Hydrophobicity, shape, and π-electron contributions during translesion DNA synthesis

Article abstract of DOI:10.1021/ja0546830

Translesion DNA synthesis, the ability of a DNA polymerase to misinsert a nucleotide opposite a damaged DNA template, represents a common route toward mutagenesis and possibly disease development. To further define the mechanism of this promutagenic proce

Full text of DOI:10.1021/ja0546830

Published on Web 12/07/2005
Hydrophobicity, Shape, and π-Electron Contributions during
Translesion DNA Synthesis
Xuemei Zhang,‡ Irene Lee,*,‡ Xiang Zhou,§ and Anthony J. Berdis*,†
Contribution from the Departments of Pharmacology and Chemistry,
Case Western ReserVe UniVersity, 10900 Euclid AVenue, CleVeland, Ohio 44106, and
Department of Chemistry, CleVeland State UniVersity, 2121 Euclid AVenue,
CleVeland, Ohio 44115
Received July 13, 2005; E-mail: ixl13@case.edu; ajb15@cwru.edu
Abstract: Translesion DNA synthesis, the ability of a DNA polymerase to misinsert a nucleotide opposite
a damaged DNA template, represents a common route toward mutagenesis and possibly disease
development. To further define the mechanism of this promutagenic process, we synthesized and tested
the enzymatic incorporation of two isosteric 5-substituted indolyl-2′deoxyriboside triphosphates opposite
an abasic site. The catalytic efficiency for the incorporation of the 5-cyclohexene-indole derivative opposite
an abasic site is 75-fold greater than that for the 5-cyclohexyl-indole derivative. The higher efficiency reflects
a substantial increase in the kpol value (compare 25 versus 0.5 s-1, respectively) as opposed to an influence
on ground-state binding of either non-natural nucleotide. The faster kpol value for the 5-cyclohexene-indole
derivative indicates that π-electron density enhances the rate of the enzymatic conformational change step
required for insertion opposite the abasic site. However, the kinetic dissociation constants for the non-
natural nucleotides are identical and indicate that π-electron density does not directly influence ground-
state binding opposite the DNA lesion. Surprisingly, each non-natural nucleotide can be incorporated opposite
natural templating bases, albeit with a greatly reduced catalytic efficiency. In this instance, the lower catalytic
efficiency is caused by a substantial decrease in the kpol value rather than perturbations in ground-state
binding. Collectively, these data indicate that the rate of the conformational change during translesion DNA
synthesis depends on π-electron density, while the enhancement in ground-state binding appears related
to the size and shape of the non-natural nucleotide.
Introduction
of dATP is that the efficiency of insertion is influenced by the
base-stacking capabilities of the incoming nucleotide.7,8 The
Translesion DNA synthesis represents the ability of a DNA
polymerase to insert opposite and extend beyond a DNA lesion.
This process is promutagenic since the polymerase typically
misinserts an incorrect nucleotide opposite the DNA lesion.1
One commonly formed DNA lesion is an abasic site that can
be produced nonenzymatically2 or through the action of DNA
repair enzymes.3 This lesion is highly mutagenic since it is
devoid of hydrogen-bonding potential. Despite the nontemplat-
ing nature of an abasic site, most DNA polymerases preferen-
tially incorporate dATP (1) opposite this lesion.4 Several
mechanisms including steric constraints5 and desolvation6 have
been proposed to account for this kinetic phenomenon. An
alternative mechanism to explain the preferential incorporation
validity of this hypothesis has been strengthened by the favorable
enzymatic incorporation of non-natural nucleotides such as
5-NITP (2) and 5-PhITP (3) opposite this DNA lesion.9,10 Both
analogues have substituent groups rich in π-electron density, a
feature that provides greater base-stacking capabilities compared
to natural dNTPs. Remarkably, the catalytic efficiency for the
incorporation of 2 and 3 opposite an abasic site is ∼1,000-fold
greater than that measured for dATP incorporation.9,10 Further-
more, replacement of the nitro or phenyl moiety with functional
groups such as -H, -F, or -NH2 that are lacking π-electron
density reduces the efficiency of insertion by 3 orders of
magnitude.11 These data collectively indicate that the presence
of π-electron density significantly enhances nucleotide incor-
poration opposite damaged DNA.
† Department of Pharmacology, Case Western Reserve University.
‡ Department of Chemistry, Case Western Reserve University.
§ Cleveland State University.
(1) (a) Wagner, J.; Etienne, H.; Janel-Bintz, R.; Fuchs, R. P. DNA Repair (Amst)
2002, 1, 159-167. (b) Pages, V.; Fuchs, R. P. Oncogene 2002, 21, 8957-
8966.
(2) Lindahl, T. Nature 1993, 362, 709-715.
Although 5-NITP and 5-PhITP are preferentially inserted
opposite an abasic site, these analogues are also incorporated
(6) (a) Vesnaver, G.; Change, C. N.; Eisenberg, M.; Grollman, A. P.; Breslauer,
K. J. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 3614-3618. (b) Goodman,
M. F.; Ceighton, S.; Bloom, L. B.; Petruska, J. Crit. ReV. Biochem. Mol.
Biol. 1993, 28, 83-126.
(3) Boiteux, S.; Guillet, M. DNA Repair (Amst) 2004, 3, 1-12.
(4) (a) Schaaper, R. M.; Kunkel, T. A.; Loeb, L. A. Proc. Natl. Acad. Sci.
U.S.A. 1983, 80, 487-491. (b) Boiteux, S.; Laval, J. Biochemistry 1982,
21, 6746-6751. (c) Efrati, E.; Tocco, G.; Eritja, R.; Wilson, S. H.;
Goodman, M. F. J. Biol. Chem. 1997, 272, 2559-2569. (d) Mozzherin, D.
J.; Shibutani, S.; Tan, C.-K.; Downey, K. M.; Fisher, P. A. Proc. Natl.
Acad. Sci. U.S.A. 1997, 94, 6126-6131.
(7) Cuniasse, P.; Fazakerley, G. V.; Guschlbauer, W.; Kaplan, B. E.; Sowers,
L. C. J. Mol. Biol. 1987, 213, 303-314.
(8) Berdis, A. J. Biochemistry 2001, 40, 7180-7191.
(9) Reineks, E. Z.; Berdis, A. J. Biochemistry 2004, 43, 393-404.
(10) Zhang, X.; Lee, I.; Berdis, A. J. Biochemistry, 2005, 44, 13101-13110.
(11) Zhang, X.; Lee, I.; Berdis, A. J. Org. Biomol. Chem. 2004, 2, 1703-1711.
(5) (a) Kool, E. T. Annu. ReV. Biophys. Biomol. Struct. 2001, 30, 1-22. (b)
Matray, T. J.; Kool, E. T. Nature 1999, 399, 704-709.
9
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J. AM. CHEM. SOC. 2006, 128, 143-149
143

A R T I C L E S
Zhang et al.
opposite all templating nucleobases with reduced efficiencies.9,10
Both nucleotides bind with high affinity (KD values of ∼20 µM)
regardless of templating position. This feature suggests that
π-electron density contribute significantly toward the ground-
state binding of a nucleotide. In fact, the lack of selectivity for
incorporation coupled with the low KD values were argued to
reflect the presence of a “nonspecific” dNTP binding site that
utilized π-π stacking interactions between the aromatic rings
of the incoming dNTP and amino acids.9 To further define this
hypothetical mechanism, we characterized the incorporation of
two isosteric analogues of 5-PhITP (3) that possess different
π-electron densities. The nucleotide containing π-electron
density is incorporated opposite an abasic site 50-fold faster
than the isosteric analogue devoid of this feature. However, the
kinetic dissociation constants for the non-natural nucleotides
are identical and indicate that π-electron density does not
directly influence ground-state binding opposite the DNA lesion.
These data are interpreted with respect to a model in which
π-electron density enhances the rate of the enzymatic confor-
mational change step required for insertion opposite the abasic
site while binding affinity is driven by the hydrophobicity and
size of the nucleotide substrate.
cooling in an ice bath and quenched with H2O. A solution of saturated
NaHCO3 was added to neutralize the reaction, from which the organic
and the aqueous phases were separated. The aqueous phase, which
contained inorganic solid, was extracted with EtOAc 4 times. The
organic phases were pooled, washed with saturated NaCl, dried over
MgSO4, filtered, and then evaporated to dryness. The residue was
purified by silica flash chromatography using hexane as the eluent to
yield 1.4 g of product. H NMR (CDCl3) δ 1.5 (6H, m, CH2), 1.8 (4H,
m, CH2), 2.6 (1H, m, CH), 6.5 (1H, s, 3-H) 7.1 (1H, d, J ) 9.0 Hz,
Ar), 7.2 (1H, s, Ar), 7.35 (1H, d, J ) 9.0 Hz, Ar), 7.50 (1H, s, Ar),
8.15 (1H, br s, NH). HiRes FAB-MS (+): Calculated mass spectra
(formula C14H17NO for M), 199.13610; Experimental mass spectra,
199.13610. C NMR (CDCl3) δ 26.36, 27.20, 35.23, 44.74, 102.46,
110.68, 118.02, 121.79, 124.22.
Synthesis of 1-(3,5-Di-O-p-toluoyl-2-deoxy-â-D-erythropentafura-
nosyl)-5-(1-hydroxyl)-cyclohexylindole (9a). To a solution of 5-(1-
hydroxylcyclohexyl)indole (7a) (0.8 g, 4 mmol) in dry acetonitrile (100
mL) was added 0.35 g of NaH (9 mmol). The reaction was stirred at
room temperature for 90 min before the addition of 2.4 g of 1-chloro-
2-deoxy-3,5-di-O-p-toluoyl-R-D-erythropentofuranose (8) (6 mmol). The
resulting mixture was further stirred at room temperature for 16 h,
filtered, and then evaporated to dryness. The crude product was purified
by silica flash column chromatography using toluene as the eluent to
yield 1.5 g of colorless oil. H NMR (CDCl3) δ 1.60-1.90 (10H, m,
CH2), 2.43 (3H, s, CH3), 2.45 (3H, s, CH3), 2.63 (1H, m, 2′-H), 2.87
(1H, m, 2′-H), 4.57 (1H, m, 4′-H), 4.63 (2H, m, 5′-H), 5.72 (1H, m,
3′-H), 6.44 (1H, t, J ) 7.5 Hz, 1′-H), 6.55 (1H, d, J ) 3 Hz, 3-H),
7.20-7.32 (6H, m, Ar), 7.50 (1H, d, J ) 9.0, Ar), 7.75 (1H, s, Ar),
7.95 (4H, m, Ar). HiRes FAB-MS (+): Calculated mass spectra
(formula: C35H37NO6 for M), 567.26209; Experimental mass spectra,
567.26003.
Experimental Section
General. Tributylammonium pyrophosphate was purchased from
Sigma. Other reagents such as sodium hydride, sodium methoxide, and
phosphoryl oxychloride were purchased from ACROS. Trimethyl
phosphate and tributylamine were dried over 4 Å molecular sieves.
DMF was distilled over ninhydrin, stored in 4 Å molecular sieves. All
NMR spectra were recorded in a Gemini-300 FT NMR spectrometer.
Proton chemical shifts are reported in ppm downfield from tetramethyl-
silane (TMS). Coupling constants (J) are reported in hertz (Hz). 31P
NMR spectra were obtained in D2O in the presence of 50 mM Tris
(PH 7.5) and 2 mM EDTA. Phosphoric acid (85%) was used as external
standard. UV absorptions were measured using Beckman DU-70
spectrometer. Both low- and high-resolution positive fast atom bom-
bardment mass spectra [FAB-MS (+)] were obtained with a Kratos
MS-25RFA spectrometer at Case Western Reserve University.
LC-MS was performed at Cleveland State University. High-resolution
electrospay mass spectrometry [HiRes ESI-MS] was performed on an
IonSpec HiRes ESI-FTICRMS at the University of Cincinnati.
Synthesis of 5-(1-Hydroxylcyclohexyl)indole (7a). 5-Bromoindole
(6) (8 g, 40 mmol) in 80 mL of anhydrous THF was added to a solution
of KH (5.89 g, 41 mmol) in 80 mL of anhydrous THF at 0 °C. The
reaction mixture was stirred for 15 min and then cooled to -78 °C.
tert-Butyllithium (60 mL, 90 mmol) prechilled to -78 °C was added
via cannula. After 30 min, cyclohexanone (8 g, 8.26 mL, 80 mmol) in
THF (40 mL) was introduced. The reaction mixture was slowly warmed
to room temperature (about 2 h) and then poured into an ice-cold
solution of 1 M H3PO4 (150 mL). The aqueous phase was extracted
with EtOAc (3 × 100 mL). The organic phases were combined and
washed with NaHCO3 solution, and then saturated NaCl. The resulting
organic layer was dried over MgSO4, filtered, and concentrated to
dryness. The resulting crystal was washed with hexane to yield 6 g of
the product. H NMR (CDCl3) δ 1.60-1.90 (10H, m, CH2), 6.5 (1H, s,
3-H), 7.1 (1H, s, Ar), 7.4 (2H, s, Ar), 7.8 (1H, s, Ar), 8.15 (1H, br s,
NH). HiRes FAB-MS (+): Calculated mass spectra (formula: C14H17NO
for M), 215.13101; Experimental mass spectra, 215.13159. C NMR
(CDCl3) δ 22.55, 25.74, 39.35, 73.43, 102.90, 110.79, 116.42, 119.53,
124.61, 127.69, 134.77, 141.08.
Synthesis of 1-(3,5-Di-O-p-toluoyl-2-deoxy-â-D-erythropentafura-
nosyl)-5-cyclohexylindole (9c). This compound was prepared and
purified by the method described for 1-(3,5-di-O-p-toluoyl-2-deoxy-
â-D-erythropentafuranosyl)-5-(1-hydroxyl)cyclohexylindole (9a) using
5-cyclohexylindole (7c) (0.8 g, 4 mmol) as the starting material. The
product was a light yellow oil, and the yield was 1.5 g. H NMR (CDCl3)
δ 1.45 (6H, m, CH2), 1.90(4H, m, CH2), 2.43 (3H, s, CH3), 2.45 (3H,
s, CH3), 2.55 (1H, m, CH), 2.65 (1H, m, 2′-H), 2.87 (1H, m, 2′-H),
4.57 (1H, m, 4′-H), 4.63 (2H, m, 5′-H), 5.72 (1H, m, 3′-H), 6.44 (1H,
dd, J ) 7.5 Hz, 6 Hz, 1′-H), 6.48 (1H, d, J ) 3 Hz, 3-H), 7.02 (1H, d,
J ) 9.0, Ar), 7.20-7.45 (7H, m, Ar) 7.95 (4H, m, Ar). HiRes ESI-MS
(+): Calculated mass spectra (formula C35H38NO5 for M + H),
552.2750; Experimental mass spectra, 552.2758.
Synthesis of 1-(2-Deoxy-r,â-D-erythropentafuranosyl)-5-cyclo-
hexenylindole (10b). To 30 mg of 1-(3,5-di-O-p-toluoyl-2-deoxy-â-
D-erythropentafuranosyl)-5-(1-hydroxyl)cyclohexylindole 9a (0.05 mmol)
in 10 mL of THF was added 15 mg of AlCl3 (0.1 mmol). The reaction
mixture was stirred at room temperature until completion as determined
by TLC (approximately 45 min). The resulting reaction intermediate
9b was used in the next step without further purification. To 9b was
then added a solution of sodium methoxide in methanol until the pH
of the reaction mixture reached >12. The reaction stirred at room
temperature for 16 h before being evaporated to dryness. The resulting
residue was purified by silica flash chromatography using dichlo-
romethane-methanol, 95:5 as eluent. A 1:1 mixture of a- and
â-anomers were obtained. The product mixture was a light purple oil
and the yield was 8 mg. H NMR (DMSO) δ 1.60 (2H, m, CH2), 1.75
(2H, m, CH2), 2.20 (3H, m, CH2 and 2′-H), 2.50 (3H, m, CH2 and
2′-H), 3.50 (2H, m, 5′-H), 3.80, 3.95 (1H, m, 4′-H), 4.29 (1H, m,
3′-H), 4.75, 4.85 (1H, m, 5′-OH), 5.28, 5.40 (1H, m, 3′-OH), 6.06 (1H,
m, CH), 6.33 (1H, t, J ) 7.0 Hz, 1′-H), 6.43 (1H, s, 3-H), 7.2 (1H, m,
Ar), 7.5 (2H, m, Ar), 7.65 (1H, s, Ar). UV (MeOH) λmax (nm): 240,
266 (ꢀ ) 8540 M-1 cm-1), 292 (sh). HiRes ESI-MS (+): Calculated
mass spectra (formula C19H24NO3 for M + H), 314.1756; Experimental
mass spectra, 314.1749.
Synthesis of 5-Cyclohexylindole (7c). To anhydrous THF in an ice-
bath was added 1.14 g (30 mmol) of LiAlH4 and then 3.6 g (28 mmol)
of AlCl3. After 5 min, 3 g of 5-(1-hydroxylcyclohexyl)indole (7a) (14
mmol) was added, and the reaction mixture was refluxed for 2 h before
9
144 J. AM. CHEM. SOC. VOL. 128, NO. 1, 2006

Incorporation of Non-Natural Nucleotides
A R T I C L E S
Synthesis of 1-(2-Deoxy-â-D-erythropentafuranosyl)-5-cyclohexyl-
indole (10c). To 1.5 g of 1-(3,5-di-O-p-toluoyl-2-deoxy-â-D-erythro-
pentafuranosyl)-5-cyclohexylindole 9c (2.7 mmol) in methanol (50 mL)
and ether (15 mL) was added solid sodium methoxide until the pH of
the solution reached 12. The mixture was stirred at room temperature
for 16 h and then evaporated to dryness. The resulting residue was
treated with 3 mL of MeOH, from which the methanolic solution was
filtered, and then evaporated to dryness. The crude product was further
purified by silica flash chromatography, dichloromethane-methanol
95:5 as eluent, to yield 570 mg of a colorless crystal as the final product.
H NMR (DMSO) δ 1.40 (6H, m, CH2), 1.80 (4H, m, CH2), 2.16 (1H,
m, 2′-H), 2.45 (1H, m, 2′-H), 2.50 (1H, m, CH), 3.50 (2H, m, 5′-H),
3.80 (1H, m, 4′-H), 4.31 (1H, m, 3′-H), 4.85 (1H, m, 5′-OH), 5.28
(1H, m, 3′-OH), 6.30 (1H, t, J ) 7.5 Hz, 1′-H), 6.45 (1H, d, J ) 3.4
Hz, 3-H), 7.01 (1H, d, J ) 9.0 Hz, Ar), 7.35 (1 H, s, Ar), 7.45 (1H, d,
J ) 9.0, Ar), 7.50 (1H, d, J ) 3 Hz, Ar). UV (MeOH) λmax (nm) 266
(ꢀ ) 6843 M-1 cm-1). Upon irradiation at 6.30 ppm for the anomeric
hydrogen, the NOE observed at 3.80 (H-4′, 0.52%) along with 2.16
(H-2′R, 3.1%), 7.45 (H-2, 3.21%), 7.50 (H-7, 1.10%). HiRes ESI-MS
(+): Calculated mass spectra (formula C19H26NO3 for M + H),
316.1913; Experimental mass spectra, 316.1899.
of the triphosphate was eluted at 74.2% B (11.3 min retention time) in
preparative reversed phase HPLC. Yield: 4%. H NMR (D2O) δ 1.25
(6H, m, CH2), 1.70 (4H, m, CH2), 2.22 (1H, m, 2′-H), 2.45 (1H, m,
CH), 2.60 (1H, m, 2′-H), 3.95 (2H, m, 5′-H), 4.05 (1H, m, 4′-H), 4.58
(1H, m, 3′-H), 6.35 (1H, dd, J ) 8.0 Hz, 6.0 Hz, 1′-H), 6.40 (1H, d,
J ) 3.4 Hz, 3-H), 7.10 (1H, d, J ) 8.3 Hz, Ar) 7.4 (3H, m, Ar). 31P
NMR (D2O/Tris) δ γ-P, -7.18; R-P, -10.55; â-P, -21.64. HiRes ESI-
MS (-): Calculated mass spectra (formula: C19H25NO12P3 for M -
H), 554.0746; Experimental mass spectra, 554.0842.
Enzymatic Characterization of the Incorporation of (4) and (5)
Opposite an Abasic Site or Templating DNA. Enzyme Assays. The
assay buffer used in all kinetic studies consisted of 25 mM Tris-OAc
(pH 7.5), 150 mM KOAc, and 10 mM 2-mercaptoethanol. All assays
were performed at 25 °C. Polymerization reactions were monitored by
analysis of the products on 20% sequencing gels as previously described
(24). Gel images were obtained with a Packard PhosphorImager using
the OptiQuant software supplied by the manufacturer. Product formation
was quantified by measuring the ratio of 32P-labeled extended and
nonextended primer. The ratios of product formation are corrected for
substrate in the absence of polymerase (zero point). Corrected ratios
are then multiplied by the concentration of primer/template used in
each assay to yield total product. All concentrations are listed as final
solution concentrations.
The kinetic parameters, kpol, KD, and kpol/KD, for nucleotide 5 were
obtained by monitoring the rate of product formation using a fixed
amount of gp43 (50 nM) and DNA substrate (1,000 nM) at varying
concentrations of nucleotide triphosphate (0.01-1 mM). Aliquots of
the reaction were quenched into 0.5 M EDTA, pH 7.4 at times ranging
from 5 to 600 s. Samples were diluted 1:1 with sequencing gel load
buffer, and products were analyzed for product formation by denaturing
gel electrophoresis. In all cases, steady-state rates were obtained from
the linear portion of the time course. Data obtained for steady-state
rates in DNA polymerization measured under pseudo-first-order reaction
conditions were fit to eq 1
Preparation of Indole Derivative Triphosphates. Phosphoryl
oxychloride (16 µL, 3 mol equiv, 0.18 mmol) was added dropwise to
a stirred and cooled (0 °C) solution of 2′-deoxynucleoside (20 mg, 0.06
mmol) and triethylamine (34 mg, 5 equiv, 0.33 mmol) in trimethyl
phosphate (0.37 mL). The reaction was monitored on TLC, and after
1.5 h, about 50% conversion had occurred. The reaction mixture was
then treated with a 0.5 M solution of tributylammonium pyrophosphate
(400 mg, 0.75 mmol) in DMF and tributylamine (0.18 mL, 0.75 mmol).
Upon stirring at room temperature for 20 min, the reaction was
neutralized with 1.0 M TEAB (15 mL) and then stirred at room
temperature for an additional 2 h. The final reaction mixture was
evaporated under reduced pressure at 32 °C to about 2 mL. The resulting
residue was purified by preparative reversed phase HPLC (300 pore
size C-18 column from Vydac, 22 mm × 250 mm) using a linear
gradient of 50% to 80% B within 14 min at a flow rate of 17.00 mL/
min (mobile phase buffer A, 0.1 M TEAB; buffer B, 50% ACN in 0.1
M TEAB). Both R and â isomers of the triphosphates were separately
collected and characterized by mass spectrum and NMR spectroscopy.
The resulting products were evaporated to 1 mL under reduced pressure
at 32 °C, redissolved in 5 mL of methanol, and then evaporated to
dryness. This process was repeated 3 times. The final product was
dissolved and stored in 10 mM Tris HCl, pH 7.5. The concentration of
the triphosphate was determined by UV absorbance of the nucleoside
at 266 nm. The extinction coefficients for 5-CEITP (4) and 5-CHITP
(5) at 266 nm are 8540 M-1 cm-1 and 6843 M-1 cm-1, respectively.
y ) mt + b
(1)
where m is the slope of the line, b is the y-intercept, and t is time. The
slope of the line is equivalent to the rate of the reaction, ν, and has
units of nM/s. Data for the dependency of ν as a function of dXTP
concentration were fit to the Michaelis-Menten equation
ν ) Vmax[dXTP]/KD + [dXTP]
(2)
where ν is the rate of the reaction, Vmax is the maximal velocity, KD is
the Michaelis constant for dXTP, and dXTP is the concentration of
non-natural nucleotide substrate. kcat is defined as Vmax/[gp43].
Pre-Steady-State Nucleotide Incorporation Assays. A rapid
quench instrument (KinTek Corporation, Clarence, PA) was used to
monitor the time courses for the incorporation of nucleotide 4 opposite
an abasic. Experiments were performed using single turnover reaction
conditions. 1000 nM gp43 exo- was incubated with 250 nM DNA in
assay buffer containing EDTA (100 µM) and mixed with variable
concentrations of nucleotide analogue (5-500 µM) and 10 mM
MgAcetate. The reactions were quenched with 500 mM EDTA at
variable times (0.005-10 s) and analyzed as described above. Data
obtained for single turnover DNA polymerization assays were fit to
eq 3
Synthesis of 1-(2-Deoxy-D-erythropentafuranosyl)-5-cyclohex-
enylindole Triphosphate (4). This compound was synthesized and
purified according to the general method described above, using 1-(2-
deoxy-D-erythropentafuranosyl)-5-cyclohexenylindole as the starting
material (10b). A mixture of R- and â-isomers was obtained. Charac-
terization of the R-isomer is provided as Supporting Information. The
â-isomer of the triphosphate was eluted at 70.9% B (9.8 min retention
time) in preparative reversed phase HPLC. H NMR (D2O) δ 1.55 (2H,
m, CH2), 1.65 (2H, m, CH2), 2.09 (2H, m, CH2), 2.20 (1H, m, 2′-H),
2.32 (2H, m, CH2), 2.65 (1H, m, 2′-H), 3.95 (2H, m, 5′-H), 4.05 (1H,
m, 4′-H), 6.10 (1H, m, CH), 6.40 (1H, m, 1′-H), 6.50 (1H, d, J ) 3
Hz, 3-H), 7.30 (1H, t, J ) 7.5 Hz, Ar), 7.40 (2H, m, Ar), 7.55 (1H, d,
J ) 7.5, Ar). HiRes ESI-MS (-): Calculated mass spectra (formula:
C19H25NO12P3 for M - H), 552.0590; Experimental mass spectra,
552.0704.
y ) Ae-kt + C
(3)
where A is the burst amplitude, k is the observed rate constant for initial
product formation, t is time, and C is a defined constant.
Synthesis of 1-(2-Deoxy-D-erythropentafuranosyl)-5-cyclohexyl-
indole Triphosphate (5). This compound was synthesized and purified
according to the general method described above, using 1-(2-deoxy-
â-D-erythropentafuranosyl)-5-cyclohexylindole (10c) as the starting
material. A mixture of R- and â-isomers was obtained. The â-isomer
Results and Discussions
Abasic sites are commonly formed DNA lesions that are
potentially mutagenic due to the lack of hydrogen-bonding
9
J. AM. CHEM. SOC. VOL. 128, NO. 1, 2006 145

A R T I C L E S
Zhang et al.
Figure 1. (A) Structures of 2′-deoxynucleoside triphosphates used or referred to in this study. For convenience, dR is used to represent the deoxyribose
triphosphate portion of the nucleotides. (B) Defined DNA substrates used for kinetic analysis. “X” in the template strand denotes any of the four natural
nucleobase or the presence of a tetrahydrofuran moiety designed to functionally mimic an abasic site.
Figure 2. Comparison of the efficiency of insertion for non-natural and natural nucleoside triphosphates opposite an abasic site.
potential. Although most DNA polymerases involved in chro-
mosomal replication preferentially incorporate dATP (1) op-
posite this lesion, the underlying mechanism accounting for its
preferential remains undefined. We proposed based upon kinetic
studies measuring the incorporation of non-natural nucleotides
that that the presence of π-electron density significantly
enhances nucleotide incorporation opposite this form of DNA
damage. In this mechanism, initial ground-state binding is
enhanced through π-π stacking interactions between the
aromatic amino acids lining the polymerase’s active site with
and the π-electrons present on the non-natural nucleotide.
Furthermore, the rate of catalysis is enhanced through stacking
interactions of the non-natural nucleotide inside the DNA helix.
To further evaluate this proposed mechanism, we characterized
the incorporation of two isosteric nucleotide analogues that
possess differences in π-electron densities. One analogue, the
5-cyclohexene derivative 4 contains limited π-electron density
by virtue of the presence of a double bond, while the 5-cyclo-
hexylindolyl derivatives 5 is devoid of π-electron density (Figure
1A). Each analogue was synthesized as shown in Scheme 1
using established protocols.11,12 Briefly, hydroxylcyclohexylin-
dole 7a was obtained using the protocol developed by Moyer
et al.12 Reduction with lithium aluminum hydride in the presence
of a Lewis acid offered cyclohexylindole 7c. The protected
hydroxycyclohexylindole nucleoside 9a and cyclohexylindole
nucleoside 9c were generated as a â anomeric isomer using the
protocol of Girgis et al.12 Dehydration of hydroxycyclohexyl-
indole nucleoside 9a, followed by deprotection of the hydroxyl
groups in the sugar ring under basic condition, offered the
cyclohexenylindole nucleoside (10b) as a mixture of R and â
anomers. Deprotection of 9c in basic condition yielded only
the â anomeric isomer of 10c. Both cyclohexenylindole and
cyclohexylindole triphosphates (4 and 5) were prepared with a
modified procedure from Zhang et al.,11 using triethylamine as
a base. For the synthesis of both triphosphates, R and â isomers
were formed and well resolved by the reversed phase C-18
column. Only â isomers were used for the kinetic study.
The ability of these nucleotide analogues to be inserted
opposite an abasic site was quantified by measuring the rate of
primer elongation using the DNA substrate depicted in Figure
1B. The studies here continue to use the exonuclease-deficient
form of the bacteriophage T4 DNA polymerase, gp43 exo-, as
the model enzyme. Initial experiments were performed under
pseudo-first-order reaction conditions in which 5 µM nucleotide
4 or nucleotide 5 was added to a preincubated solution of 50
nM gp43 exo- and 1 µM 13/20SP-mer in the presence of 10
mM Mg2+. As a positive control, experiments were also
performed under identical conditions except that 500 µM dATP
(1) was used as a natural nucleotide. Representative data
provided in Figure 2 indicate that both non-natural nucleotides
4 and 5 are incorporated more effectively compared to natural
purine substrate 1. However, it is also clear that the cyclohexene
derivative 4 is incorporated faster opposite the lesion compared
(12) (a) Moyer, M. P.; Shiurba, J. F.; Rapoport, H. J. Org. Chem. 1986, 51,
5106-5110. (b) Girgis, N. S.; Cottam, H. B.; Robins, R. K. J. Heterocycl.
Chem. 1988, 25, 361. (c) Loakes, D.; Brown, D. M.; Linde, S.; Hill, F.
Nucleic Acids Res. 1995, 23, 2361-2366.
9
146 J. AM. CHEM. SOC. VOL. 128, NO. 1, 2006

Incorporation of Non-Natural Nucleotides
A R T I C L E S
Scheme 1 a
a Reagents and conditions: (i) (1) KH, THF; (2) t-BuLi, THF; (3) Cyclohexanone; (4) 1 M H3PO4. (ii) LiAlH4, AlCl3, THF. (iii) NaH, CH3CN. (iv)
AlCl3, THF. (v) NaOMe/Methanol. (vi) (1) Trimethyl phosphate, 0 °C, POCl3, Et3N; (2) Bu3N, (Bu3NH)2H2P2O7 in DMF, 10 min; (3) 1 M Et3N‚H2CO3.
to the cyclohexyl derivative 5. Although these data suggest that
π-electron density is important for effective insertion opposite
the lesion, they cannot be used to unambiguously determine if
the enhancement in insertion reflects an increase in binding
affinity or through an increase in the rate of the conformational
change step required for phosphoryl transfer.
To evaluate this question, KD and kpol values were measured
for both non-natural nucleotides during incorporation opposite
the lesion. Since the rates of nucleotide 4 incorporation were
extremely fast (>100 nM/sec), a rapid quench instrument13 was
used to accurately monitor the kinetics of nucleotide incorpora-
tion. Experiments were performed using single turnover reaction
conditions in which 1 µM gp43 exo- was incubated with 350
nM 13/20SP-mer in assay buffer containing EDTA (100 µM)
and mixed with variable concentrations of nucleotide analogue
(5-75 µM) and 10 mM Mg2+. Figure 3A shows representative
data for the concentration dependency of nucleotide 4 on the
rate constant in primer elongation. All time courses were fit to
the equation for a single-exponential process to define kobs, the
rate constant in product formation. The plot of kobs versus the
concentration of nucleotide 4 is hyperbolic (Figure 3B), and a
fit of the data to the Michaelis-Menten equation14 yields a kpol
value of 25.3 ( 1.9 s-1 and a KD value of 5.1 ( 1.7 µM. Similar
analyses were performed monitoring the incorporation of
nucleotide 5 opposite the abasic site. Data for the rate of
nucleotide incorporation as a function of nucleotide concentra-
tion were fit to the Michaelis-Menten equation to yield a kpol
value of 0.46 ( 0.03 s-1 and a KD value of 6.2 ( 1.3 µM.
Values are summarized in Table 1.
Figure 3. (A) Dependency of 5-CE-ITP concentration on the apparent burst
rate constant as measured under single turnover conditions. 5-CE-ITP
concentrations were 1 µM (O), 2.5 µM (b), 5 µM (0), 10 µM (2), 20 µM
(9), 40 µM (+), and 70 µM (4). The solid lines represent the fit of the
data to a single exponential. (B) The observed rate constants for 5-CE-ITP
insertion (b) were plotted against 5-CE-ITP concentration and fit to the
Michaelis-Menten equation to determine values corresponding to Kd and
The fast kpol value of ∼25 s-1 measured for the incorporation
of nucleotide 4 opposite an abasic site rivals that of 120 s-1
and 50 s-1 measured for the incorporation of the non-natural
kpol
.
nucleotides 29 and 3,10 respectively. In contrast, there is a 50-
fold reduction in the kpol value of compound 5 that clearly
reflects the effects of removing π-electron density during
translesion DNA synthesis. The difference in kpol values
(13) Johnson, K. A. Methods Enzymol. 1995, 249, 38-61.
(14) Ferscht, A. Enzyme Structure and Mechanism; W. H. Freeman and
Company: New York; pp 98-101.
9
J. AM. CHEM. SOC. VOL. 128, NO. 1, 2006 147

A R T I C L E S
Zhang et al.
Table 1. Summary of Kinetic Rate and Dissociation Constants for
the Insertion of 5-Cyclohexeneindolyl-2′-deoxyriboside
Triphosphate and 5-cyclohexylindolyl-2′-deoxyriboside
Triphosphate Opposite Templating and Nontemplating
Nucleobases
the absence of π-electron density. Thus, while the rate of the
conformational change during translesion DNA synthesis de-
pends on π-electron density, the enhancement in ground-state
binding appears related to the size and shape of the non-natural
nucleotide.
1
1
dXTP
template
kpol (s-
)
KD
(µM)
kpol/KD (M-1 s-
)
5-CE-ITP
5-CE-ITP
5-CE-ITP
5-CE-ITP
5-CE-ITP
5-CH-ITP
5-CH-ITP
5-CH-ITP
5-CH-ITP
5-CH-ITP
abasic
A
C
G
25.1 ( 1.5
4.6 ( 1.0
11 ( 1
5 460 000
4810
20 000
1160
1120
74 200
7360
To further evaluate this mechanism, kinetic rate, and dis-
sociation constants for the insertion of nucleotides 4 and 5 were
measured opposite each of the four natural templating nucleo-
0.051 ( 0.002
0.24 ( 0.02
12 ( 1
0.029 ( 0.003
0.076 ( 0.005
0.46 ( 0.03
0.095 ( 0.003
0.077 ( 0.007
0.011 ( 0.001
0.018 ( 0.005
25 ( 7
bases. As summarized in Table 1, the catalytic efficiency (kpol
/
T
63 ( 12
6.2 ( 1.3
13 ( 2
abasic
A
C
G
T
KD) for both compounds is ∼300-5,000-fold lower than that
for insertion opposite the abasic site. The lower catalytic
efficiency is caused by a large reduction in kpol values rather
than a perturbation in KD values. The reduced kpol values are
caused by a decrease in the rate of the conformational change
step that likely results from the inability of these large, bulky
nucleotides to pair with the templating nucleobase. It is also
noteworthy that the KD values for nucleotides 4 and 5 are in
the low µM range and relatively independent of templating
position. This kinetic phenomenon was previously observed with
other 5-substituted indolyl deoxyriboside triphosphates such as
2 and 3 that contain π-electron density.9,10 The lack of specificity
coupled with low KD values was proposed to reflect the presence
of a “non-specific” dNTP binding site that utilized π-electron
interactions of the incoming dNTP with aromatic amino acids
of the DNA polymerase.9 The data obtained with nucleotide 4
are consistent with this model. However, the low KD value
measured for nucleotide 5 argues that physical parameters
independent of π-electron density such as desolvation and/or
size can influence dNTP binding.
31 ( 9
2480
470
720
23 ( 4
25 ( 10
measured for 4 versus 5 corresponds to a ∆∆G of 2.32 kcal/
mol. This value most likely reflects the energetic contributions
of π-electron density toward facilitating the conformational
change step. It is unlikely that desolvation accounts for this
energetic difference since the calculated log P values for the
nucleobase portion of 4 and 5 are essentially identical at 3.15
and 3.63, respectively. Although size constraints may influence
the dynamics of the conformational change step (vide infra), it
is unlikely that size alone dictates this rate constant, since the
surface area for the nucleobase portion of 4 and 5 are nearly
identical at 236.4 Å2 and 240.9 Å2, respectively.
Although the kpol values vary significantly for nucleotides 4
and 5, their respective KD values for incorporation opposite the
abasic site are essentially identical (compare 4.6 versus 6.2 µM,
respectively). This result is surprising since it was predicted
that the KD for nucleotide 5 would be significantly higher since
it lacks significant π-electron density. The identity in binding
affinity suggests that efficient dNTP binding can occur even in
These data are used to redefine our original model for
translesion DNA synthesis outlined in Figure 4 that accounts
for the role of π-electron interactions as well as other biophysical
Figure 4. Proposed model for the enzymatic insertion of non-natural nucleotide insertion opposite DNA. The first step represents the binding of dNTP to
the polymerase/DNA complex (KD). The second step represents the conformational change preceding phosphoryl transfer (kpol) required to place the triphosphate
moiety in close proximity with the positively charged amino acids as well as to stack the nucleobase portion of the incoming dNTP into the hydrophobic
environment of the interior of the duplex DNA. The final step represents the phosphoryl transfer step required for elongation of the primer strand (kchem).
9
148 J. AM. CHEM. SOC. VOL. 128, NO. 1, 2006

Incorporation of Non-Natural Nucleotides
A R T I C L E S
constraints such as size and desolvation. An essential caveat of
this revised model is that the templating nucleobase is oriented
in a conformation that prevents direct hydrogen-bonding contacts
between the templating base and the incoming nucleotide during
the initial binding event. This supposition is supported by the
structures of several DNA polymerases bound with nucleic acid
in the absence and presence of an incoming dNTP.15 Indeed,
these structures provide physical evidence that direct Watson-
Crick pairing interactions are precluded due to the expected
orientation of the template. In our model, the orientation of the
templating nucleobase in an extrahelical position creates a
transient “void” in the DNA that provides a functional mimic
of an abasic site (Figure 4, step A). It is easy to envision that
large, bulky nucleotides such as compounds 4 and 5 can easily
fill the “void” produced by the transiently formed abasic site
intermediate. Since both molecules are similar in shape and size,
their KD values are essentially identical and independent of the
templating nucleobase. In fact, the low binding constant
measured for insertion opposite a truly functional abasic site is
also consistent with this mechanism. Unlike binding affinity,
however, the kpol value is highly dependent upon the presence
of a templating nucleobase. We argue that the kpol step (Figure
4, step B) represents the conformational change step required
to place the templating base in an interhelical conformation that
then allows for proper alignment of the primer-template required
for the phosphoryl transfer step (kchem) (Figure 4, step C). When
a templating base is present, the rate of this conformational
change step is slowed since the shear bulk of the non-natural
nucleotide hinders the facile repositioning of the templating
nucleobase from an extra- into an intrahelical conformation. At
an abasic site, however, the rate of this conformational change
is significantly increased since the lack of a templating
nucleobase circumvents the need for this repositioning. In fact,
large non-natural nucleotides such as 4 and 5 should both be
effectively inserted opposite the abasic site since both would
provide size and shape complementarity to adequately fill the
void. However, the model invoking steric fit is inadequate since,
despite having similarities in shape and size, nucleotide 4 has
a kpol value that is 55-fold faster than that for nucleotide 5. The
most discernible difference between compounds 4 and 5 is with
respect to π-electron density. Thus, while dNTP binding
opposite the DNA lesion appears to be driven by shape/size
constraints, the rate of the conformational change is linked with
the presence of π-electron density at the 5 position of the
modified indole.
Acknowledgment. The research was supported in part by
funds from the NIH (CA118408) to A.J.B. and the Presidential
Research Award to I.L.
Supporting Information Available: Detailed characterization
of isomeric nucleotide triphosphate. This material is available
free of charge via the Internet at http://pubs.acs.org.
(15) (a) Franklin, M. C.; Wang, J.; Steitz, T. A. Cell 2001, 105, 657-667. (b)
Hogg, M.; Wallace, S. S.; Doublie, S. EMBO J. 2004, 23, 1483-1493.
Johnson, S. J.; Taylor, J. S.; Beese, L. S. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 3895-3900. Li, Y.; Korolev, S.; Waksman, G. EMBO J. 1998,
24, 7514-7525.
JA0546830
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