DNA conformational changes at the primer-template junction regulate the fidelityof replication by DNA polymeraseKausiki Dattaa, Neil P. Johnsonb,c, and Peter H. von Hippela,1

aInstitute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403-1229; bCentre Nationale de la RechercheScientifique, Institut de Pharmacologie et de Biologie Structurale, 205 route de Narbonne, F-31077 Toulouse, France; and cUniversité Paul Sabatier,F-31077 Toulouse, France

Contributed by Peter H. von Hippel, August 19, 2010 (sent for review July 21, 2010)

Local conformational changes in primer-template (P/T) DNA are in-volved in the selective incorporation of dNTP by DNA polymerases(DNAP). Here we use near UV CD and fluorescence spectra of pairsof base analogue probes, substituted either at the primer terminusor in the coding region of the template strand, to monitor and in-terpret conformational changes at and near the coding base of thetemplate in P/T DNA complexes with Klenow fragment (KF) DNAPas the polymerase moves through the nucleotide addition cycle.Incoming dNTPs and rNTPs encounter binary complexes in whichthe 3′-end of the primer shuttles between the polymerization(pol) and exonuclease (exo) sites of DNAPs, even for perfectly com-plementary P/T DNA sequences. We have used spectral changes ofprobes inserted in both strands to monitor this two-state distribu-tion and determine how it depends on the formation of ternarycomplexes with both complementary (“correct”) and noncomple-mentary (“incorrect”) NTPs and on the local sequence of the P/TDNA. The results show that the relative occupancy of the exo andpol sites is coupled to conformational changes in the P/T DNA ofthe complex that are partially regulated by the incoming NTP. Wefind that the coding base on the template strand is unperturbed bythe binding of incorrect dNTPs, while binding of complementaryrNTPs induces a novel template conformation. We conclude that,in addition to its editing function, primer strand occupancy ofthe 3′-exo site may also serve as a regulatory checkpoint for accu-rate dNTP selection in DNA synthesis.

DNA editing ∣ Klenow DNA polymerase ∣ primer partitioning in DNAPactive sites ∣ low energy circular dichroism ∣ base analogues 6-MI and 2-AP

Error rates during DNA synthesis for replicative and somerepair DNA polymerases (DNAPs) are in the range of 1 in105 to 108 (1, 2). While the inherent nucleotide selectivity thatis thought to reflect Watson–Crick base pairing of the incomingdNTP with the templating base and the “steric fit” of the resultingbase pair into the catalytic polymerase (pol) site of the DNAPscontribute significantly to maintain such extraordinary accuracy,fidelity is improved further by the 3′-exonuclease (exo) activitythat removes mismatches from the growing primer terminus (2).Thus controlling the distribution of primer-template (P/T) DNAbetween the pol and exo sites of replicative DNAPs is essential foraccurate DNA synthesis. Here we report evidence for a role forthis distribution in regulating replication fidelity that transcendsthe editing function in KF of Escherichia coli DNAP I.

Structures of binary DNAP-P/T and ternary DNAP-P/T-dNTPcomplexes have been obtained for a variety of A-family DNApolymerases (3–6). All share a common architecture resemblinga “right hand”: including a “thumb” subdomain that binds P/TDNA, a “fingers” domain that interacts with the incoming dNTP,and a “palm” domain containing the highly conserved residues ofthe polymerase active site (5). Until recently it was thought thatDNAPs cycle between two distinct conformational states duringDNA synthesis: a binary “open” conformation in which thefingers are farther away from the active site and the P/T DNA

(shown schematically* in Fig. 5C) and a ternary “closed” confor-mation in which the fingers close around the P/T DNA and theincoming dNTP to form the catalytically active complex (Fig. 5E)(5, 6). Recent studies, including this one, suggest that structurallydistinct and mechanistically important intermediate states existas well.

Crystallographic studies (3, 5) have shown that the 3′-end ofthe primer strand of P/T DNA can bind to and occupy eitherof the catalytic sites (which are separated by about 30 Å), formingpol or exo complexes. In addition three distinct conformationshave been observed for the coding base, n (5) in DNAP. Thusn may occupy the “preinsertion” site in the open binary complex(Fig. 5C); this is a pocket between the conserved O and O1helices of the fingers domain. When bound in this pocket then base is “flipped out” of the DNA helical axis and is not acces-sible to the incoming dNTP. n can also occupy the “insertion” sitein the closed ternary complex prior to catalysis (Fig. 5E); herethe n base is stacked next to the 3′ primer terminus and formsa Watson–Crick base pair with the incoming dNTP. Significantglobal motions of the O-helices are required to transfer the n basefrom the preinsertion site to the insertion site (5). Finally, follow-ing phosphodiester bond formation and DNA translocation,n occupies the “postinsertion” site, while the newly extended3′ primer terminus occupies the pol active site in preparationfor the next round of synthesis.

Recent kinetic studies have suggested that a conformationalchange prior to “finger-closing”may be involved in an early check-point for correct dNTP incorporation (7–10). The conformationalchanges in the DNAP or the P/T DNA that might be involved inthis checkpoint have not been defined. In the homologous T7RNA polymerase (RNAP) the coding base in a preinsertion siteappears to hydrogen bond with the incoming complementaryrNTP, thereby forming an “open ternary” complex (11, 12). Asimilar conformation has been proposed, though not observed,to explain the kinetic selection of dNTP by DNAP (13–15).Following Waksman (15), we refer to such a positioning of thecoding base as an “intermediate preinsertion” site conformation(Fig. 5D), to distinguish it from the preinsertion site conformationin the binary DNAP complex (Fig. 5C) in which the coding base isnot accessible to incoming dNTPs (5).

Author contributions: K.D., N.P.J., and P.H.v.H. designed research; K.D. performed research;K.D. and N.P.J. analyzed data; and K.D., N.P.J., and P.H.v.H. wrote the paper.

The authors declare no conflict of interest.

*Fig. 5 shows a schematic representation of the various conformational states of the P/TDNA in the DNAP binary and ternary complexes that appear to be critical for thenucleotide selection pathway, based both on results from this study and from theavailable crystal structures. In this section we use this figure to introduce backgroundaspects of the structural characteristics of these complexes. New findings are consideredin Discussion.

1To whom correspondence should be addressed. E-mail: petevh@molbio.uoregon.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1012277107/-/DCSupplemental.

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To investigate the structural origins of this kinetic checkpointwe have monitored local conformational changes within the P/TDNA during dNTP selection by means of fluorescence and nearUV circular dichroism (CD) spectra changes for adjacent pairsof 2-aminopurine (2-AP, an adenine analogue) (11) or 6-methylisoxanthopterin (6-MI, an analogue for guanine) (16) bases atspecific positions within the P/T DNA. Previously we used thistechnique to map local conformations at specific positions withinthe primer DNA bound at either the pol or the exo active site ofKF, and to measure the distribution of the primer terminusbetween these two enzymatic sites (17). Here we probe the localconformations of both the primer terminus and the template nbase bound to KF in the presence of various potential dNTPand rNTP substrates to further define structural aspects of pos-sible checkpoint mechanisms in DNAP fidelity control.

ResultsUsing the Near UV CD Spectra of 6-MI Dimers to Probe DNA Confor-mation Changes at the Coding Base Within Polymerase-P/T DNAComplexes. We introduce a novel base analogue probe, the 6-MIdimer (two adjacent 6-MI bases on the same DNA strand), tomonitor the local conformations of the coding base of P/T DNA.The fluorescence properties of single 6-MI (Fig. 1) bases havebeen used previously to study the behavior of guanine baseswithin DNA (16). The structure of the bp formed by 6-MI withcytosine is shown in Fig. 1. We note that the 6-MI dimer has asignificantly larger low-energy CD signal per mole residue(Fig. 1B) than do the 2-AP and pyrrolo C (PC, a cytosine analo-gue) probes that we used in our earlier studies (11, 18).

Fig. 1 A and B show characteristic CD spectra, at wavelengthsbelow or above 300 nm, of the 6-MI dimer (denoted as gg) inwhich the probes are either unpaired and partially stacked in

single-stranded (ss) DNA, or fully base-paired and stacked ineither double-stranded (ds) DNA or an RNA-DNA hybrid withina P/Tconstruct. A bimodal signal centered at the absorption max-imum is characteristic of reporter bases stacked in the B-formdsDNA conformation, while partially unstacked probe conforma-tions display a positive or negative CD band, depending on theirchiral environment (11). The CD signal of the 6-MI dimer iscentered in the 320–390 nm wavelength region of the near UVspectrum and is therefore well removed from the larger globalsignals below 300 nm that arise from the canonical bases andbps. We note that the 230–300 nm CD spectra are essentially un-changed by the substitution of 6-MI bases for guanine, showingthat the 6-MI dimer probe fits well into the cooperative duplexconformations of both dsDNA and RNA-DNA hybrids (comparedotted and solid spectra in Fig. 1A). Somewhat larger differenceswere observed in the ssDNA spectra, suggesting that the noncoo-perative stacking of adjacent 6-MI probes in ssDNA may differsomewhat from the stacking of pairs of G bases when the confor-mation is not “cooperatively locked in” by a fully duplex structure.Additional characterization of 6-MI dimer probes as GG repla-cements (Fig. S1) is summarized in SI Text and will also be pub-lished elsewhere in more extended form (Datta et al., ms inpreparation).

Distribution of the P/T DNA Primer Strand Between the pol and exoSites of KF in the Binary Complex. Using 2-AP dimer probes inthe primer strand as a monitor, we have previously established ex-perimental conditions that form “end-state” binary KF-P/T DNAcomplexes in which the 3′-end of the primer strand is bound en-tirely in either the pol or the exo site (17). Here 6-MI dimer probeslocated at positions n;nþ 1 are used to characterize template

A B

Fig. 1. CD spectra of 6-MI modified single-stranded DNA and duplex DNA–DNA and RNA–DNA constructs. The molecular structures for the canonicalG∶C bp and the 6-MI∶C bp are shown at the top. The P/T DNA construct usedis shown above the graphs and the positions of the 6-MI dimer probes areindicated as “gg”. n denotes the position of the template coding base, whilethe positive and negative numbers following n represent bases positionedeither downstream or upstream from the n base, respectively. The 16-merprimer (top) strand was either ssDNA, forming a DNA–DNA duplex (red) withthe 6-MI modified template (bottom) strand (dark green), or RNA, resultingin an RNA–DNA heteroduplex (dark blue) of the same sequence. Panel Ashows the high energy CD spectra of the constructs that are dominatedby the canonical bases. Panel B shows the low-energy CD spectra of the6-MI analogue dimers within the constructs. The solid lines represent 6-MImodified constructs while the dashed lines denote the correspondingunmodified oligonucleotides with identical sequence.

A B

C

Fig. 2. CD spectra of 6-MI labeled P/T DNA constructs in binary complexeswith KF DNAP. Low-energy CD spectra of the KF-DNA binary complex with (A)the upper construct in Ca2þ buffer; (B) the upper construct in EDTA buffer;and (C) the lower (three terminal mismatches) construct in Ca2þ buffer.

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strand conformations at the n base in these end-state complexes.Fig. 2C shows CD spectra for the construct and the complex inCa2þ buffer, in which the 3′-primer terminus of a P/T DNA con-struct with three terminal mismatches is bound exclusively at theexo site. Fig. 2B shows spectra in EDTA buffer (containing 2 mMEDTA and no added divalent cations), in which the primer of afully base-paired P/T DNA construct is totally bound in the polsite (see ref. 17 and SI Text). In contrast, and as shown previouslywith 2-AP dimer probes positioned at the end of the primer strand,the 3′-ends of fully complementary P/T DNA constructs in Ca2þbuffer are distributed between the two active sites (Fig. 2A).

With the primer terminus fully bound in the exo site, theseresults show that the CD signal for this template-substituted com-plex resembles closely that obtained with the free DNA construct(Fig. 2C), suggesting that in the binary exo complex the local con-formation of the n;nþ 1 template bases are unperturbed relativeto the free construct. In contrast, with the primer strand bound inthe pol site, the CD spectrum of the template-substituted dimerprobe looks very different, exhibiting a peak near 360 nm (Fig. 2B,pink trace) that suggests that the 6-MI probes at the coding baseare significantly unstacked under these conditions (11). This re-sult doubtless reflects the local distortion of the template strandthat is required to place the coding (n) base in the preinsertionsite of the binary DNAP pol complex (5) and confirms that thisdistorted conformation also forms in solution at the templatebase of a fully complementary binary DNAP-P/T DNA complex.

These results demonstrate unequivocally that the templatebases at and near coding base n assume very different local con-formations when the primer terminus is bound in the pol or theexo site of KF, providing important insight into how both strandsof the P/T DNA are handled by KF during both synthesis andediting. Furthermore, Fig. 2A shows that the CD signal of 6-MIresidues in a matched P/T DNA construct in the presence ofdivalent Ca2þ appears to be a linear combination of thesespectra (pink trace), showing that the two-state character of thepol–exo distribution can be established and monitored by usingprobes in either strand. We quantified this equilibrium distribu-tion by deconvoluting the CD spectrum of the 6-MI dimer in thetemplate strand as previously described for 2-AP dimer probesplaced at the 3′-end of the primer strand (17).

Experiments using P/T DNA constructs containing either GG(or gg) at the n;nþ 1 template positions or AA (or 2-AP dimer,denoted as aa) at the 3′-end of the primer gave identical 70% exoto 30% pol distributions (solid black fits; Fig. 3 A and B). Hence,as long as the overall sequence of the P/T DNA was not changed,these different analogue probes at these positions yielded thesame distribution of primer ends between the exo and pol sites,again confirming that these base analogues can effectively replacetheir canonical counterparts in a variety of contexts.

In contrast, changing the sequence of the template DNAimmediately downstream of the P/T junction does significantlyinfluence this distribution, with AA or TA bases in the n;nþ 1 po-sitions decreasing the fraction of primer ends in the exo site to40%, compared with 70% exo for GG bases in the same positions(Fig. 3A). This result may argue that “stiffer” sequences contain-ing GG (or gg) at the n;nþ 1 positions in the template strand aremore difficult to distort in placing the n base into the preinsertionsite, thus favoring a higher population of primer ends in the exosite for GG sequences relative to sequences containing AA or TAin these positions. We note that all the P/T DNA constructs con-taining the 2-AP dimer probe with varied bases in the n and nþ 1template positions displayed the same low-energy CD spectra inthe absence of KF (Fig. 3A, Inset), demonstrating that the changedspectral signals observed in the binary complexes result fromdifferent conformations of the P/T DNA construct that dependon the binding of the primer terminus to the two different catalyticsites of KF DNAP. We expect (see also ref. 17) that sequence

alterations elsewhere in the P/T DNA construct will also perturbthe two-state pol to exo primer binding distribution.

Local DNA Conformations Within Ternary Complexes. We have alsomonitored conformational changes in the P/T DNA frameworkinduced by adding NTP substrates to binary complexes to formternary (KF-DNA-NTP) complexes. The addition of the correctdNTP (dCTP) to the binary complex provoked a large increase inthe depth of the CD trough near 340 nm, with similar spectrabeing obtained for DNA constructs with either a 3′-deoxy (3′-H)or a 3′-OH nucleotide residue positioned at the 3′-primer end(see Fig. 4 A and B). The use of a nonextendable primer allowedus to capture the CD signal of bases n;nþ 1 within a closed andcatalytically active DNAP complex, where coding base n presum-ably occupies the insertion site (Fig. 5E) (5, 6). The addition ofrCTP (the correct ribo-NTP) resulted in a much smaller change inthe CD signal (Fig. 4A), although the direction of change was thesame as that observed with dCTP. The addition of dCTP to a bin-ary complex formed with the 3′-OH P/Tconstruct resulted in thegradual appearance of a bimodal signal with a peak at 380 nm(Fig. 4B, Inset, and Fig. S2), while the spectrum of the constructcontaining the 3′-H primer (Fig. 4A) remained unchanged underidentical conditions. Furthermore, no time-dependent changes inthe CD spectrum of the n;nþ 1 bases relative to the binary com-plex were observed in the presence of incorrect dNTPs or rNTPs(Fig. S2). Hence, the change in CD signal with dCTP as a functionof time must reflect the slow incorporation of cytosine. The use ofCa2þ rather than Mg2þ in these experiments slows the reactionrate (7) sufficiently to permit time-dependent monitoring of thechain extension process.

A B

Fig. 3. Measuring the equilibrium distribution of the primer terminusbetween the pol and exo sites in binary complexes using dimer probes placedeither at the primer terminus or at the coding base. Low-energy CD spectrafor KF-P/T DNA complexes containing either (A) 2-AP or (B) 6-MI probes atthe indicated positions. The constructs used are above each panel, andthe positions of the 2-AP and 6-MI dimer probes are indicated as “aa” and“gg”, respectively. nðAÞ, nðTÞ, nðGÞ and nðgÞ identify the n base (A,T,G and 6-MI,respectively) in the various constructs. The black traces represent the bestfits of the corresponding experimental curve to a linear combination of theexo-mode and pol-mode spectra. Inset in A shows the CD spectra of the freeprimer-labeled constructs and demonstrates that these spectra are notaltered by changing the n base.

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Unlike complementary dCTP or rCTP, the incorrect dNTPsand rNTPs did not induce any significant rearrangement ofthe n;nþ 1 bases in the template (Fig. 4A and B). Furthermore,primer extension experiments using radioactively labeled primerDNA with a 3′-OH terminus, performed under the same condi-tions used for the spectroscopic measurements, showed negligiblemisincorporation in the presence of noncomplementary dNTPsand rNTPs (Fig. 4E). Hence our spectroscopic measurementsreveal P/T conformational changes that appear to correlate withdiscrimination between correct and incorrect dNTPs and rNTPsby the KF-P/T DNA complex.

Additional experiments were performed to probe the n − 1;nand nþ 1;nþ 2 template positions in binary and ternary KFcomplexes (Fig. 4C and Fig. S3). The addition of dCTP (thecorrect dNTP) resulted in the characteristic trough at 340 nmin the CD spectrum of probe residues at the n − 1;n templatepositions. This signal is similar to that observed for the ternarycomplex with the n;nþ 1 construct in the presence of the correctdNTP (Fig. 4A and B). Due to the extendable nature of theprimer strand used for probing the n − 1;n positions, we alsoobserved the time-dependent evolution of the CD spectrum, re-flecting the slow incorporation of cytosine at the primer terminus(Fig. 4C, Inset). The characteristic trough was not observed withthe nþ 1;nþ 2 construct when the correct dNTPwas added to thisKF-P/T DNA complex (Fig. S3). Therefore, the CD trough at340 nm observed for constructs with probes in the n − 1;n andn;nþ 1 template positions must reflect a local conformation inwhich the n base occupies the active site in a Watson–Crickbase-paired state with the correct dNTPwithin theKF closed tern-ary complex. The ternary complex involving the n − 1;n constructwith added incorrect dNTPs showed significant unstacking of the

probe bases relative to the binary complex (Fig. 4C), while no dif-ference was observed with the n;nþ 1 construct (Fig. 4A and B).

Complementary fluorescence and CD experiments were alsoperformed with constructs containing 2-AP dimer probes at theprimer terminus to monitor the effects of NTPs on base pairing atthe duplex terminus of P/T DNA. The results were fully suppor-tive of the data presented above and are presented in SI Text,including Fig. S4 and Fig. S5.

DiscussionAccurate replication is accompanied by local conformationalchanges of the DNA framework of DNA-DNAP complexes.We monitor these changes by taking advantage of relationshipsbetween local nucleic acid conformations and the low-energyCD spectra of base analogue probes placed at specific positionswithin the P/T DNA. Using the 2-AP dimer probe we have re-cently (17) shown in solution that the terminal bases of the primerstrand bind in an extended conformation in the exo site, whileremaining stacked when bound at the pol site of KF, as expectedfrom crystal structures of homologous A-family DNA poly-merases (3, 6). The low-energy CD spectra of the 6-MI dimer alsoprovide characteristic signals for stacked and unstacked bases(Fig. 1) and extends the repertoire of dimer probes that canbe used to monitor local DNA conformations (11, 18). Herewe report DNA conformational changes associated with dNTPselection, using P/T constructs carrying 2-AP dimer probes atthe 3′-end of the primer strand or 6-MI dimer probes locatedat or near the coding base position (n) of the template strand.

In addition to the conformational changes induced in the pri-mer strand, our results show that the conformation of the templatestrand in the binary KF-P/T DNA complex is also significantlychanged when the primer strand terminus moves from the polto the exo site (Figs. 2 and 3). The template strand bases at posi-tions n and nþ 1 are largely unstacked (CD peak at 360 nm)when the primer is bound at the pol site, consistent with assumedpositioning of template coding base n in the preinsertion site inthis complex (Fig. 5C). In contrast, when the primer terminusis bound in the exo site (Fig. 5A) the bases at positions n;nþ 1of the template strand are stacked, showing a bimodal (exciton-coupled) CD spectrum. In this configuration the template strandmay interact with the RRRY motif (19), a recently postulatedtemplate binding sequence in KF that is physically separate fromthe pol site (5).

DNA Conformations in Ternary Complexes Respond to NTP Binding.We [and others (17, 20, 21)] have observed that under most con-ditions the primer strand at P/T DNA junctions populates boththe pol and exo sites of binary DNAP complexes, even in theabsence of a terminal mismatch. DNA binding in the exo site isusually thought to be involved in the replication pathway onlyafter covalent misincorporation; that is, in the context of primerediting to remove an incorrect 3′ terminal base. However, becauseincoming NTPs encounter binary complexes in which P/T DNA isin a dynamic equilibrium between the pol and exo binding sites, itis possible that this equilibrium could participate in an early struc-tural checkpoint for NTP selection.

The intense CD trough near 340 nm was observed only in thepresence of the correct dNTP (Fig. 4), which stabilizes the closedconformation of the ternary DNAP complex with the template nbase in the insertion site and base-paired with the incoming dCTP(Fig. 5E). In this conformation the primer strand forms a duplexwith the template DNA (Fig. S5), thereby positioning the α-phos-phate of the incoming dNTP for an in-line nucleophilic attack onthe 3′-OH of the primer terminus (2). We find no indication ofthe formation of a closed ternary complex in the presence ofincorrect dNTPs (Fig. 4), in agreement with published kineticstudies (7, 14, 22).

A B C

D E

Fig. 4. Near UV CD spectra of the n;nþ 1 and n − 1;n template basepositions during dNTP selection. Low-energy CD spectra of KF-P/T binaryand KF-P/T-NTP ternary complexes with P/T constructs containing: (A) anonextendable 3′-deoxy primer terminus with 6-MI at the n;nþ 1 positions;(B) an extendable primer terminus with 6-MI at n;nþ 1 positions; and (C) anextendable primer terminus with 6-MI at n − 1;n positions. Insets show time-dependent changes in the low-energy CD in the presence of complementarydCTP, due to its incorporation at the primer terminus. Gels show the productsof primer-extension reactions with either (D) the nonextendable or (E) theextendable constructs with 6-MI at the n;nþ 1 positions under the samereaction conditions used for the CD experiments. Quantitative estimatesof the products formed with the extendable primer after 60 min of reactionare also shown.

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The CD spectrum of the template n;nþ 1 bases in the presenceof noncomplementary dNTPs is different from that observed inthe presence of either dCTP or rCTP (Fig. 4A). In ternary com-plexes with noncomplementary dNTPs, the probe residues atpositions n − 1;n become more unstacked (Fig. 4C), which couldreflect sequestration of n base into the preinsertion site. However,probes positioned at n;nþ 1 did not exhibit any significant changerelative to the respective binary complexes (Fig. 4 A and B), sug-gesting that bases at these positions display the same conforma-tional equilibrium with the pol site as in the binary complex. Thisapparent discrepancy may reflect the extent of primer shuttlinginto the pol site for the two constructs. The terminal bp at theP/T junction (position n − 1) is G•C in the construct in whichthe n − 1;n positions were probed and A•T for the construct usedfor probing the template n;nþ 1 positions, suggesting theG•C bp might stabilize duplex DNA bound in the polmode morethan A•T. Finally, although the template n;nþ 1 bases do notshow any change in the equilibrium distribution in the presenceof incorrect dNTP, 2-AP fluorescence quenching indicates thatthe primer terminus of the same construct may partially shift to-ward the pol site (Fig. S5 A and B). In this hypothesis, binding ofincorrect dNTP could cause less stable P/T junctions (terminalA•T bp) to assume a conformation where the P/T DNA hasmoved partway toward the pol site, perhaps without completesequestration of the n base into the preinsertion site (Fig. 5B).The presence of such an intermediate transition state at the“crossroads” between replication and editing has been suggestedearlier for other proof-reading-proficient DNAPs (23, 24). Herewe report the possible utilization of such a transient state in thenucleotide selection pathway of DNA replication.

The presence of saturating concentrations of the correct ribo-NTP (rCTP) also significantly quenches the 2-AP dimer probe,consistent with the transfer of the primer terminus from theexo site, where it is unwound, to a duplex with template DNA.However, this transfer occurs only at much higher rCTP concen-trations than required with dCTP (Fig. S5). The template n;nþ 1bases also stack more on rCTP binding, although the CD signal isnot characteristic of the closed complex formed with correctdCTP (trough at 340 nm). The CD spectra of the ternary complexwith rCTP could have a small component of signal from theclosed complex. Alternatively, the binding of rCTP could drivethe n;nþ 1 bases into an arrangement such as the intermediatepreinsertion configuration (Fig. 5D), which could contribute sucha signal. In either case complementary rNTP binding moves thecoding base from the preinsertion site, most likely to a positionwhere it can make H-bond contact with the n base but it cannotmove the n base into the insertion site (compare brown and bluetraces Fig. 4A). It has been previously reported that complemen-tary rNTP inhibits finger-closing within the complex (7), perhapsdue to a clash of the conserved “steric gate” residues with theribose sugar of the rNTP (25). Here we show directly that thecoding base does not transfer to the insertion site in the presenceof complementary rNTP.

Possible Structural Role of the exo–pol Site Distribution of the 3′ Pri-mer Terminus as a Fidelity Checkpoint in DNA Replication. Taken to-gether these results suggest that initial discrimination againstincorrect nucleotides by KF is achieved in two steps, both ofwhich precede the final finger-closing process. NTPs first encoun-ter the binary DNAP complex and modulate the partitioning of

A B C

D E

Fig. 5. A schematic overview of the disposition and occupancy of the active sites of DNA polymerase in binary (A, B, and C) and ternary (D and E) complexes.The figures for the open binary pol (5C) and closed ternary complexes (5E) are adapted from ref. 5, while that of the open binary exo complex (5A) is based onthe structure in ref. 3. Cartoons representing the conformations of the various KF-DNA complexes in the reaction are shown at the top of each panel, and blow-ups of the conformations of the DNA bases are shown just underneath. KF is shown in green, while the polymerase subsites are color coded as follows: yellow(insertion site); gray (preinsertion site); pink (intermediate site between pol and exo active sites); and cyan (exo site). T, F, and P represent the thumb, fingers,and palm subdomains, respectively. The primer and the template DNA strands are shown in red and blue, respectively, and the coding base (n) is shown in black.The incoming dNTP/rNTP is shown in purple. In the open binary complex (5C), n is bound at the preinsertion site (between the O andO1 helices) and a conservedtyrosine (Tyr, orange) blocks access to the insertion site (shown in yellow). Formation of the closed conformation (5E) in the presence of the complementarydNTP (purple) involves rearrangement of the O and O1 helices, which simultaneously blocks the template preinsertion site and unblocks the insertion site.These rearrangements move the coding base (n) of the template to the insertion site, where it pairs with an incoming dNTP. Incoming dNTP occupies theintermediate preinsertion site (5D) in a conformation previously proposed for T7 RNAP (12, 15).

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the P/T DNA to the pol site. This partitioning is also dependenton the stability of the bp at the P/T junction, leading to selectivestabilization of the coding base at the preinsertion site. The dy-namics of the partitioning of the primer strand between the poland exo active sites has been studied extensively in the context ofproofreading. However, the regulatory role of NTPs in theseevents remains to be examined (see SI Text). Here we show thatthe binding of various NTPs promotes the depopulation of theexo site in the following order of increasing effectiveness: incor-rect dNTPs or rNTPs < correct rNTP < correct dNTP (Fig. S5),as would be expected if partitioning of the primer terminusbetween the exo and pol sites were coupled to NTP selection.Binding of complementary rNTP also modifies the coding baseconformation (Fig. 4A), while incorrect dNTPs do not have thiseffect, indicating that they may be discriminated against in thepreinsertion site prior to this conformational change. As arguedabove, the CD spectral changes that accompany rCTP bindingcould be explained by formation of a complex with the templatecoding base in an intermediate preinsertion site (Fig. 5D), whereit could pair with the complementary rNTP as proposed forRNAP (12). These changes in P/T DNA conformation are doubt-less accompanied by conformational changes in the polymerase,which may also be involved in an early kinetic checkpoint fordNTP selection (7, 15, 22).

These results suggest that the role of the exo site goes consid-erably beyond its enzymatic proofreading function. Binding ofthe primer in the exo site partially unwinds the DNA duplex andcould thus block the translocation of the replication complex, reg-ulating the advance of the polymerase. For example, interactionsbetween PCNA and DNAP from Pyrococcus furiousos appear todecrease the binding of primer strands at the exo site and stabilizethe P/T DNA duplex within the polymerase, thereby favoringprocessive elongation (26). In addition, partitioning of the primerterminus between the polymerase and exonuclease active sitesmodulates translesion synthesis by E. coli DNA Pol II (27). Poly-merase features that are involved in control of the occupancy of

the exo site are beginning to be investigated by protein engineer-ing (27). Not all DNA polymerases have an exo binding mode;repair polymerases in the X and Y families and certain A-familypolymerases that perform translesion and mutagenic DNA synth-esis lack proofreading exonuclease activity (28). It is possible thatprimitive replicative DNAPs may have stabilized unwound P/TDNA as a means of placing replication under the control of trans-acting protein or NTP components as shown here, and that suchregulatory mechanisms subsequently evolved to produce 30 → 50exonuclease activity and replicative editing.

Materials and MethodsMaterials. Unlabeled DNA oligonucleotides were purchased from IntegratedDNA Technologies (Coralville, IA). 6-MI-labeled DNA oligonucleotides werefrom Fidelity Systems (Gaithersburg, MD), while 2-AP modified oligonucleo-tides were purchased from Operon (Huntsville, AL). P/T DNA constructs wereprepared as described (17). The clone (plasmid pXS106) for the exo− D424Aderivative of KF, and the host (CJ 376) cells were gifts from Catherine Joyce(Yale University). The protein was expressed and purified as described pre-viously (17). Unless otherwise stated, all experiments were performed at25 °C in Ca2þ buffer (20 mM Hepes (pH 7.9), 50 mM sodium acetate,5 mM CaðOAcÞ2, and 1 mM DTT) at equimolar (3 μM) concentration ofDNA and KF and 0.5–2 mM concentrations of dNTP and rNTP substrates.

Spectroscopic Procedures. The fluorescence and CD spectra were measured asdescribed in SI Text and (17). The CD spectra shown are reported as εL-εR inunits of M−1 cm−1 per mole of 6-MI (for 6-MI dimer probes between 300 and450 nm) or permole of nucleotide residues (for canonical DNA bases between230 and 300 nm).

Polymerase activity assays. Polymerase activities weremeasured with P/T DNAconstructs labeled at the 5′-end with γ-P32-ATP and the DNA products wereanalyzed as described in SI Text and (17).

ACKNOWLEDGMENTS. This work was supported by National Institutes ofHealth grant GM-15792 (to P.H.v.H) and by salary support for N.P.J. by theCentre Nationale de la Recherce Scientifique. P.H.v.H is an American CancerSociety Research Professor of Chemistry.

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