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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:
[email protected].
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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