Dna polymerase selectivity: Sugar interactions monitored with high-fidelity nucleotides

Article abstract of DOI:10.1002/1521-3773(20011001)40:19<3693::AID-ANIE3693>3.0.CO;2-O

Do enzyme - sugar interactions contribute to DNA-polymerase fidelity? With the novel chemical probes 1 (R=CH₃)₂), which are used by a DNA polymerase more selectively than the natural counterpart (R=H), insights are obtained into the

Full text of DOI:10.1002/1521-3773(20011001)40:19<3693::AID-ANIE3693>3.0.CO;2-O

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Nanayakkara, C. Gonzalez, M. Challacombe, P. M. W. Gill, B. G.
Johnson, W. Chen, M. W. Wong, J. L. Andres, M. Head-Gordon, E. S.
Replogle, J. A. Pople, Gaussian, Inc., Pittsburgh, PA, 1998. Fluorine:
basis set 6-31‡G(d,p), gold: relativistically corrected 6s5p3d basis set
and pseudopotentials for 60 core electrons (Institut für Theoretische
Chemie, Universität Stuttgart).
impact of DNA polymerase interactions with the 2'-deoxy-
ribose moiety and the participation of these interactions in
processes which contribute to fidelity. Crystal structures of
DNA polymerases together with enzyme mutation studies
suggest that the sugar moiety of the incoming triphosphate is
fully embedded in the nucleotide binding pocket and under-
goes essential interactions with the enzyme.[1a, 2] Here we
report a functional strategy to monitor steric constraints in
DNA polymerases that act on the sugar moiety of an incoming
nucleoside triphosphate within the nucleotide binding pocket.
We found that novel modified nucleotide probes are sub-
strates for a DNA polymerase with significantly increased
selectivity compared to their natural counterpart. Through
use of these high-fidelity nucleotides in functional investiga-
tions we could show that enzyme ± sugar interactions are
involved in DNA-polymerase fidelity mechanisms.
[51] MP2 calculation with PDZ basis set and effective core potentials for
the heavy elements: K. O. Chiste, D. A. Dixon, D. Mc Lemore, W. W.
Wilson, J. A. Sheekey, J. A. Boatz, J. Fluorine Chem. 2000, 101, 151 ±
153.
[52] R. J. Gillespie, K. C. Moss, J. Chem. Soc. A 1966, 1170.
[53] H. Schumann, W. Genthe, E. Hahn, M.-B. Hossein, D. van der Helm,
J. Organomet. Chem. 1986, 28, 2561 ± 2567.
[54] G. Sheldrick, Program for Crystal Structure Solution, Universität
Göttingen, 1986.
[55] G. Sheldrick, Program for Crystal Structure Refinement, Universität
Göttingen, 1993.
In order to sense interactions of DNA polymerases with the
sugar moiety of incoming triphosphates we introduced alkyl
labels at the 4'-position in the 2'-deoxyribose moiety in such a
way that they do not interfere with hydrogen bonding,
nucleobase pairing, and stacking. We designed steric probes
1a ± d by substituting the hydrogen atom at the 4'-position of
thymidine triphosphate (TTP) with alkyl groups of different
size (Scheme 1).
DNA Polymerase Selectivity: Sugar
Interactions Monitored with High-Fidelity
Nucleotides**
T
T
Daniel Summerer and Andreas Marx*
PPPO
H
PPPO
R
O
O
1a:
1b:
1c:
1d:
R = CH3
R = CH2CH3
R = CH(CH3)2
R = CH2CH(CH3)2
The essential prerequisite of any organism is to keep its
genome intact and to accurately duplicate it before cell
division. All DNA synthesis required for DNA repair,
recombination, and replication depends on the ability of
DNA polymerases to recognize the template and correctly
insert the complementary nucleotide. In a current model,
fidelity is achieved by the ability of DNA polymerases to edit
nucleobase pair shape and size.[1] This model is supported by
crystal structures of DNA polymerases that suggest the
formation of nucleotide binding pockets, which exclusively
accommodate Watson ± Crick base pairs.[2] Nevertheless,
structural data of DNA polymerases complexed with the
DNA substrates and a noncanonical triphosphate, which
would be very helpful for gaining insights into the causes of
error-prone DNA synthesis, have at present not been
reported. Thus, functional studies of DNA polymerases with
nucleotide analogues have been shown to be extremely
valuable.[1] Since most functional studies have focused on
nucleobase recognition processes,[1] little is known about the
OH
TTP
OH
1a-d
O
P
O
O
PPP:
T: thymine
O
O
O
P
P
O
O
O
Scheme 1. Thymidine-5'-triphosphate (TTP) and the steric probes 1a ± d.
The synthesis of nucleosides 4a and 4b with different
synthetic strategies (see Scheme 2) has been reported pre-
viously.[3, 4] However, these methods are not suitable for the
synthesis of all the targeted compounds and the formation of
undesired by-products was observed in the synthesis of 4a.[3]
Thus, we developed a more suitable access to the target
compounds. Our synthesis started with the known alcohol 2,
which is easily accessible as described recently.[5] Compound 2
was converted into the methylated thymidine 4a by func-
tional-group interconversions (Scheme 2). Alkylated nucleo-
sides 4b ± d were synthesized from known compounds[5] by
employing Wittig reaction, desilylation, and subsequent
reduction of the aliphatic double bond. Next, nucleosides
4a ± d were converted into the desired triphosphates 1a ± d
following standard procedures.[6] In order to gain insights into
potential effects of the modifications on the sugar conforma-
tion we performed conformation analysis based on coupling
constants deduced from the 1H NMR data by employing
described methods.[7] We found only small differences in 1a ±
d compared to natural TTP, which indicates that similar sugar
conformations are present in solution (see Supporting In-
formation).
[*] Dr. A. Marx, Dipl.-Chem. D. Summerer
Â
Kekule-Institut für Organische Chemie und Biochemie
Universität Bonn
Gerhard-Domagk-Strasse 1, 53121 Bonn (Germany)
Fax : (‡ 49)228-73-5388
E-mail: a.marx@uni-bonn.de
[**] This work was supported by a grant from the Deutsche Forschungs-
gemeinschaft. We thank Professor Dr. Michael Famulok for his
continuing support.
Supporting information for this article is available on the WWW under
http://www.angewandte.com or from the author.
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thymidine analogue adjacent at the primer 3'-end,[9]
to monitor polymerase function. Figure 1A shows
the pattern of insertion of thymidine-5'-triphosphate
(TTP) and 1a ± d catalyzed by KF . The results
obtained reveal that 1a ± d are functionally compe-
tent, although with varied efficiencies.
In order to quantify these observations we deter-
mined the insertion efficiencies (vmax/KM; vmax ˆ the
maximum rate of the enzyme reaction, KM ˆ the
Michaelis constant) of the thymidine analogues by
investigation of single-nucleotide insertion at vari-
ous nucleotide concentrations under single complet-
ed hit and steady-state conditions as recently de-
scribed.[8, 10±12] The quantitative analyses revealed
that KF incorporates 1a and 1b with virtually the
same efficiency as unmodified TTP (Table 1). Thus,
T
T
T
TBDPSO
HO
TBDPSO
I
HO
O
O
O
b), c)
f), g)
i), j)
a)
H3C
OTBS
2
OTBS
3
OH
4a
d)
T
T
T
T
TBDPSO
HO
HO
HO
O
O
e)
h)
OTBS
6
OH
4b
T
TBDPSO
O
O
OTBS
5
T
T
TBDPSO
TBDPSO
O
O
OH
k)
OTBS
7
4d
T
TBDPSO
O
Table 1. Steady-state analyses for canonical nucleoside triphos-
phate insertion. The data presented are averages of duplicate or
triplicate experiments. Further experimental details are de-
scribed in Supporting Information.
O
O
O
m), n)
l)
OH
4c
OTBS
OTBS
9
8
Nucleoside
triphosphate
KM
[mm]
vmax
[min  10
vmax/KM
1
3
1
]
[m 1 min
]
O
O
P
O
T
T
TTP
1a
1b
1c
1d
0.11 Æ 0.05
20 Æ 3
58 Æ 4
29 Æ 5
85 Æ 5
76 Æ 1
180000
150000
140000
1800
O
P
O
O
P
O
R
HO
R
O
O
0.40 Æ 0.01
0.21 Æ 0.01
47 Æ 4
o)
O
O
O
OH
1a-d
OH
4a-d
241 Æ 22
320
Scheme 2. Syntheses of 1a ± d. a) Ph3P, I2, imidazole, C6H6, 508C, 85%; b) Pd/C, H2,
EtOH, EtOAc, NEt3; c) TBAF, THF, 79% (two steps); d) oxidation;[5] e) CH3PPh3Br,
nBuLi, THF, 78 to 258C, 99%; f) TBAF, THF; g) Pd/C, H2, CH3OH, 88% (two steps);
h) (CH3)2CHPPh3I, nBuLi, THF, 78 to 258C, 83%; i) TBAF, THF; j) Pd/C, H2,
CH3OH, 94% (two steps); k) alkylation and oxidation;[5] l) CH3PPh3Br, tBuOK, THF,
258C, 91%; m) TBAF, THF; n) Pd/C, H2, CH3OH, 84% (two steps); o) POCl3, 1,8-
bis(dimethylamino)naphthalene, (CH3O)3PO, 08C, then (nBu3NH)2H2P2O7, nBu3N,
DMF, then 0.1m aqueous (Et3NH)HCO3, 23 ± 68%. TBDPS ˆ tert-butyldiphenylsilyl,
TBS ˆ tert-butyldimethylsilyl, TBAF ˆ tetrabutylammonium fluoride, THF ˆ tetra-
hydrofuran, DMF ˆ N,N-dimethylformamide.
it appears that 4'-methyl and -ethyl groups are nicely
accommodated within the nucleotide binding pocket
of KF . Further increase in the bulk of the probes
caused a marked decrease in the insertion efficiency
of 1c and 1d, presumably due to a steric clash since
the modifications do not cause significant perturba-
tion of the sugar pucker conformation.
We tested the effect of probes 1a ± d on the Klenow
fragment (KF ) of E. coli DNA polymerase I (exo -mutant),
an enzyme extensively used as a model for investigations into
intrinsic DNA-polymerase mechanisms and function.[1] We
used a gel-based single nucleotide insertion assay,[8] where an
adenine in the template strand codes for the insertion of a
Since the ratio of nucleotide-insertion efficiencies opposite
a complementary to a noncomplementary nucleobase is a
measurement of DNA-polymerase fidelity,[8, 10, 11] we next
investigated the ability of KF to catalyze non-Watson ± Crick
nucleobase pair formation. We focused on 1a and 1b, since
only these are inserted as efficiently as TTP by KF in
Figure 1. Nucleoside triphosphate insertion catalyzed by KF . A) Insertion encoded by adenine (A) in the template strand. Conditions: Primer/template
complex (50 nm), KF (2 nm), 378C, 5 min. B) Nucleoside triphosphate insertion encoded by guanine (G) in the template strand. Conditions: Primer/
template complex (50 nm), KF (4 nm), 378C, 5 min. C) Mismatch extension. Conditions: Primer/template complex (50 nm), KF (2 nm), 378C, 20 min. In
each case, the nucleotide concentrations [mm] are shown in the figure. dCTP ˆ 2'-deoxycytidine-5'-triphosphate, T*TP ˆ TTP analogues. Further
experimental details and the DNA sequences employed are described in the Supporting Information.
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canonical base pair formation and thus they should be ideally
suited for use in the monitoring of differential enzyme
interactions between insertion and misinsertion events. As-
says of nucleotide incorporation opposite G, T, and C revealed
that KF misinserts TTP and 1a, b opposite G with the
highest efficiency.[9, 13] Strikingly, as is already apparent from
the results shown in Figure 1B, 1a and 1b exhibit dramatically
decreased misinsertion efficiency. Steady-state kinetic analy-
ses revealed an approximately 100-fold decrease in misinser-
tion efficiency using the 4'-alkylated probes 1a, b compared
to TTP (Table 2).
In conclusion, the selectivity of nucleotide insertion by a
DNA polymerase can be significantly increased by modified
sugar moieties. Our results strongly implicate the involvement
of differential DNA-polymerase interactions with the sugar in
processes that contribute to the fidelity of DNA synthesis.
Furthermore, our studies provide a new functional and
general method to monitor steric constraints in nucleotide
binding pockets of DNA polymerases. Further analyses with
such steric probes, both at the functional and structural level
should reveal more insights into mechanisms of DNA
polymerase selectivity.
Received: May 14, 2001 [Z17101]
Table 2. Steady-state analyses for nucleoside triphosphate misinsertion
and mismatch extension. The data presented are averages of duplicate or
triplicate experiments. Further experimental details are described in
Supporting Information.
[1] Recent reviews and commentaries: a) T. A. Kunkel, K. Bebenek,
Annu. Rev. Biochem. 2000, 69, 497 ± 529; b) E. T. Kool, J. C. Morales,
K. M. Guckian, Angew. Chem. 2000, 112, 1046 ± 1068; Angew. Chem.
Int. Ed. 2000, 39, 991 ± 1009; c) T. A. Kunkel, S. H. Wilson, Nat. Struct.
Biol. 1998, 5, 95 ± 99; d) U. Diederichsen, Angew. Chem. 1998, 110,
1745 ± 1747; Angew. Chem. Int. Ed. 1998, 37, 1655 ± 1657; e) M. F.
Goodman, Proc. Natl. Acad. Sci. USA 1997, 94, 10493 ± 10495.
Nucleoside
triphosphate
KM
[mm]
vmax
[min  10
vmax/KM
1
3
1
]
[m 1 min
]
misinsertion:
TTP
1a
1b
22 Æ 0.2
16 Æ 1
730
5
7
Â
[2] a) S. Doublie, S. Tabor, A. M. Long, C. C. Richardson, T. Ellenberger,
65 Æ 5
0.34 Æ 0.02
1.7 Æ 0.1
Nature 1998, 391, 251 ± 258; b) Y. Li, S. Korolev, G. Waksman, EMBO
J. 1998, 17, 7514 ± 7525; c) J. R. Kiefer, C. Mao, J. C. Braman, L. S.
Beese, Nature 1998, 391, 304 ± 307; d) H. F. Huang, R. Chopra, G. L.
Verdine, S. C. Harrison, Science 1998, 282, 1669 ± 1675; e) H. Pelletier,
M. R. Sawaya, A. Kumar, S. H. Wilson, J. Kraut, Science 1994, 264,
1891 ± 1903.
228 Æ 5
mismatch extension:
TTP
1a
1b
19 Æ 1
80 Æ 17
40 Æ 5
36 Æ 2
1900
40
100
2.9 Æ 0.1
4.0 Æ 0.3
[3] C. O.-Yang, W. Kurz, E. M. Eugui, M. J. McRoberts, J. P. H. Verhey-
den, L. J. Kurz, K. A. M. Walker, Tetrahedron Lett. 1992, 33, 41 ± 44.
[4] I. Sugimoto, S. Shuto, S. Mori, S. Shigeta, A. Matsuda, Bioorg. Med.
Chem. Lett. 1999, 9, 385 ± 388.
[5] A. Marx, P. Erdmann, M. Senn, S. Körner, T. Jungo, M. Petretta, P.
Imwinkelried, A. Dussy, K. J. Kulicke, L. Macko, M. Zehnder, B.
Giese, Helv. Chim. Acta 1996, 79, 1980 ± 1994.
Further experiments also showed that the analogues 1a and
1b were not as well inserted opposite T or C as unmodified
TTP was (see Supporting Information). These results clearly
show that nucleotide-insertion selectivity is increased by
substitution of the hydrogen atom at the 4'-position of the
sugar with bulkier alkyl groups.
Â
[6] T. Kovacs, L. Ötvös, Tetrahedron Lett. 1988, 29, 4525 ± 4528; recent
review: K. Burgess, D. Cook, Chem. Rev. 2000, 100, 2047 ± 2059.
[7] L. J. Rinkel, C. Altona, J. Biomol. Struct. Dyn. 1987, 4, 621 ± 648.
[8] S. Creighton, L. B. Bloom, M. F. Goodman, Methods Enzymol. 1995,
262, 232 ± 256.
The second critical determinant of intrinsic DNA-polymer-
ase fidelity is the capacity to extend from mismatched primer/
template ends.[1a, 8, 11] Here again, 1a and 1b should be ideally
suited to monitor differential DNA-polymerase interactions
with the sugar moiety in mismatch extension events. We
investigated extension from template-T/primer-G, T/T, and
T/C by using KF to extend from a mismatched base at the
primer 3'-end by incorporation of TTP or 1a, b. The most
efficient extension was from T/G for all thymidine triphos-
phate derivatives (data shown for T/G in Figure 1C).[9, 13] The
results of quantitative analyses of T/G extensions are pre-
sented in Table 2. KF extends a T/G mismatched primer end
with probes 1a, b significantly less efficiently than with TTP.
Again, this is in contrast to the results in Table 1 where TTP
and 1a, b extension of a perfectly matched primer were
equivalent. Our results indicate that nucleobase pair mis-
matches at the 3'-end of a primer/template complex trigger
unfavorable enzyme interactions with the sugar moiety of an
incoming nucleoside triphosphate and prevent inadvertent
sealing of a base substitution in the nascent DNA strand.
Indeed, this mechanism may be considered essential for
proofreading, and may allow a pause for other functions such
as intrinsic exonuclease activity or dissociation of the DNA-
replication complex.
[9] Detailed experimental procedures as well as DNA sequences applied
in the in vitro replication assays are provided in the Supporting
Information.
[10] M. S. Boosalis, J. Petruska, M. F. Goodman, J. Biol. Chem. 1987, 262,
14689 ± 14696.
[11] M. F. Goodman, S. Creighton, L. B. Bloom, J. Petruska, Crit. Rev.
Biochem. Mol. Biol. 1993, 28, 83 ± 126.
[12] Quantitative investigations towards the action of 4'-modified triphos-
phates on HIV-1 reverse transcriptase and cellular DNA polymerases
have been reported recently. 4'C-Azidothymidine triphosphate: M. S.
Chen, R. T. Suttmann, E. Papp, P. D. Cannon, M. J. McRoberts, C.
Bach, W. C. Copeland, T. S.-F. Wang, Biochemistry 1993, 32, 6002 ±
6010; 4'C-acetylated thymidine triphosphate: a) A. Marx, M. Spichty,
M. Amacker, U. Schwitter, U. Hübscher, T. A. Bickle, G. Maga, B.
Giese, Chem. Biol. 1999, 6, 111 ± 116; b) A. Marx, M. Amacker, M.
Stucki, U. Hübscher, T. A. Bickle, B. Giese, Nucleic Acids Res. 1998,
26, 4063 ± 4067.
[13] Results obtained with TTP agree with recently published data:
a) C. M. Joyce, X. C. Sun, N. D. F. Grindley, J. Biol. Chem. 1992, 267,
24485 ± 24500; b) S. S. Carroll, M. Cowart, S. J. Benkovic, Biochem-
istry 1991, 30, 804 ± 813; c) K. Bebenek, C. M. Joyce, M. P. Fitzgerald,
T. A. Kunkel, J. Biol. Chem. 1990, 265, 13878 ± 13887.
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