First publ. in: Journal of Biological Chemistry ; 287 (2012), 17. - pp. 14099–14108
Amino Aeid Templating Meehanisms in Seleetion of
Nueleotides Opposite Abasie Sites by a Family A DNA
Polymerase*~
Samra Obeid*§l, Wolfram Welte§~, Kay Diederichs § ~, and Andreas Marx*§2
From the Departments of *Chemistry and ~Biology and the §Konstanz Research School Chemical Biology, University of Konstanz,
Universitätsstrasse 70,078457 Konstanz, Germany
Bacl<ground: Abasie sites are the most frequent DNA lesions and are often bypassed by incorporating an adenosine opposite
that lesion.
Results: We determined structures of DNA polymerase in eomplex with different nucleotides opposite an abasie site.
Conclusion: Interaction ofthe in coming nucleotide with a single amino acid governs nucleotide selection opposite abasie sites.
Significance: This work furthers the understanding of the bypass of a mutagenic lesion by DNA polymerases.
Cleavage of the N-glyeosidie bond that eonneets the nucleobase to the backbone in DNA leads to abasie sites, the most
frequent lesion under physiologieal eonditions. Several DNA
polymerases preferentially ineorporate an A opposite this
lesion, a phenomenon termed /lA-rule." Aecordingly, KlenTaq,
the large fragment of Thermus aquaticus DNA polymerase I,
ineorporates a nucleotide opposite an abasie site with efficiendes of A > G > T > C. Here we provide struetural insights into
eonstl'aints of the aetive site during nucleotide seleetion opposi te an abasie site. Lt appears that these eonfines govern
the nucleotide selection mainly by interaetion of the ineoming
nucleotide with Tyr-671. Depending on the nucleobase, the
nucleotides are differently positioned opposite Tyr-671 resulting in different alignments of the funetional groups that are
required for bond formation. The distanees between the lX phosphate and the 3' -primel' terminus inereases in the order
A < G < T, whieh follows the order of ineorporation effideney.
Additionally, a binary KlenTaq strueture bound to DNA eontaining an abasie si te indicates that binding of the nucleotide
triggers a remukable rearrangement of enzyme and DNA template. The ability to resolve the stacking arrangement might be
dependent on the intrinsie properties of the respeetive nucleotide eontributing to nucleotide seleetion. Furthermore, we studied the ineorporation of a non-natural nucleotide opposite an
abasie site. The nucleotide was often used in studying stacldng
effeets in DNA polymerization. Here, no interaetion with Tyr761 as found for the natural nucleotides is observed, indieating a
different reaetion path fOl' this non-naturalnucleotide.
* This
work wa s supported by the Deutsche Forschungsgemeinschaft
through a grant in 5PP 1170.
0 This article contains supplementa l Figs. 51-57 and Tab le 51 .
The atomic coordinates and structure factors (codes 3RR8, 3RRG, 3RRH, 3RR7,
and 3T3F) have been deposited in the Pro tein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
(hrrp:/lwww.rcsb.org/).
1
2
Supported by funding from the Zukunftskolleg, University of Konstanz.
To whom correspondence should be addressed: Dept. of Chemistry and
Konstanz Research 5chool Chemical Biology, University of Konstanz, Universitätsstrasse 10, D 78457 Konstanz, Germany. Tel.: 49-7531-885139; Fax:
49-7531-885140; E-mail: [email protected].
The most eommon DNA damage under physiologieal eonditions eonsists of abasie sites resulting from spontaneous
hydro lysis of the N-glycosidic bond between the sugar moiety
and the nucleobase in DNA (1) . Beeause the genetic information is lost by th'e cleavage of the nucleobase, abasie sites bear a
high mutagenie potential (2- 4). In most ca ses, the lesion is
removed by DNA repair systems using the si ster strand to guide
for incorporation ofthe right nucleotide. However, undetected
lesions, 01' those form ed du ring S phase, pose achallenge to
DNA polymerases and block replication (5, 6). Several studies
indicated the mutagenic potential ofthese lesions in transiesion
synthesis, whieh is more pronounced in animal as compared
with bacterial eells presumably due to higher transiesion synthesis in eukaryotes (4, 7, 8). Interestingly, it has been shown
that in human cells, an adenosine (A) is preferentially incorporated opposite abasie sites (4). Further in vitro and in vivo studies ofDNA polym erases from family A (including human DNA
polymerases y and f)) and B (including human DNA polymerases Ci, E, and 8) in the presence ofthe stabilized tetrahydrofuran abasfc site analog F (su pp lemental Fig. SI ) have shown
that purines, in particular adenosine, and to a lesser extent
guanosine, are most frequently incorporated opposite the
lesion. The strong preference for adenosine has been termed
"A-rule" (7- 20).
A set of studies coneerning the behavior ofDNA polymerases
from different sequenee families showed that there are multiple
meehanisms to overeome an abasie site. Most transiesion DNA
polymerases follow various loop-out m echanisms (11, 21 - 25) .
Thereby, the nudeotide selection is int1uenced by the followin g
upstream templating bases resulting in deletions and eomplex
mutation speetra. Reeently, an amino acid templating mechanism was found for the "error-free" bypass of an abasie site by
the yeast Revl DNA polymerase member of family Y (26).
Beeause guanine is d eaved most frequently (2), the preference
of Rev1 for dCMP incorporation opposite an abasie site represents the "best guess."
However, the determinants of the A-rule are still eontroversially discussed. Struetural and functional studies have added
signifieantly to our understanding of the basic meehanisms of
14099
Konstanzer Online-Publikations-System (KOPS)
URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-192120
transiesion synthesis by DNA polymerases (27, 28). Superior
stacking as weil as solvation properties of adenine have been
discussed as the driving force behind adenine selection (17,
29 -:H). However, previously reported structures of Kien Taq,
the large fragment of Thermus aquaticus (Taq) DNA polymerase I, which is a member of the sequence family A, suggests that
this enzyme follows the A-rule by applying an amino acid templating mechanism (19, 20). Thereby, interaction with Tyr-671
seems to be the basis of the prefere nce of purines. It facilitates
nudeotide ineorporation by mimieking a pyrimidine nudeobase direeting for purine incorporation opposite abasie sites
due to the enhaneed geometrie fit to the aetive site (32, 33). The
erucial role of this tyrosine in transiesion synthesis, whieh is
highly conserved throughout evolution in DNA polymerase
family A from bacteria to humans (34), was further probed by
analysis of site-direeted mutations (19) . Interestingly, when the
six-membered ring of tyrosine was mutated to the bicydic
indole of tryptophan, the A-rule was shifted to a "C/T -rule,"
further supporting geometrie factors as determinants of nudeotide selection opposite an abasie site.
Still, several aspects remain to be darified to fully understand
the mechanism of nudeotide selection opposite an abasie site
by DNA polymerases from the family A. These aspects indude
the overall conformation of the enzyme-DNA complex in the
binary state prior to nudeotide bin ding. Furthermore, the order
of nudeotide incorporation efficiency opposite abasie sites by
DN A polymerases such as Kien Taq is A, G, T, and e. The mechanistie understanding for this observation is sparse due to the
lack of structural data. Here, we report a structure of a binary
complex of KlenTaq DNA polymerase bound to the primer/
template bearing an abasie site lesion. Additionally,.we present
several ternary structures ofKlenTaq caught incorporating different nucleotides opposite an abasie site, providing insights
into the preference order of nucleotide incorporation efficiency
opposite this lesion.
EXPERIMENTAL PROCEDURES
Pro tein and Oligonucleotides
Protein expression and purification were conducted as
described (35). In brief, an Escherichia coU codon-optimized
KlenTaq gene (amino acids 293- 832 of Taq gene; purchased
from GeneArt) was doned into a pET-21b vector without any
purification tags and expressed in E. coli strain BL21 (DE3) . Of
note, codon optimization resulted in changes within the gene
sequence without affecting the amino acid sequence. After heat
denaturation and ultracentrifugation, a PEI precipitation was
performed. The resulting material was purified by anion
exchange (Q Sepharose) chromatography followed by size-exclusion chromatography (Superdex 75) in 20 mM Tris HCI, pH
7.5, 150 mM NaCI, 1 mM EDT A, 1 mM ß -mercaptoethanol.
Oligonudeotides were purchased from Metabion or Thermo
Scientific. The dideoxy cytosine modified primel' was synthesized on an Applied Biosystems 392 DNA/RNA synthesizer
using 5' -dimethoxytrityl-2' ,3' -dideoxy cytosine modified Nsuccinoyl-Iong chain alkylamino controlled pore glass, which
was purchased from Gien Research. The synthesized oligonucleotide was purified by preparative PAGE on a 12% polyacryl-
14100
amide gel containing 8 M urea (DMT-OFF). The nucleotide
dNITp 3 was synthesized starting from dNI purchased from
Berry & Associates Inc., according to published procedures
(36).
Crystal/ization and Structure Determination
KlenTaq (buffer: 20 mM Tris HCI, pH 7.5, 150 mM NaCI, 1
mM EDT A, 1 mM ß -mercaptoethanol) was incubated in the
presence of DNA primel' (5' -d(GAC CAC GGC GC)-3'), an
abasie site F containing template (5' -d(AA.A FNG CGC CGT
GGT C)-3'); N represents the templating base directing the
processing of ddGTP, ddTTP, 01' ddCTP.
Kien TaqF_G_r- The crystallization was set up using purified
KlenTaq (11 mg/mi), DNA template/primer duplex, and
ddGTP in a molar ratio of 1:3:50 and in the presence of 20 mM
MgCl 2. The crystallization solution was mixed in a 1:1 ratio
with the reservoir solution containing 0.05 M sodium cacodylate (pH 6.5), 0.2 M NH 4 0Ac, 0.01 M Mg(OAc)z' and 25% PEG
8000.
Kien TaqF_c_ lI- The crystallization was set up as for
Klen TaqF_G_ 1except using Kien Taq (11 mg/ml):DNA template/
primel' duplex:ddGTP = 1:3:50 in the presence of 20 mM
MgCl z. The reservoir solution contained 0.05 M sodium cacodylate (pH 6.5), 0.2 MNH 4 0Ac, 0.01 MMg(OAc)2' and 28% PEG
8000.
J([en Taq F-./- The crystallization was set up as for J([en Taq f' -G- I
except using KlenTaq (11 mg/ml):DNA template/primer
duplex:ddTTP = 1:3:50 in the presence of 20 mM MgCI 2 . The
reservoir solution contained 0.05 Msodium cacodylate (pH 6.5),
0.2 MNH 4 ÜAc, 0.01 M Mg(OAc)2' and 28% 'PEG 8000.
Kien TaqF-binary-lI- The crystallization was set up as for
KlenTaqF_G_1exceptusingKlenTaq (11 mg/ml):DNA template/
primel' duplex:ddCTP = 1:1.5:60 in the presence of 20 mM
MgCl z. The reservoir solution contained 0.1 M Tris HCI (pH
8.0), 0.2 M Mg(formiate)z' and 15% PEG 8000.
KlenTaqF_b'"ary-The crystallization was set up using purifiedKlenTaq (8 mg/mI), a template (5' -d(AAA FGG CGC CGT
GGT C)-3'), and a previously synthesized primel' with a dideoxy cytosine at its 3' -end, in a molar ratio of 1:1.4 and in the
presence of 20 mM MgCl z. The crystallization solution was
mixed in 1:1 ratio with the reservoir solution containing 0.1 M
Tris HCI (pH 7.5), 0.2 M Mg(formiate)2' and 12% PEG 8000.
Kien TaqF_NI- The crystallization was set up as for
KlenTaqF_binat-y except using KlenTaq (8 mg/ml):DNA tem plate/primer duplex = 1:1.4 and in the presence of 20 mM
MgCl z. The reservoir solution containedO.l M Tris HCI (pH
7.0),0.2 MMg(formiate)z' and 18% PEG 8000. The crystals were
soaked overnight by adding 0.5 ILI of dNITP (20 mM) solution.
The occupancy ofthe bound dNITP was refined to 0.77.
Crystals were produced by the hanging drop vapor diffusion
method by equilibrating against 1 ml of the reservoir solution
für 5 days at 18 "e. The crystals were frozen in liquid nitrogen.
3The abbreviations used are: dNITP, 5-nitroindolyl -2'-deoxyriboside 5'triphosphate; ddTTP, dideoxythymidine 5'-triphosphate; r.m.s.d., root
mean square deviation; dNIMP, 5-nitroindoyl-2'-deoxyriboside 5'-monophosphate; ddGTP, dideoxyguanosine 5'-triphosphate; ddCTP, dideoxycytidine 5' -triphosphate; ddATP, dideoxyadenosine 5 '-tri phosphate;
ddNTP, dideoxynucleoside 5' -tri phosphate.
B
Datasets were eolleeted at beamline PXI (X06SA) of the Swiss
Light Souree (Paul Scherrer Institute, Villigen, Switzerland), at
a wavelength of 1.000 or 0.900 Ausing a PILATUS 6M deteetor.
Data reduetion was performed with the XDS paekage (37, 38).
The structures were solved by differenee Fourier teehniques
using KlenTaq wild type (Protein Data Bank (PDB) 3LWL) as
model. Refinement was performed with PHENIX (39), and
model rebuilding was done with COOT (40) . Figures were
made with PyMOL (41) .
Prim er Extension Assays
Prim er extension was performed as deseribed (19) . In brief,
for incorporation opposite F, 20 ,.d of the J(lenTaq reaetions
eontained 100 nM primer (5' -d(CGT TGG TCC TGA AGG
AGG ATA GG)-3'), l30 nM template (5' -d(AAA TCA FCC
TAT CCT CCT TCA GGA CCA ACG TAC)-3'), 100 !.LM
dNTPs in buffer (20 mM Tris HCI, pH 7.5, 50 mM NaCI, and 2
mM MgCI 2 ), and 500 nM of the respeetive J(lenTaq polymerase.
Reaetion mixtures were incubated at 37 oe. Incubation times
are provided in the respeetive figul'e legends. Primel' was
labeled using [y- 32 P1ATP aeeording to standard teehniques.
Reactions were stopped bythe addition of45 !.LI of stop solution
(80% (v/v) formamide, 20 mM EDTA, 0.25% (w/v) bromphenol
blue, 0.25% (w/v) xylene cyanol) and analyzed by 12% denaturing PAGE. Visualization was performed by phosphorimaging.
Enzyme Kinetics
The rates of single turnovers in pre-steady-state kinetics
were determined as described (19) . In brief, 15 !.LI of radio labeled primer/template complex (200 nM) and DNA polymerase
(2 mM) in reaction buffer (see primel' extension assay) were
rapidly mixed with 15 !.LI of a dNTP solution in reaction buffer
at 37"e. Quenching was achieved by adding previously
described stop solution. For reaction times longer than 5 s, a
manual quench was performed. The analysis of dNTP incorporation opposite to the abasie site primel' (tor sequenees, see
"Primer Extension Assays") and templates (for sequenees, see
"Primer Extension Assays") were applied. Quenehed sam pies
were analyzed on a 12% denaturing PAGE followed by phosphorimaging. For kinetic analysis, experimental data were fit by
nonlinear regression using the pro gram GraphPad Prism 4. The
data were fit to a single exponential equation: (eonversion) =
A X (1 - exp( -kobs t)) . The observed catalytie rates (kobJ were
then plotted against the dNTP concentrations used, and the
data were fit to a hyperbolie equation to determine the J(d of the
ineoming nucleotide. The incorporation efficieney is given by
kpo/J(d'
RESULTS
Overall Strueture of J(lenTaq Complexes in Presenee of an
Abasie Site-All J(len Taq crystals grew in the same space group
and very sim ilar cell parameters (supplemental Table Sl) as
those reported earlier (19, 20, 35, 42- 45). Thus, signifieant differenees in crystal packing fOt'ces aeting on the active site
should be negligible. Nevertheless, parts of the finger domain
differ in their mobility (as measured by temperature faetors)
between the different struetures. This is a consequenee of
the struetural differences, whieh were induced in the aetive site
c
o
FIGURE 1. Structure of KlenTaqF.blnaG! (blue) . A, template staeking assemb ly
of An + 2 and An + 3 in KlenTaq F_binory' Ihe abasie site analog Fn + 1 is loeated
extrahelically. B, the stiekand surfaee depietion highlights the template staeking arrangement. C, top view of the primer/template staeking arrangement.
0, hydrogen-bondi ng network of the amino acid side ehains Tyr-671 and
Glu-615 with the template strand. E, same arrangement as in A for the superimposed struetures of KlenTaq F_bln,ry (blue) and KlenTaqbin ary (brown) .
assembly by the primer/template eomplexes and respective
nucleotides that were investigated. In the following, the aetive
sites for the various eomplexes are shown and deseribed in
detail.
Strueture of J(lenTaq in Binary Complex with DNA Duplex
Containing an Abasie Site (J(lenTaqF_binct/.)-To address the
issue, ifthe overall eonformation ofthe enzyme-DNA complex
is affeeted by an abasie site prior to nucleotide binding, we crystallized J(lenTaq bound to primer/template duplex eontaining
an abasie site following a similar strategy as reported reeently
(19,20, 35,43,46) . The structure was solved by differenee Fourier techniques at aresolution of 1.9 A(slIppiemental Table SI) .
J(lenTaqF _bil1Ol'Y is very similar to one reported for J([enTaq in a
binary eomplex bound to undamaged DNA duplex (PDB
4KTQ; J([enTaqbinary) (35) as reflected by an r.m.s.d. for C"
atoms of 0.60 A (Fig. 1). However, remarkable struetural
ehanges were observed for the single-stranded 5' -overhang of
the DNA template, which is rotated around the helical axis.
Thereby, the lesion is flipped out of a developing DNA duplex
(Fig. 1A) . This eonformation is stabilizedby staeking of the
5'-upstream nucleobases of the template strand on the top of
the primer/template duplex (Fig. 1, Band C) and a distinet
hydrogen- bonding network of amino acid residues Glu-615
and Tyr-671 with the DNA template (Fig. ID). In contrast, in
J([enTaqbinary' the baekbone rotates 5' to the DNA duplex, redirecting the remainder ofthe single-stranded template out ofthe
DNA polymerase aetive site (Fig. IE) .
J([enTaq with ddGTP Opposite an Abasie Site (J([enTaqF_G_p
J([enTaqF_C_II)- 1'0 gain insights into the struetural basis for
the preferenee of adenosine over guanosine of J([enTaq in
bypassing an abasie site, we crystallized J([enTaq as a ternary
eomplex bound to a primer/tem plate duplex and ddGTP oppo-
14101
A
F
E
G
3'-end
closed
FIGURE 2. Structure of KlenTaqF _G_, (black) and KlenTaqF_G_1I (gray), A, stabilization network of ddGTP in KlenTaq F.G_" The amino acid side chains
Arg -587 and Tyr-671 are labeled . Gray, red, and black dashed lines indicate hydrogen -bonding interactions, cation -7T interaction, and distance (A),
respectively. B, the same as in A for KlenTaq F.G.II' C, top view ofthe nascent ba se pair opposite F (KlenTaq F_G.,). ln the front, the incoming ddGTP opposite
Tyr-671 is depicted. The first nucleobase pair of the primer/template terminus is shown as transparent. 0, same view as in C for KlenTaqF .G.II' E, the 0
helices from the superimposition of KlenTaq F.G_1I (gray), KlenTaq F_A(purpie), KlenTaqc_G (green), and KlenTaq op. n (gold) are highlighted . F, comparison of
the nascent base pairs of KlenTaqF .G.1I (gray), KlenTaqF.A (purpie), and KlenTaqc' G (green) . G, the incoming ddNTPs of KlenTaqF _G.1I (gray), KlenTaqF .A
(purpie), and KlenTaq c.G (green) and the respective 3' -prim er terminus are shown . The arrow indicates the displacement ofthe a-phosp hate regarding
to the 3' -primer terminus.
site the abasie site. Two structures were reproducibly found in
several crystallization trials and solved by difference Fourier
techniques at resolutions of 23- 2.4 Ä (KZenTaq,, _G_, and
KZenTaq F.G_II) (supplemental Tab te S I). The obtained structures show conformational heterogencity in the active site
region. In particular, we found two different orientations ofthe
incoming ddGTP (Fig. 2, A and B, and suppl emental Fig. S2, A
and B), However, the overall conformation ofthe two ddGTPtrapped structures is very similaI' to the ternary complex of
KlenTaq harboring ddATP opposite an abasie site (PDB 3LWL;
KlenTaq".A; r.m.s.d. for Ca of 0.42 Ä) (19), showing the same
remarkable structural changes as compared with the undamaged case (KlenTaq bound to an undamaged primer/template
duplex processing a ddGTP; PDB 1QSS; KlenTaqc_G ) (42) .
Thus, the conformation of the 0 helix leaves the active site
more open, similar to the one in Kien Taq F_A' and somehow
between the open (PDB 2KTQ; KlenTaqupel1) (35) and closed
(KlenTaqc _G) conformation s in the reported ternary complexes
(Fig. 2E). Like in KlenTaqF_A' we found that the abasi c site is
intrahelically located and that Tyr-67l is positioned opposite
the incoming ddGTP at the place that is L1sually occupied by the
templating nudeobase (Fig. 2A) . However, remarkable differences between KlenTaqF.A and KlenTaqF_G., appear in the
positioning and interaction pattern of the incoming triphosphates (Fig. 2A and supplemel1tal Fig. S3, A and B). In
KlenTaqF_G_JI Tyr-671 stabilizes ddGTP via hydrogen bonds
between the Tyr-67l hydroxyl group and the NI of guanine,
whereas in KlenTaqF_A' the N3 of adenine interacts with Tyl'67l (Fig. 2A and supp lem cn tal Fig. S3A ). Furthermore, an
important stabilizing factor of the incoming nudeotide is Arg587. In KlenTaqF_A' Arg-587 forms a hydrogen bond to the N7
of adenine (sLIppIementa l Fig. S3A ). However, in KlenTaq F_G_JI
14102
ddGTP is stabilized by cation-7T interaction with Arg-587.
Because this type of interaction strongly depends on the distri bution of the electron density in the aromatic ring system, there
is in the case of purines a d eal' preference for arginine to position itsclf bclow 01' above the imidazole five-atom ring system
(47). Indeed, this is observed in KlenTaq F _ G ~ 1 for Arg-587 (Fig.
2, A and C) . The binding arrangement of ddGTP causes a misalignment of the a-phosphate, resulting in a large distance of
the a -phosphate to the 3' -primel' terminus (8.7 Ä) . Hence,
KlenTaq F_G_1 might represent an initial binding event of the
incoming triphosphate (Fig. 2A) , In comparison, the guanine
base in KlenTaqF_G.1I is rotated around the N-glycosidic bond,
and the N7 of guanine points to the hydroxyl group ofTyr-67l
(Fig. 2, Band D , and suppl emental Fig. S2B ). Thereby, the sugar
moiety is realigned, resulting in a shortened distance between the
a -phosphate and 3'-primer terminus of about 6.9 Ä. However, the
reorientation of the nudeobase positions the six-membered heterocyde above Arg-587 (Fig. 2, Band D), a tess tiwored arrangement observed only in rare cases (47). In summary, the distance
between 3' -OH lInd the a -phosphate increases in the following
direction KlenTaq F_A < KlenTaqF_G_1I < KlenTaqF_G _, that are all
longer than the one observed in the canonical case (Fig. 2G).
Pyrimidines Opposite an Abasie Site- Pyrimidine nudeotides are incorporated opposite an abasie site by KlenTaq with
pOOl' efficiencies (19) , Furthermore, we demonstrated that the
purine preference could be switched to pyrimidine preference
by a single site-directed mutagenesis of Tyr-67l to Trp (19) .
T his suggests that the low incorporation efficiencies of pyrim idines relies on a specific interaction with this amino acid resi due. To get structural insights, we crystallized KlenTaq in the
presence of a primer/template duplex and ddTTP, The structure was solved by difference Fourier techniques at aresolution
A
3'-end
FIGURE 3. Structure of KlenTaqF _T (yellow).A, stabilization network ofddTIP. The amino acid side chains Arg-s87, Phe-667, and Tyr-671 are labeled. Gray and
black dashed fines indicate hydrogen-bonding interactions and distance (A), respectively. B, the 0 helices from the superimposition of KlenTaqF_T (ye/law),
KlenTaqF_A (purpie), KlenTaqA_T (magenta), and KlenTaqblnart (gold) are highlighted. C, the incoming ddNTPs of KlenTaq F_T (ye/low), KlenTaqF _A (purpie), and
KlenTaqA_T (berry) and the respective 3' -primer terminus are shown. The arrow indicates the displacement of the a-phosphate regarding the 3' -primer
terminus.
A
c
FIGURE 4. Structure of KlenTaqF_NI (orange). A, stabilization network of dNITP. The amino acid side chains Arg-s87, Phe-667, and Tyr-671 are labeled. Black
dashed lines indicate distance (A). B, the 0 helices from the superimposition of KlenTaq F_NI (cyan), KlenTaqF_A (purpie), Kien Taqc_G(green), and KlenTaqblna,y (gold)
are highlighted. C, the incoming ddNTPs of KlenTaqF_NI (cyan) and KlenTaq c_G (green), the respective Mg 2+ ions, and the respective catalytically relevant
residues Asp-610, Tyr-611, and Asp-78s are shown.
of 1.8 A (KIen TaqF_T) (suppl emental Table 51). Although
kinetic studies eluddated that thymidine incorporation opposite an abasie site is unfavorable, we observed abound thymidine triphosphate in the active site (Fig. 3A and suppl emcntal
Fig. S2C). Once more we obtained a semiclosed enzyme conformation as can be seen from the superimposition of this
structure with KlenTaq A_T (PDB lQTMi processing a canonical base pair with an incoming ddTTP) (42), KlenTaq F_AI and
KlenTaqopell (Fig. 3B). The 0 helix does not pack against the
nascent base pair, a110wing Tyr-671 to position itself between
the incoming ddTTP and the abasie site as is observed for Tyr671 in KlenTaqF_A (Fig. 3A and supplem cnta l Fig. S3A ). The
ddTTP is stabilized via H -bonding between the NI of thymidine and the hydroxyl group of Tyr-671 as we11 as 1T-stacking intcractions to Phc-667. In contrast to KlenTaq F_A in
KlenTaqF_TI Arg-587 is released from the stabilization pattern
of the incoming triphosphate, as observed for the purinetrap'p ed structures, while still interacting with the phosphate
backbone of the 3' -primer terminus. The resulting thymidine
tyrosine pair is accommodated in the active site in a way that
results in a misalignment of the O'-phosphate and thereby positions the sugar moiety on top of the 3' -primer terminus (Fig.
3C).
The same approach as described before was used to co-crysta11ize Klen Taq in the presence ofa primer/template duplex and
ddCTP (K{enTaq F_ billo"y_II)' Howcvcr, in a11 conductcd
approaches, only the formations of binary complexes of
KlenTaq and the prim er/te mplate were obtained. These structures are very similar to the binary structure KlenTaqF_bllla,'Y
(supplemental Fig. S4).
Nucleotide Analog dNITP Ineorporation Opposite an Abasie
Site- Base stacking capability was suggested to playa dedsive
role in influendng the incorporation emciency opposite an abasie site (17, 31, 48 - 50). Intriguingly, nucleotides that bear
nudeobase surrogates with strong staeking but laeking hydrogen-bonding eapability such as dNITP (Fig. 5A) are incorporated opposite abasie sites with higher efficieney as their natural
countcrparts. Thcrcforc, non-natural nuclcotidcs such as
dNITP were used to investigate abasie site bypass as a probe for
staeking. To get insights into how dNITP is proeessed opposite
an abasie site by KlenTaq, we performed soaking experiments
with crystals of a binary eomplex of KlenTaq in the presenee of
an abasie site and dNITP. The strueture was solved by difference Fourier techniques at aresolution of 1.9 A (supp lcm enta l
Tab le 5J and supp lemental Fig. S2D). In the presence ofdNITP,
the enzyme is aeeumulated in a dosed and produetive eomplex,
vcry similar to reportcd eanonieal cases (e.g. PDB IQTMi
K{enTaqA _Ti r.m.s.d. for Ca of 0.53 A) and in eontrast to
K{enTaq F_A (Fig. 4·, A and B) . In disparity with the structures of
KlenTaq complexed with natural nucleotides opposite an abasie site, Tyr-67I is released from its stacking interaetion to the
tcmplate strand and providcs spacc for thc incoming dNITP
(Fig. 4A) . The hydrophobie nucleotide analog perfectly stacks
on the developing DNA duplex, resulting in a proper alignment
of the O'-phosphate and recruitment of two catalytiea11y essential magnesium ions (Fig. 4C and supplem ental Fig. S5).
Furthermore, kinetics show that dNIMP is incorporated
more efficiently than any other natural nucleotide by KlenTaq
opposite an abasie site resulting in the fo11owing order of incorporation cffidcncics (kpo/Kd ): NI » A > G » T > C (Fig. SC
and suppl emen tal Fig. S6A ). Due to the decrease in Kd and
simultaneous increase in kpoll dNIMP shows a 22-fold increase
in ineorporation effideney as eompared with dAMP. Interestingly, if KlenTaq mutant Y671 W is used, we obtained the following incorporation emdency (kpo/Kd ) order: NI > C > T »
A > G (Fig. 5D and slIpp iementa l Fig. S6B ). Therefore, one can
assllme that Tyr-671 does not inflllence the ineorporation of
14103
B
~d NTP
5'- ... TAGG
3'- ... ATCC FACT
c
KTQwt
N:-
NI
time :
A
T
G
C
__________
24 nt
23 nt
N/F-dNTP
K. [~M]
T-dATP'
F-dATP'
15.3±4.7
516±44
149±35 '19.7±1.1
2.73±0.23
7,80±1.12
F-dN ITP
k"", [s" x1 0'2]
D
k.JK. [M"'s" x1 0"']
3373
1.83
39.5
KTQ Y671W
NI
N: time :
A
G
T
C
__________
24 nt
23 nt
N/F-d NTP
T-dATP'
F-dCTP'
F-dATP'
F-dNITP
363±104
845±161
514±182
97.8±24.4
91.6±15.2
0.92±0.12
0.10±0.02
0.24±0,02
25.2
0.11
0.02
0.25
FIGURE 5. Nucleotide incorporation opposite an abasie site analog F.
A, structure of nucleotide analog dNITP. 8, partial primer/template sequence
used in primer extension experiments. C. single nucleotide (nt) incorporation
of KlenTaq wild type opposite F for 1, 10, or 60 min, respectively. The respective dNTP is indicated. Transient kinetic analysis of nucleotide incorporation
opposite T/F by KlenTaq wild type was performed. " kinetic data were first
reported in Ref. 19.0, single nucleotide incorporation of KlenTaq Y671W
mutant opposite F for 10,60, or 120 min, respectively. The respective dNTP is
indicated. Transient kinetic analysis was performed as in (for KlenTaq Y671W.
dNIMP, whereas it clearly direets the ineorporation of the natural nucleotides.
DISCUSSION
General Aspeets- Although the bypass of a non-instruetive
lesion by DNA polymerases is extensively studied, the meehanisms are not fully understood yet. Besides initial binding ofthe
nucleotide and the ehemieal reaction, noneovalent intermediates with energetie minima are erucial for the reaetion pathway.
These intermediates are believed to be responsible tor preselecti on of nucleotides and represent kinetic checkpoints explaining the overall aeeuraey and fldelity ofthese enzymes (51 -53).
The present structural study provides further in sights into how
KlenTaq DNA polymerase, a member of sequence family A
DNA polymerases, is able to bypass abasie sites (Table 1). Independent of the incoming natural nucleotide opposite the abasie
site, the obtained ternary eomplexes show two notieeable alterations as eompared with the eanonical eases. Firstly, it is always
observed that Tyr-671 is placed opposite the incoming nucleoti des, and seeondly, the enzyme adopts a semiclosed eonformation . Sueh a conformation was also associated with mismateh
14104
incorporation (54) . However, FRET studies ofKlenow fragment
DNA polymerase in the presenee of mismatches indicate that
along with the open form, the closed state is also partially oeeupied (55). Because erystallographie data only provide single
snapshots, it is likely that in solution, there is also a distribution
of the populations between the open and closed state even in
the presenee of an abasie site. Similarly to nucleotide discrimination between matehed and mismatched nucleotides, DNA
polymerases might also undergo several eonformational
changes as prescleetion steps bcfore preeeding thc phosphoryl
transfer in the presenee of an abasie site. Therefore, the
observed conformations might represent various local energy
minima along the reaetion coordinate. Along these lines, computer simulations of the fidelity ofT7 DNA polymerase support
the hypothesis that the formation of mismatches mayaiso
occur with high energy barriers from a partially open protein
conformation (56) . In general, there is an ongoing discussion
whether the chemical step demands similar eonformational
states processing a correet 01' incorrect nucleotide (52-54, 57).
However, the ineoming nucleotides opposite an abasie site in
the herein depicted structures are not properly aligned for the
phosphoryl transfer. This suggests either that arearrangement
to the dosed conformation has to oecur or that the incorporation step proceeds from the semiclosed conformation after
appropriate eonformational ehanges of the nucleotide. Both
scenarios support the observed overall poor incorporation etliciencies opposite abasie sites (19) .
Structure of Binary Complex Containing Abasie Site and
KlenTaq- In the binary eomplex of KlenTaq bound to an abasie site, we observed the 5' -upstream template strand staeking
on the primer/template duplex. Nucleotide binding forees the
release of the template strand from its staeking arrangement
and triggers a remarkable rearrangement of the bound DNA
template rather than a reorganization of the enzyme (supplemental rig. S7). Furthermore, the ability to resolve the staeking
arrangement might be dependent on the intrinsie properties of
the respective nudeotide, such as the stacking and solvation
ability of the nucleobase. It remains to be clucidated whether
this arrangement is sequenee-specific.
Nucleotide Seleetion Opposite Abasie Sites of KlenTaq,
Adenosines versus Guanosines- The struetural da ta offer valuable clues how nucleotide seleetion is performed opposite a
non-instructive lesion and moreover support the previous
kinetie analysis.of abasie site bypass by Klen Taq (19) . In the ease
of an ineoming guanosine, we obtained several crystal structures showing the ineoming triphosphate in two different orientations. This heterogeneity was not observed in the presenee
of the other nucleotides. In KlenTaqr _G_, and KlenTaqr_(; _II'
Tyr-671 is filling the spaee ofthe vaeant nucleobase of the abasic site and is located opposite the incoming ddGTP and
thereby roughly mimies the geometry of a naseent nucleobase
pair. Of note, such a selection of purines mediated by Tyr-671 is
also observed for KlenTaqF_A' However, in KlenTaqF_G_ 1 and
KlenTaqF _G_ Ii' the interaetion with Tyr-671 causes misalignment of the a-phosphate resulting in enlarged distanees to the
3' -primer terminus (Figs. 2F and 6, A and B). The different
steric eonstraints of the purine struetures aeeount for the preference of adenosine over guanosine. In detail, the exocydic
TABLE 1
Summary of KlenTaq structures bound to an abasie site analog F
F - A
F - T
F - G-I
F - G-II
Y67 1 positioned
opposite ddATP
Y67 1 positioned
opposite ddGTP
Y67 1 pos itioned
opposite ddGTP
Y671 positioned
opposite ddTTP
H-bond N3-ddATP OH-Y671
H-bond N1-ddGTP OH-Y671
H-bond N7-ddGTP OH -Y67 1
H-bond N1 -ddTTP OH-Y67 1
F - binary
F - NI
Y671 is released from
stacking in teraction to
the template strand
Y671 is released from
stacking interaction to
the temp late stra nd
R587 stabilizes the
R587 stabilizes the
R587 stab ilizes the
R587 stabilizes the
R587 sta bilizes the
R587 stabilizes the
phosphate-backbone of phosphate-backbone of phosphate-backbone of phosphate-backbone of phosphate-backbone of phosphate-backbone of
the 3'-primer term inus the 3'-primer terminus the 3'-primer term inus the 3'-primer terminus the 3'-primer terminus the 3'-primer terminus
H-bond N7-ddATP R587
cation-TI interaction
between 5-cycl ic ring
and R587
cation-TI interaction
between 6-cycl ic ring
and R587
F located intra helically
F located intrahelica lly
F loca ted intra helica lly
F located intra helica lly
1 Mg'+ ion is complexed by ddATP
Pa - 3'-primer
termi nus: 5.9 A
a-P - 3'-primer
terminus: 8.7 A
a-P - 3'-primer
terminus: 6.9 A
1 Mg'+ ion is complexed by ddTTP
a-P - 3'-p rimer
terminus: 7.5 A
PDB-ID: 3LWL
PDB-ID: 3RR8
PDB-ID: 3RRG
PDB-ID: 3RRH
F located extrahelically F located intrahelica lly
2 Mg'+ io n are complexed by ddN ITP
a-P - 3'-primer
terminus: 3.8 A
PDB-ID: 3RR7
PDB-ID: 3T3F
FIGURE 6. Geometry fit to active site. A, aetive site assembly in the presenee of an abasie site analog F: The naseent base pair of Tyr-671 and ddGTP
(KlenTaqF_G_I) is depieted_The surfaee ofthe surrounding aetive site residues is shown in gray. B, sa me as in A for KlenTaq F_G_II' C, aetive site assembly in the ease
of an undamaged template showing the na seent base pair of dC and ddGTP (KlenTaqc_G)' 0 , same as in A for KlenTaqF_T' E, same as in A for KlenTaq F_A.F, same
as in ( for KlenTaqF_NI showing the na seent base pair of Fand dNITP. A- F, the arrows indieate the positioning of the ,,-phosphate and the 3' -primer terminus,
respeetively.
C 2 -NH 2 group of guanine prevents the same arrangement of
the nudeotide, as was fuund for adenosine (Table 1) . Oue tu the
steric res trietion of the active site, the assembly of the sterically
more dem anding guanosine results in an alignm ent with an
enlarged distance between the ex-phosphate and the 3' -prim er
terminus in comparison with adenosine. Interestingly, the
complex showing the st ro nger cation-7T interaction between
the Arg-587 and the five- membered ring (Klen TaqF _G_') rcsults
in an arrangem ent of t he ddGTP obviating a possible attack at
the ex-phosphate. T he doser orientation of the nudeotide
toward the 3' -primer terminus suggests that the structure of
KlenTaqF_G_ II for ming a less st able cation-7T interaction (47)
may represent one step ahead on the reaction coordinate in
comparison with K[enTaqr_G_" In condusion, we find that the
distance between the ex- phosphates and the 3' -primer terminus
(see Table 1, 8.7- 6.9 A fo r guan osines versus 5_9 A for adeno-
14105
sine) corrclates well with the measured incorporation effidendes. This indicates that active misalignment of the incoming
guanosines governs their unfavorable processing by KlenTaq in
comparison with adenosines.
Nucleotide Selection Opposite Abasie Sites by KlenTaq,
Purines versus Pyrimidines- To shine light on the iricorporation mechanisms of the less favored pyrimidines, we investigated a structure bearing an incoming ddTTP opposite an abasic site. The DNA polymerase interacts with the incoming
nucleotide by hydrogen bonding of Tyr-671 with the nucleobase. This results in a nucleoside triphosphate conformation
where the sugar moiety is positioned above the 3' -prim er terminus instead of the a-phosphate (Figs. 3C and 6D). In this
scenario, all components of the active site are assembled and
organized in a topological and geometrical arrangement that
does not allow the enzyme to proceed with the chemical step,
explaining the very low incorporation efficiency. In contrast to
purines, ddTTP shows low B-factors (supplemental Table SI),
whieh indieates that it is well stabilized opposite Tyr-671. Thus,
the tightly bound thymidine probably stalls the DNA polymerase in a "nonproductive" complex by active misalignment,
whereas the purine nucleobases enhance the geometrie fit to
the active site, resulting in an arrangement with shortened distances between the a-phosphate and the 3' -primer terminus as
compared with the pyrimidine base (Figs. 2G and 6, Band E).
Nucleobase Staeking and Abasie Site Bypass- Superior stacking and solvation properties of adenine have been discussed as a
driving force behind adenine selection opposite abasie sites (17,
29 -31). The nucleotide dNITP contains a nucleobase surrogate lacking hydrogen-bonding capability but increased stacking ability in comparison with thcir structural congener
purines. These properties build the basis for the motivation to
employ the nucleotide in abasic site bypass (17, 31,48 - 50). We
find, in contrast to the results obtained with nucleotides containing the natural nucleobases, that bin ding of the nucleotide
analog dNITP readily allows the enzyme to change'its active site
conformation in a closed, productive complex that is similar to
those found for undamaged DNA substrates. The accumulation of the enzyme in a productive conformation is clearly supported by the kinetic data, whieh show a signifieant increase in
incorporation effidency of dNIMP as compared with dAMP
opposite the abasic site. Furthermore, the drcumstance that
dNlTP is processed with high er effidency than the favored
nucleotides of either KlenTaq wild type or KlenTaq mutant
Y671 W shows that Tyr-671 apparently lost its selection criteria
beeause both the dNlTP and the Tyr-671 residues are not eompatible with the geometrie constraints of the active site. Our
results suggest that due to its increased stacking ability, the
nucleobase surrogate imposes active site conformations that
differ signifieantly from those induced by the natural nucleobases. Consequently, results that are obtained by usage of
dNITP instead of the natural nucleotides should be interpreted
with eaution toward their significanee in abasie site bypass by
natural nucleotides. It is noteworthy that dNITP is known as a
chain terminator (59, 60) as well as universal base (61) and has
shown no preference for ineorporation opposite one of the four
natural nucleobases. Again, enforeement of aberrant enzyme
14106
and DNA conformations due to the strong stacking ability of
dNITP might be the origin of the observed properties.
Taken together, the present struetural study of KlenTaq in
complex with different nucleoside triphosphates opposite an
abasie site reveals that natural nucleotides are bound, due to
interaetion with Tyr-671 and the geometrie confines of the
aetive site, in positions that require additional conformational
ehanges for proper alignment to fadlitate the ehemical step.
The degree of misalignment, measured as distanee of the
a -phosphate to the 3' -primer terminus, paralleIs the order of
ineorporation effidency of KlenTaq opposite an abasie site.
These conformational changes might be aceompanied by barriers of varied energy explaining the observed decline in nucleotide incorporation efficiency from A and G to T and C as well
as the strong block of the abasic site lesion. The resulting pausing of DNA synthesis might allow the DNA polymerase to be
replaeed by repair systems that use the si ster strand for errorfree repair.
The human DNA polymerase () and )' are members of the
sequence family A as KlenTaq . Interestingly, the human DNA
polymerase () also obeys the A-rule (18), and its intrinsieally
error-prone abasie site bypass might eontribute to somatie
hypermutation of Ig genes (62) . Furthermore, the role of Tyr671 in nucleotide selection opposite an abasie site is highlighted. Tyr-671 is highly eonserved in family A DNA polymerases (19, 34) and is known to be involved in the
discrimination process between canonieal and noncanonical
base pairs (63- 65). Along these lines, Leob and co -workers (63)
demonstrated that Tyr-671 is essential for maintaining Taq
DNA polymerase I activity. Mutations at that position are
hardly tolerated and compromise activity with the exception of
phenylalanine. Nevertheless, mutations of the homolog residue
Tyr-766 of the Klenow fragment from E. eoli DNA polymerase
I to serine or alanine result in significant decrease in fidelity
(64) . Interestingly, mutation of the corresponding Tyr-955 in
human DNA polymerase )' has been attributed to progressive
external ophthalmoplegia, stressing its importance in accurate
function ofDNA polymerases (58, 66) .
Acknowledgments- We thank the beamline stajf oJ the Swiss Light
Source (SLS) and Konstanz Research School Chemical Biology Jor
support.
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