymerase/exonuclease balance on atemplate/primer DNA structure

To study if mutations at residues Phe128 andGlu161 affect the polymerase activity of theenzyme, the hybrid molecule 15mer/21mer wasused as a defined template/primer structure inwhich the primer sp1 is 50-labelled (see Materialsand Methods). Since this hybrid molecule is asubstrate for both, 30-50 exonucleolysis and 50-30

polymerisation activities, the assay allows thedirect study of the dynamic equilibrium betweenboth activities, by analysing the minimum dNTPsconcentration required to get a productiveelongation of the primer strand. In the absence ofnucleotides, the only products that can be detectedare those produced by the exonucleolytic digestionof the primer strand. In these conditions, the differ-ent patterns and extent of degradation obtainedreflect the level of 30-50 exonuclease activity of thedifferent mutants with respect to the wild-type.As it can be observed in Figure 2, mutant deriva-tives showed a similar degradation pattern as thewild-type enzyme, indicating that mutations,under these conditions, did not interfere withsuch an activity. On the other hand, by addingincreasing amounts of dNTPs, exonucleolysis isprogressively competed, favouring polymerisation.The concentration of dNTPs required to obtain anefficient elongation can be used to define the pro-ductive Pol/Exo ratio shown by the mutantenzymes, which is independent on the amount ofenzyme/DNA complexes formed (see Materialsand Methods). Thus, if one of the above-mentionedactivities were specifically affected, such equi-librium would be displaced towards polymerisation(low exonuclease activity) or towards exonucleolysis(low polymerisation activity). As shown in Figure 2,the mutant polymerases displayed a similar dNTPrequirement (20/100 nM) as the wild-type enzymefor a net polymerisation balance (Table 1). Theseresults allow us to conclude that the mutationsintroduced did not affect the polymerase activity ofthe enzyme, at least under conditions in whichpolymerisation is DNA-primed.

Using limiting amounts of mutant DNApolymerases, we could study specifically its 30-50

exonuclease activity on such template primerstructure, through a wide range of times. Undersuch conditions, mutant F128Y showed a 30-fold

Figure 1. Multiple amino acidsequence alignment of two seg-ments corresponding to the 30-50

exonucleolytic domain of proof-reading DNA polymerases belong-ing to family B (eukaryotic-type)that use a protein as primer.Nomenclature and sequence refer-ences are according to de Vegaet al.35 Numbers indicate the pos-ition of the first aligned amino acidwith respect to the N terminus ofthe respective DNA polymerase.Highly conserved residues of the(S/T)Lx2h and Exo III motifs, S, L,Y and D, are white on a black back-ground. Conserved aromatic resi-dues from (S/T)Lx2h motif andacidic residues from Exo III motifhave a grey background. Mutationsites at f29 DNA polymerase resi-dues F128 and E161 are markedwith asterisks.

f29 DNA Polymerase Phe128 Interaction with TP 87

reduction on this activity (see Table 1). Taking intoaccount its wild-type Pol/Exo ratio, which isindependent on the amount of enzyme, we couldconclude that the mutation is affecting the generalinteraction with the dsDNA but not specifically toany of the above-mentioned activities. In the caseof mutants E161Q and E161A, they showed a twoand fivefold reduction in this activity, respectively(see Table 1). However, such a decrease was notpronounced enough to affect their Pol/Exobalance. A similar result was obtained whenssDNA was used as substrate (see Table 1).

DNA-binding stability of f29 DNApolymerase mutants

To test the capacity of mutant derivatives torecognise efficiently the primer terminus structure,

we performed gel shift assays, using as substratethe same 15mer/21mer molecule described aboveand Mg2þ as metal ion. Under such conditions, thewild-type enzyme forms a stable complex compe-tent for DNA replication, giving rise to a uniqueretarded band (see Figure 3) whose intensitydepends on the amount of enzyme added. As itcan be observed in Figure 3, mutant polymerasesF128A, E161Q and E161A were able to interactwith the DNA to a similar extent as the wild-typeenzyme. However, under such conditions, nostable interaction between mutant polymeraseF128Y and the 15mer/21mer substrate wasdetected. This result indicates that mutation F128Yimpairs the normal interaction with the DNAsubstrate. On the other hand, a slight difference inthe mobility of the complex formed betweenmutants E161Q, E161A and dsDNA can be

Table 1. Enzymatic activities of wild-type and mutant derivatives of f29 DNA polymerase

f29 DNA polymerasea

Phe128 Glu161

Parameter assayed Substrate Wt F128Y F128A E161Q E161A

30-50 exonuclease dsDNA (15mer/21mer) 100 3 90 35 20ssDNA (15mer) 100 8 62 51 52

Enzyme/DNA bindingb dsDNA (15mer/21mer) 100 ND 81 55 59ssDNA (15mer) 100 ND 43 26 32

Pol/Exo ratioc 15mer/21mer, dNTPs 20/100 20/100 20/100 20/100 20/100f29 TP-DNA replication f29 TP-DNA, TP, dNTPs 100 20 2 109 77f29 TP-DNA amplification f29 TP-DNA, TP, SSB, DBP, dNTPs 100 3 20 120 130M13 DNA replication Primed M13 DNA, dNTPs 100 105 52 115 105f29 TP-DNA initiation f29 TP-DNA, TP, dATP 100 ,1 ,1 67 53TP-deoxynucleotidylation TP, dATP 100 ,1 ,1 62 31

ND, not detected.a Numbers indicate the average percentage of activity relative to the wild-type enzyme obtained from several experiments.b Analysed by gel retardation assay on low-ionic strength polyacrylamide gels.c Numbers indicate the dNTP concentration (in nM) required to efficiently elongate the 15mer primer until the 20mer position.

Figure 2. Pol/Exo balance of mutants at residues F128 and E161 of f29 DNA polymerase. The assay was carried outas described in Materials and Methods using 32P-labelled hybrid molecule 15mer/21mer as primer/template DNA andthe indicated concentration of dNTPs. Polymerisation or 30-50 exonuclease activity is detected as an increase ordecrease, respectively, in the size (15mer) of the 50-labelled primer. The productive Pol/Exo ratio and the relativepolymerisation for each mutant polymerase are indicated in Table 1.


observed. This could be interpreted as a partialdistortion of the binding to the 30-50 exonucleaseactive site. When ssDNA was used as substrate,results yielded by mutant DNA polymerasesparalleled those obtained with dsDNA (see Table1), suggesting that residue Phe128 could beoriented to a common binding site for both kindsof substrates.

Mutations introduced at Phe128 of f29 DNApolymerase impair f29 TP-DNA replication

Replication of bacteriophage f29 TP-DNAinvolves the TP-primed initiation at both DNAends, a special activity of f29 DNA polymerasethat catalyses the template-directed formation ofa covalent complex between the viral TP and50-dAMP (initiation step) and the subsequentelongation, via strand displacement, of theinitiation complex to produce full-length f29DNA (19285 bp).2,3

To test if the mutations introduced in f29 DNApolymerase were playing any role in the protein-primed DNA replication process, we used aminimal f29 TP-DNA replication system, makinguse only of f29 TP-DNA, primer TP and DNApolymerase. As it can be observed in Figure 4(a),the efficiency displayed by mutants F128Y andF128A with respect to the wild-type enzyme wasreduced five and 50-fold, respectively, whereasmutants E161Q and E161A had essentially a wild-type activity (see also Table 1).

Similarly, in f29 TP-DNA amplification assays,which more closely resemble the in vivo situation(since in addition to primer TP and DNApolymerase, f29 ssDNA binding protein (SSB)

and f29 DBP in the presence of limiting amountsf29 TP-DNA were used (see Materials andMethods), both mutants, F128Y and F128A, werealso severely affected (Figure 4(b)) in comparisonwith the wild-type enzyme (30- and fivefold,respectively) (see also Table 1). On the other hand,mutants in residue E161 displayed a wild-typeactivity.

Strand displacement coupled topolymerisation capacity of mutant f29DNA polymerases

As mentioned above, f29 DNA polymerase hasto couple strand displacement to processive DNAsynthesis, in the absence of accessory proteins orhelicases.12 To analyse whether the impairment

Figure 4. (a) F29 TP-DNA replication by wild-typeand mutant f29 DNA polymerases. The assay wascarried out as described in Materials and Methods inthe presence of 30 ng wild-type (wt) or mutant f29DNA polymerases. After incubation for five and tenminutes at 30 8C, relative activity values were calculated(see Table 1) and the length of the synthesized DNAwas analysed by alkaline agarose gel electrophoresis.The migration position of unit length f29 TP-DNA isindicated. (b) f29 TP-DNA amplification. The assay wascarried out as described in Materials and Methods, inthe presence of 5 ng wild-type or mutant f29 DNA poly-merases, 5 ng f29 TP and 10 mg each f29 DBP and f29SSB. After incubation for 90 minutes at 30 8C, sampleswere processed and the amplified DNA was analysedby alkaline agarose gel electrophoresis as described inMaterials and Methods. The migration position of unitlength f29 TP-DNA (19285 bases) is indicated.

Figure 3. Gel retardation of dsDNA by wild-type ormutant f29 DNA polymerases. The assay was carriedout as described in Materials and Methods, using50-labelled 15mer/21mer synthetic hybrid as substrate,in the presence of 2.5 ng of either wild-type or mutantf29 DNA polymerases. Samples were analysed by poly-acrylamide gel electrophoresis. Bands correspond to freeDNA and to the DNA polymerase/dsDNA complexdetected by autoradiography.


shown by mutant polymerases F128Y and F128A inreplicating f29 TP-DNA were due to a defect intheir strand displacement capacity, we performeda DNA-primed M13 replication assay (seeMaterials and Methods), in which f29 DNApolymerase starts DNA replication from the 30-OHend of an oligonucleotide hybridised to the circularM13 ssDNA molecule. The first replication rounddoes not require strand displacement, but once itis completed, the polymerase encounters the50-end of the primer, requiring the coupling ofpolymerisation to strand displacement to continueDNA synthesis. As it can be observed in Figure 5,mutants at residue E161 replicated M13 DNA withthe same efficiency as the wild-type enzyme, as itwas the case in the f29 TP-DNA replication assays.Mutant polymerase F128Y replicated M13 DNA asefficiently as the wild-type enzyme. However, thesize of the replication products obtained with themutant polymerase F128A reveals that it is affectedin the polymerisation velocity during the firstreplication cycle. Thus, when this mutant polymer-ase starts the second round of replication, forwhich strand displacement is required, the velocitydefect is aggravated with respect to the wild-typeenzyme. Addition of f29 SSB restored the replica-tion velocity of F128A mutant polymerase (notshown). f29 SSB keeps the ssDNA template in theoptimal conformation for DNA elongation by dis-rupting secondary structures of the ssDNA regionsand reducing the energy demand required for theunwinding process coupled to DNApolymerisation.28 This could explain the partialrecovery displayed by F128A mutant polymerasein its amplification activity (in the presence ofSSB) with respect to f29 TP-DNA replication (inthe absence of SSB), suggesting a partial involve-ment of this residue in the strand displacementmechanism of f29 DNA polymerase.

TP–dAMP formation (initiation) of mutantpolymerases affected in f29 TP-DNA replication

To start f29 DNA replication, the DNA polymer-ase has to form an heterodimer with a free TPmolecule. Once the replication origin is recognisedby this complex, f29 DNA polymerase catalysesthe template-directed insertion of dAMP onto thehydroxyl group of Ser232 of the TP, using thesecond dTMP of the DNA strand as template. Asshown in Figure 6, mutants at residue F128 dis-played a dramatically reduced TP-primedinitiation capacity, whereas the activity of mutantsat residue E161 was slightly affected (Table 1).Since initiation of f29 DNA replication is a tem-plate-directed event, the initiation defect ofmutants at residue F128 could be the consequenceof a weak affinity for the template DNA. Thispossibility could be tested because f29 DNA poly-merase can catalyse the deoxynucleotidylation ofTP in the absence of template.29 Under these con-ditions, the activity of the mutant derivatives atresidue F128 of f29 DNA polymerase was as lowas in the case of templated TP–dAMP formation(see Figure 6 and Table 1). The activity of mutantsat residue E161 was similar to that obtained in thepresence of TP-DNA. Thus, the low initiationcapacity displayed by the mutant polymerases atresidue F128 could be explained by a defect in theinteraction with the primer TP. The ability ofmutant polymerases F128Y and F128A to interactwith the TP was tested by using an interferenceassay (see Materials and Methods) in which wild-type and mutant polymerases compete for alimited amount of TP. As a control, f29 DNApolymerase mutant D249E, which is catalyticallyinactive but has a normal capacity to interact withTP, was used. As shown in Figure 7, the inhibition

Figure 5. Strand-displacement coupled to M13 DNAreplication by wild-type and mutant DNA polymerases.Replication of primed-M13 DNA was carried out asdescribed in Materials and Methods using 40 mMdNTPs and 100 ng of either wild-type or mutant DNApolymerases. After incubation for the indicated times at30 8C, relative activity values were calculated fromdNMP incorporation (see Table 1). The position of unit-length M13 DNA is shown at the right.

Figure 6. Formation of the TP–dAMP complex cata-lysed by wild-type or mutant f29 DNA polymerases inthe presence or absence of f29 TP-DNA. The reactionswere carried out as described in Materials and Methods.The template-dependent reaction (top) was carried outin the presence of 10 mM MgCl2, 0.5 mg TP-DNA, 7.5 ngTP and 15 ng wild-type or mutant f29 DNA poly-merases. Incubation was for three minutes at 30 8C. Thetemplate-independent reaction (bottom) was performedin the presence of 1 mM MnCl2, 15 ng TP and 30 ngwild-type or mutant f29 DNA polymerases. Incubationwas for 120 minutes at 30 8C. Samples were analysed bySDS-PAGE and autoradiography. The band correspondsto the TP–dAMP initiation complex. Quantification wasby densitometric analysis of the band corresponding tothe labelled TP–dAMP complex, detected byautoradiography.


profile obtained with this mutant paralleled thetheoretical one. Contrarily, the wild-type enzymewas poorly competed by F128Y, probably reflectinga defective interaction with TP. On the other hand,although mutant F128A showed an interferencecapacity, it was slightly lower than that of mutantD249E. The above results are in agreement with apossible involvement of residue F128 of f29 DNApolymerase in making contacts with the TP.

To confirm these results, the interaction ofmutant polymerases F128Y and F128A with TPwas directly analysed by glycerol gradient centri-fugation, as described in Materials and Methods.Wild-type f29 DNA polymerase (66 kDa) and TP(31 kDa) form an heterodimer of 96 kDa and thetwo proteins co-sediment in the same fractions. Incontrast, mutant polymerase F128Y and TP elutedseparately as monomers, indicating a TP-bindingdefect. Under the same assay conditions, mutantpolymerase F128A was still able to form a complexwith TP (results not shown).

Transition from protein-priming to DNA-priming in f29 DNA replication

Due to the defective interaction with TP dis-played by mutant polymerases F128Y and F128A,it was interesting to study the transition step fromthe initiation mode to the elongation mode duringthe first steps of f29 TP-DNA replication. For thistransition study, we performed a truncatedelongation assay (see Materials and Methods). Pro-viding dATP as the only nucleotide, the wild-typeenzyme gave rise to TP–dAMP and TP–(dAMP)2,as expected, since the longer product TP–(dAMP)3

is degraded by the 30-50 exonuclease activity(Figure 8). In the presence of dATP, dGTP anddTTP, elongation with the wild-type polymerasemainly occurs up to TP–(dNMP)8 and TP–

(dNMP)11, the expected sizes considering thesequences of the replication origins (see Figure 8,upper panel). About 29% of the initiation productswere elongated by the wild-type enzyme duringthe transition step (Figure 8). However, in the caseof mutant polymerase F128Y only 4% of theinitiation products were elongated, and essentiallyno elongation could be observed with mutant poly-merase F128A. Moreover, the relative proportion ofTP–(dAMP)2 with respect to TP–dAMP, was muchlower with the mutant polymerases F128Y andF128A than in the case of the wild-type enzyme.The very low amount of elongated products bymutant polymerases cannot be a consequence oftheir 30-50 exonuclease activity, since very poor orno accumulation of TP–(dAMP)2 was observed.These results reflect a defect of the mutant deriva-tives at residue Phe128 in the transition from theinitiation to the elongation mode of replication. Infact, the reduced (F128Y) and nearly completeabsence (F128A) of TP–(dAMP)2 could suggestimpairment in performing the sliding-backmechanism.

Discussion

F29 DNA polymerase is a relatively smallenzyme which has served as model for the studyof the relationship between structure and functionfor more than one decade.15 It shares with othereukaryotic-type (family B), Pol I-like (family A)and Pol III DNA polymerases several regions ofamino acid homology at their 30-50 exonucleasedomain.30 In this domain, the Exo I, Exo II andExo III motifs are the most important ones, sincethey contain the four acidic residues acting asligands for the metal ions responsible for catalysis,and the Tyr residue which orients the attackingwater molecule.30 – 32 In the specific case of family BDNA polymerases, there is also a conserved Lysresidue belonging to the Kx2h motif playing anauxiliary catalytic role by orienting the carboxylatemoiety of the Asp residue of the Exo III motif.33 Inaddition, these motifs also contain conservedresidues specifically involved in stabilising thessDNA to the 30-50 exonuclease active site, as a Thrfrom Exo I,34 a His, Tyr, Asn and Phe from ExoII,22,35 and the Ser and Leu residues from the(S/T)Lx2h motif.22

On the other hand, it has been possible toidentify several residues belonging to those motifs,which are specifically conserved among the DNApolymerases that use a protein as primer.20,21 Here,we have studied the role of two of these residuesof f29 DNA polymerase (Phe128 and Glu161,belonging to the (S/T)Lx2h and Exo III motifs,respectively) in its synthetic activities, protein-priming and DNA polymerisation.F29 DNA replication is a complex process in

which the same DNA polymerase has to perform,first initiation using a protein as primer, and thenDNA-primed polymerisation, without dissociation

Figure 7. Interference assay for TP binding. Reactionswere carried out as described for the template-indepen-dent initiation assay using a limited amount of TP anddifferent amounts (12.5 ng, 25 ng, 50 ng and 100 ng) ofmutant DNA polymerases. As a control for 100%inhibition, f29 DNA polymerase mutant D249E, whichis catalytically inactive but has an intact capacity to inter-act with TP, was used. The inhibition profile obtainedwith this mutant paralleled the theoretical one. TheTP–dAMP formed in the different competition con-ditions relative to that formed in the absence ofcompetition (100%) is indicated.


from the replicating genome.2,3 The first step in f29DNA replication requires the interaction between aDNA polymerase and a free TP molecule, formingan heterodimer able to recognise the origins ofreplication located at both ends of the DNA mol-ecule, probably through protein–protein inter-actions between the TP and the DNA polymeraseof the heterodimer and the parental TP covalentlylinked to the DNA ends.36,37 Initiation of replicationstarts by the covalent linkage of dAMP onto thehydroxyl group of Ser232 of the primer TP. Thisreaction (initiation) is catalysed by the DNA poly-merase directed by the second dTMP at the 30-endof the template strand. By means of a sliding-backmechanism, the TP–dAMP initiation complexslides-back one position recovering the first 30-dAMP of the template. DNA polymerase remainsbound to the TP until nine dNMP residues have

been incorporated (transition stage). Afterwards,dissociation of the DNA polymerase/TP complextakes place, and elongation occurs coupled tostrand displacement, giving rise to fully replicatedf29 DNA molecules.

The mutations introduced at residues Phe128and Glu161 did not interfere with the DNA poly-merase capacity to perform DNA-primed syntheticreactions, as shown by the wild-type Pol/Exobalance displayed by the mutants. On the otherhand, mutant polymerase F128Y showed a defectin stabilising the DNA substrate, being necessaryhigher amounts of enzyme to get a wild-typebehaviour, when short DNA substrates were used.Interestingly, as the length of the protruding tem-plate strand was increased, the binding defect dis-appeared (as reflected in the M13 DNA replicationassay). This fact could indicate an auxiliary role of

Figure 8. Analysis of the tran-sition step of f29 TP-DNA replica-tion. The assay was performed asdescribed in Materials andMethods, using 10 mM MgCl2,0.5 mg TP-DNA, 15 ng TP, 30 ngwild-type or mutant DNA poly-merases and 5 mM of the indicateddNTP. After incubation for fiveminutes at 30 8C, the differenttransition products were detectedand analysed by high resolutionSDS-PAGE. Completely replicatedmolecules remain at the interphasewith the stacking gel. The lengthof the different transition productsand the position correspondingto full-length f29 TP-DNA areindicated.


Phe128 of f29 DNA polymerase in making con-tacts with the dsDNA substrate during bothpolymerisation and exonucleolytic activities, par-ticularly during the steps in which there are fewcontacts between DNA polymerase and theremaining ssDNA template, that is, during termi-nation of replication. A longer template ssDNAcould be interacting with DNA polymerase surfaceresidues, improving the stability of the binding, asit occurs during the replication of f29 TP-DNA.

Interestingly, mutant polymerases F128Y andF128A displayed a low efficiency in replicatingand amplifying f29 TP-DNA. In the case of mutantpolymerase F128Y, such a defect cannot be relatedwith a defective strand displacement, as deducedfrom its capacity to perform rolling circleelongation using a DNA-primed M13 molecule.Mutant F128A showed a slightly diminishedstrand displacement capacity, that was recoveredwhen M13 DNA was covered by f29 SSB, asituation resembling the in vivo f29 TP-DNA repli-cation. In fact, the activity of mutant polymeraseF128A was partially recovered in a f29 TP-DNAamplification assay that contains f29 SSB withrespect to the so-called f29 TP-DNA replicationassay that does not contain the SSB protein.

The analysis of the protein-primed initiation steprevealed that mutant polymerases F128Y andF128A were severely affected in such an activity,both in the absence and in the presence of f29TP-DNA. This defect could be related with amoderately (F128A) or severely (F128Y) decreasedcapacity to form a stable and/or functional DNApolymerase/TP heterodimer, as deduced frominterference and glycerol gradient assays. In thecase of mutant polymerase F128A, although it wasstill able to interact stably with the TP, its poorinitiation activity could reflect the importance ofresidue Phe128 to make functionally activeTP/DNA polymerase complexes, being importantnot only the strength of the interaction, but alsothe correct orientation of TP in the polymerisationactive site of the enzyme. The defective interactionwith the TP displayed by mutants at residuePhe128 could explain their impairment in the tran-sition between protein-priming and DNA-priming.The mutant derivatives were extremely inefficientin performing such a step giving rise to a stop ofthe replication, mainly at the TP–dAMP stage. Infact, the very low amount of TP–(dAMP)2

obtained, suggests that contacts provided by resi-due Phe128 could be crucial not only to performthe transition between both priming modes(protein and DNA-priming), but also for thesliding-back mechanism during the initiation off29 TP-DNA replication. On the other hand,mutations introduced at residue Glu161 did notalter the capacity of the enzyme to carry out anyof the specific activities catalysed by the subgroupof protein-priming DNA polymerases. Thus, thehigh degree of conservation of this residue amongsuch DNA polymerases could suggest a structuralmore than a functional role.

To date, no crystal data are available for anyprotein-priming DNA polymerase. Thus, ourworking model is based on the extrapolation tothe resolved three-dimensional structure of otherDNA polymerases belonging to family A (Klenowfragment of Pol I, Pol Ik,38 Thermococcus aquaticusDNA polymerase,39,40 bacteriophage T7 DNApolymerase,41 and Bacillus stearothermophilus DNApolymerase42) and to family B (bacteriophageRB69 DNA polymerase,43 Thermococcusgorgonarius,44 Pyrococcus kodakaraensis DNApolymerase,45 Thermus litoralis DNA polymerase46

and Desulfurococcus sp. DNA polymerase47). All ofthem show a similar structural and functionalbimodular organisation, with a N-terminal domaincontaining the 30-50 exonuclease activity (or, as inthe case of T. aquaticus and B. stearothermophilusDNA polymerases, a vestige of such an activity),and the C-terminal domain containing the 50-30

polymerisation one. The main difference betweenthe A and B-family DNA polymerases is the rela-tive orientation of the 30-50 exonuclease domainwith respect to the polymerisation one.43 On thebasis of: (1) the fact that TP and DNA bind to theDNA polymerase through a common bindingcleft;20 (2) that DNA polymerase is unable to simul-taneously bind both TP and DNA;20 and (3) thenecessity for the TP to reach the polymerisationactive site, it was proposed that TP interacts withthe DNA polymerase through its primer cleftformed by the thumb, fingers and palmsubdomains.20 Although we have been able toidentify only one f29 DNA polymerase residue(Asp332 from the TPR-1 region, specific forprotein-priming DNA polymerases) exclusivelyinvolved in making contacts with the TP,18 someof the more than 40 residues of such a cleft whichare involved in stabilising the DNA/DNA poly-merase interaction have been demonstrated toplay a dual role in binding also the TP. Amongthem, the f29 DNA polymerase residues Arg434and Thr438 belonging to the highly conservedTx2GR motif,16 as well as Tyr226 and Gly229 fromthe YxGG motif17 in the C-terminal domain. Inaddition, the isolated expression of the C-terminaldomain of f29 DNA polymerase resulted in areduced capacity to interact with the TP,19

suggesting that residues located at the N-terminaldomain of the DNA polymerase are contributingto the stable formation of the TP/DNA polymeraseheterodimer. Thus, we have identified f29 DNApolymerase residues Tyr59, His61, Phe65 andPhe69 belonging to the Exo II motif,20,21 and Ser122and Phe128 from the (S/T)Lx2h motif (de Vegaet al.20; this paper) that play a dual role in bindingTP and DNA substrates. These results support theproposal that during the transition step the con-tacts of the DNA polymerase with the TP arereplaced by interactions with the nascent DNA,leading to a DNA-primed elongation process.

Although the use of a TP as primer could be, apriori, the easiest way to replicate the origins oflinear genomes, it has revealed as one of the steps


of replication showing the major complexity. Theinteraction between TP and a protein-primingDNA polymerase is rather complex, since it has tobe stable enough to be maintained during the firststeps of the DNA replication and, on the otherhand, it has to be dynamic to allow the progressivechange of substrates (TP and DNA) within theDNA polymerase without dissociating from theTP-DNA molecule. To solve the problem, severalDNA polymerase residues have evolved to carryout such a transition by means of their ability tosequentially interact with both substrates. Thisstudy, together with the ones mentioned above,show that the TP-binding residues are dispersedin the primary structure of the f29 DNA polymer-ase. However, they might be conforming a specificsubdomain in the three-dimensional structure ofprotein-primed DNA polymerases, involved in theefficient interaction and orientation of the primerTP.

Materials and Methods

Nucleotides and proteins

Unlabelled nucleotides were purchased fromPharmacia P-L Biochemicals. [a-32P]dATP (3000 Ci/mmol) was obtained from Amersham International plc.f29 TP was purified as described.48 The wild-type f29DNA polymerase was purified from E. coli NF2690 cellsharbouring plasmid pJLw2, as described.27 f29 DNApolymerase site-directed mutants, F128Y/A andE161Q/A, were purified as described.27 f29 SSB andf29 dsDNA-binding protein (p6), obtained fromf29-infected B. subtilis cells, were purified asdescribed.49,50

DNA templates and substrates

f29 DNA was obtained by proteinase K-treatment ofphage particles in the presence of SDS,51 phenol extrac-tion and ethanol-precipitation. M13mp18 ssDNA washybridised to the universal primer in the presence of0.2 M NaCl and 60 mM Tris–HCl (pH 7.5), and theresulting molecule was used as a primer/template toanalyse processive DNA polymerisation coupled tostrand displacement by f29 DNA polymerase. TP-con-taining f29 DNA (f29 TP-DNA) was obtained asdescribed.52

30-50 Exonuclease assays

The incubation mixture contained, in 12.5 ml, 50 mMTris–HCl (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol,4% (v/v) glycerol and 0.1 mg/ml bovine serum albumin(BSA). 1.2 nM of either 50-labelled sp1 or the hybridmolecule sp1/sp1c þ 6 was used as ssDNA and dsDNAsubstrate, respectively. The amount of DNA polymeraseadded (6 nM) was adjusted to obtain linear conditions.Samples were incubated at 25 8C for the indicated timesand quenched by adding 3 ml of gel loading buffer. Reac-tions were analysed by electrophoresis in 20% (w/v)polyacrylamide gels in the presence of 8 M urea, and bydensitometry of the autoradiographs. Total degradationwas obtained by calculating the number of catalytic

events giving rise to each degradation. From these data,the catalytic efficiency of each mutant derivative (indi-cated in Table 1) was calculated relative to wild-typef29 DNA polymerase.

Polymerase/exonuclease-coupled assay

The hybrid molecule 15/21mer contains a six nucleo-tide long 50-protruding end, and therefore, the primerstrand (15mer) can be used both as substrate for the30-50 exonuclease activity and also for DNA-dependentDNA polymerisation. The incubation mixture contained,in 12.5 ml, 50 mM Tris–HCl (pH 7.5), 10 mM MgCl2,1 mM dithiothreitol, 4% glycerol, 0.1 mg/ml BSA,0.180 ng of 50-labelled 15/21mer, 20 ng of either wild-type or mutant f29 DNA polymerase (200 ng in thecase of mutant F128Y) and the indicated concentrationof the four dNTPs. After incubation for five minutes at25 8C, the reaction was stopped by adding EDTA up to10 mM. Samples were analysed by 8 M urea–20% (w/v)PAGE and autoradiography. Polymerisation or 30-50 exo-nuclease are detected as an increase or decrease, respect-ively, in the size (15mer) of the 50-labelled primer.

DNA gel retardation assays

50-Labelled oligonucleotides sp1 and sp1/sp1c þ 6were used as ssDNA and dsDNA substrates, respect-ively, to analyse the interaction of either the wild-typeor mutant f29 DNA polymerase. The incubation mixturecontained, in a final volume of 20 ml, 12 mM Tris–HCl(pH 7.5), 1 mM EDTA, 20 mM ammonium sulphate,0.1 mg/ml BSA, 1.2 nM of either sp1 or sp1c þ 6 and2.5 ng of either wild-type or mutant f29 DNA polymer-ase. After incubation for five minutes at 4 8C, the sampleswere subjected to electrophoresis in 4% polyacrylamidegels (80:1, monomer:bis), containing 12 mM Tris–acetate(pH 7.5) and 1 mM EDTA, and run at 4 8C in the samebuffer at 8 V/cm, essentially as described.53 After auto-radiography, f29 DNA polymerase–dsDNA complexeswere detected as a mobility shift (retardation) in themigrating position of the labelled DNA. Quantitation ofthe DNA-binding capacity of the wild-type f29 DNApolymerase versus its mutant derivatives was carriedout by densitometry of the retarded band.

Replication assay (protein-primed initiation pluselongation) using f29 TP-DNA as template

The incubation mixture contained, in 25 ml, 50 mMTris–HCl (pH 7.5), 10 mM MgCl2, 20 mM ammoniumsulphate, 1 mM dithiothreitol, 4% glycerol, 0.1 mg/mlBSA, 20 mM each dCTP, dGTP, dTTP and [a-32P] dATP(2 mCi), 0.5 mg of f29 TP-DNA, 15 ng of purified TP and30 ng of either wild-type or mutant f29 DNA polymer-ase. After incubation for the indicated times at 30 8C,the reaction was stopped by adding 10 mM EDTA–0.1%(w/v) SDS, and the samples were filtered throughSephadex G-50 spin columns. Relative activity was cal-culated from the Cerenkov radiation corresponding tothe excluded volume. For size analysis, the labelledDNA was denatured by treatment with 0.7 M NaOHand subjected to electrophoresis in alkaline 0.7% (w/v)agarose gels, as described.54 After electrophoresis, theposition of unit-length f29 DNA (19285 bases) wasdetected by ethidium bromide staining, and then thegels were dried and autoradiographed. For analysis ofthe transition products, 5 mM of the indicated dNTPs


were used. Samples were subjected to a 12% SDS-PAGEgel (360 mm £ 280 mm £ 0.5 mm) to obtain enoughresolution to distinguish TP bound to the first elongationproducts.

f29 TP-DNA amplification assay

The incubation mixture contained, in 25 ml, 50 mMTris–HCl (pH 7.5), 10 mM MgCl2, 20 mM ammoniumsulphate, 1 mM dithiothreitol, 4% glycerol, 0.1 mg/mlBSA, 80 mM each dCTP, dGTP, dTTP, and [a-32P]dATP(1 mCi), 5 ng of f29 TP-DNA, 5 ng of either wild-type ormutant f29 DNA polymerase, 5 ng of TP, 10 mg of f29SSB and 10 mg of f29 p6. After incubation for 90 minutesat 30 8C, the samples were processed and the amplifiedDNA was analysed by electrophoresis in alkaline agarosegels, as described.55 After electrophoresis, the position ofunit length f29 DNA was detected by ethidium bromidestaining.

Replication of primed M13 DNA

The incubation mixture contained, in 25 ml, 50 mMTris–HCl (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol,4% glycerol, 0.1 mg/ml BSA, 40 mM each dCTP, dGTP,dTTP and [a-32P]dATP (1 mCi), 0.25 mg of primedM13mp8 ssDNA, and 100 ng of either wild-type ormutant f29 DNA polymerase. After incubation for theindicated times at 30 8C, the samples were processedand the synthesised DNA was quantitated and analysedas described above for the f29 TP-DNA amplificationassay. After electrophoresis, unit length M13mp8ssDNA was detected by ethidium bromide staining, andthen, gels were dried and autoradiographed.

TP–dAMP formation (protein-primed initiation assay)

The incubation mixture contained, in 25 ml, 50 mMTris–HCl (pH 7.5), 10 mM MgCl2, 20 mM ammoniumsulphate, 1 mM dithiothreitol, 4% glycerol, 0.1 mg/mlBSA, 0.2 mM dATP [a-32P]dATP (2 mCi), 0.5 mg of f29TP-DNA, 7.5 ng of purified TP and 15 ng of either wild-type or mutant f29 DNA polymerase, and incubated forthree minutes at 30 8C. For template-independentinitiation assay, f29 TP-DNA was omitted, 15 ng of TPand 30 ng of either wild-type or mutant f29 DNA poly-merase were added, 1 mM MnCl2 was used instead ofMgCl2, and the incubation was for 120 minutes at 30 8C.The reactions were stopped by adding 10 mM EDTAand 0.1% SDS, filtered through Sephadex G-50 spincolumns, and further analysed by SDS-PAGE asdescribed.52 Quantitation was done by densitometricanalysis of the labelled band corresponding to theTP–dAMP complex, detected by autoradiography.

Interference assay for TP binding

Reactions were carried out as described for the tem-plate-independent initiation assay, using a limitingamount of TP and different proportions of a mixture ofwild-type and mutant DNA polymerases. f29 DNApolymerase mutant D249E (catalytically inactive but dis-playing a normal interaction with the TP) was used as apositive control for the interference assay, as previouslydescribed.56 The amounts of proteins used were as fol-lows: 5 ng of TP, 25 ng of wild-type DNA polymerase,and either 25 ng, 50 ng, 100 ng or 200 ng of each mutantderivative (D249E, F128Y/A, E161Q/A). In all cases, the

incubation was for three hours at 4 8C. After incubation,reactions were stopped and analysed as indicated forthe protein-primed initiation assay.

Analysis of the interaction between TP and DNApolymerase mutants by glycerolgradients centrifugation

The incubation mixture contained, in 150 ml, 50 mMTris–HCl (pH 7.5), 1 mM dithiothreitol, 20 mMammonium sulphate, 3 mg of TP and 6 mg of eitherwild-type or mutant DNA polymerase. After incubationfor 30 minutes at 4 8C, samples were loaded onto thetop of a continuous 15–30% glycerol gradient (4 ml) inthe presence of 50 mM Tris–HCl (pH 7.5), 20 mMammonium sulphate, 0.4 M NaCl, 1 mM EDTA and7 mM 2-mercaptoethanol, and centrifuged at 4 8C for 24hours at 58,000 rpm in a Beckman TST 60.4 rotor.Gradients were fractionated and subjected to SDS-12%PAGE. Gels were stained after electrophoresis withCoomassie Blue.

Acknowledgements

This investigation was aided by research grant5R01 GM27242-23 from the National Institutes ofHealth, by grant PB98-0645 from the DireccionGeneral de Investigacion Cientıfica y Tecnica, andby an institutional grant from Fundacion RamonAreces. I.R. was a pre-doctoral fellow of theConsejo Superior de Investigaciones Cientıficas.

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Edited by J. Karn

(Received 31 July 2002; received in revised form 4 October 2002; accepted 7 October 2002)


Documents

φ29 DNA Polymerase Residue Phe128 of the Highly Conserved (S/T)Lx2h Motif is Required for a Stable and Functional Interaction with the Terminal Protein