Remarkable sensitivity to DNA base shape in the DNA polymerase active site

Article abstract of DOI:10.1002/anie.200504296

(Figure Presented) Shaping up: DNA polymerase I can distinguish easily and with high sensitivity between nucleobases that have the same size but differ in shape. The shape, altered through variation in the position of the halogen substituent(s), plays mor

Full text of DOI:10.1002/anie.200504296

Communications
sugar–phosphate backbone in experiments by Marx and co-
workers.[10]
However, the influence of steric effects in polymerases
has remained in question, in part owing to recent experiments
by Berdis and co-workers,[11] and by Engels, Kuchta, and co-
workers.[12] Berdis and co-workers studied a set of 5-substi-
tuted indoles as deoxynucleoside triphosphate derivatives and
observed low selectivity for incorporation of the analogues by
T4 DNA polymerase opposite the four natural DNA bases.
Engels, Kuchta, and co-workers tested compounds with the
related benzimidazole skeleton by using the Klenow poly-
merase and reported little relationship between different
substitution patterns and selectivity between natural DNA
bases. In both studies, it was concluded that sterics do not play
an important role in the activities observed. Thus size and
shape effects and their contribution to DNA polymerase
fidelity remain in active debate.
Enzyme Fidelity
DOI: 10.1002/anie.200504296
Remarkable Sensitivity to DNA Base Shape in the
DNA Polymerase Active Site**
We have undertaken a series of studies to begin to address
the steric hypothesis in a systematic way. An earlier report
evaluated the effects of increasing DNA base size while
keeping the shape constant by using nonpolar thymine shape
mimics (toluene, difluoro-, dichloro-, dibromo- and diiodo-
toluene) paired opposite adenine.[13] These compounds varied
in size over a small 1.0 Š range, yet experiments showed that
Herman O. Sintim and Eric T. Kool*
The mechanisms of DNA synthesis by DNA polymerase
enzymes have come under intensive study recently because of
their close biological connections to cancer.[1] Replicative
polymerases, such as Pol I and Pol III in bacteria, must there were large differences in efficiency and fidelity across
synthesize DNA with high fidelity to avoid deleterious
mutations.[2] Repair polymerases, on the other hand, operate
with lower fidelity and serve to replicate DNA beyond
damaged and mismatched sites, keeping cells alive while
other enzymes act to correct the damage and prevent
oncogenic mutations from proliferating.[3] Because of the
importance to human health, a focus of research in many labs
has been on the factors that govern fidelity in these
enzymes.[4]
Recent studies have suggested that steric effects can have
strong influences in DNA polymerase enzymes. In early
studies with nonpolar nucleoside isosteres,[5] it was observed
that purinelike molecular shapes can be selectively paired
with pyrimidine shapes,[6] and a large pyrene nucleobase
analogue was shown to be selectively replicated against a very
small (abasic) nucleoside.[7] Experiments by Hirao and co-
workers[8] have lent support to a strong contribution from
sterics in DNA replication, and studies by Shultz, Romesberg,
and co-workers[9] with methylated and fluorinated synthetic
bases have shown differing efficiency with changes in
substitution. Furthermore, steric effects in polymerase
active sites have also been observed on the level of the
the series.[13c] However, steric effects depend not only on size,
but also shape—that is, size at specific positions in a molecule.
Herein, we investigate the effects of nucleobase shape in the
DNA polymerase I active site through variation in the
positions of substitution of H, F, Cl, and Br atoms on the
toluene skeleton. We find surprisingly large kinetic effects
resulting from subtle changes in substituent size and location.
Design of this new series started with the known 2,4-
difluorotoluene deoxyriboside,[5] a nearly perfect isostere of
thymidine, and in particular, the recent variant 2,4-dichloro-
toluene deoxyriboside[13a] (6, Figure 1), which has virtually the
same shape but is slightly larger. Both of these molecules are
highly active with replicative DNA polymerases and encode
replication opposite adenine.[13c,14] To test whether it is shape,
size, or other properties of such thymidine-mimicking com-
pounds (and by inference, thymidine itself) that make them
active, we have now altered the shapes in regular increments
through the substitution of larger and smaller groups along
the periphery of the toluene ring. The sets of varied molecular
shapes are shown in Figure 1 and Figure 2. Comparison of
compounds 1–3 preserves the size but varies the position of a
single chlorine substituent, whereas compounds 4–6, with
varied double substitution, helps to determine the degree to
which shape effects are important for the remarkable
thymine-like behavior of the 2,4-isomer in pairing and in
replication.[13c] Finally, comparisons of four-compound sets
allows for slow variation in size at the hypothetically
important 2- and 4-positions separately (Figure 2). For
example, the series 1!9!6!10 incrementally increases the
size of the 4-position with H, F, Cl, and Br atoms,[13a,15] and the
3!7!6!8 series addresses effects at the 2-position in the
same way.
[*] Dr. H. O. Sintim, Prof. Dr. E. T. Kool
Department of Chemistry
Stanford University
Stanford, CA 94305-5080 (USA)
Fax: (+1)650-725-0259
E-mail: kool@stanford.edu
[**] This work was supported by the US National Institutes of Health
(GM072705).
A generalized strategy for the preparation of the new C-
glycosides involved reacting the appropriate lithiated halotol-
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Figure 2. Two series of nonpolar thymidine analogues with gradually
varied shape. Circles highlight the position with increasing steric
demand. A) Structures of deoxyribosides 7–10. B) Space-filling models
showing gradual increases in size at the 2-position (3!7!6!8) and
separately at the 4-position (1!9!6!10). Models are shown with a
methyl group replacing deoxyribose. C) Expected bond lengths at the
variable positions for the two series.[28]
Figure 1. Structures of nonpolar thymidine analogues with systemati-
cally varied shape and constant size. Compounds 1–3 have a single
chlorine substituent at varied positions along the Watson–Crick pairing
edge, while 4–6 have two substituents. Space-filling models are shown
with methyl group replacing deoxyribose.
uene (generated through lithium–halogen exchange) with
disiloxanediyl-protected deoxyribonolactone[16] 11 followed
by triethylsilane reduction under Lewis acid catalysis (see the
Supporting Information). We prepared the 5’-dimethoxytrit-
yl-3’ phosphoramidite derivatives (see the Supporting Infor-
mation) and incorporated them into synthetic 12mer and
28mer oligonucleotides by using an automated synthesizer.
The 5’-triphosphate derivatives were also prepared following
published procedures.[17]
To investigate the small shape effects for pairing in DNA,
we evaluated the relative pairing stabilities of compounds 1–
10 placed opposite each of the four natural DNA bases in a
12mer duplex context[18] (see the Supporting Information).
Optically monitored melting studies were used to evaluate
stabilities at pH 7.0 in a buffer solution containing Mg2+
(10 mm) and Na+ (100 mm). The results showed that, despite
their differences in shape, these ten molecules have quite
similar pairing properties in this sequence context. With
natural bases as partners in the 12mer duplex, compounds 1–
10 were all destabilizing relative to a natural T–A pair (also
observed previously for 6[13b] and other low-polarity com-
pounds[18–20]) and generally displayed little pairing selectivity
among the natural partners. With the exception of the 3-
substituted analogues (2, 4, and 5) and the 4-chloro compound
3, some of the new analogues showed a small but significant
preference (by approximately 2–48C in Tm) for adenine as
partner. In our previous studies with 2,4-dihalotoluene base
analogues[13b] and studies of others,[9] a similar small prefer-
ence for adenine was also observed; this may be due to the
stronger stacking of adenine relative to other DNA bases.[21]
Interestingly, two cases, the 2,4-dichloro compound 6 and the
2-chloro-4-bromo compound 7, showed a stronger preferen-
ces for adenine over a mismatch (5–78C); this may be owing
to a small degree of thymine-like shape preference opposite
adenine conferred by the backbone. However, the selectivity
was lower than that of thymine (88C) and the analogues were
both considerably destabilizing overall (by 128C and greater),
consistent with their low polarity and lack of significant
hydrogen bonding ability. Overall, despite their differences in
shape, these ten molecules have quite similar pairing proper-
ties in this sequence context, which suggests that the DNA
backbone confers relatively low shape selectivity.
To determine the DNA polymerase shape selectivity, the
properties of the shape-varied compounds in the active site of
KF exonuclease-deficient (exoꢀ) polymerase were then
tested. Preliminary gel analyses of single-nucleotide insertion
reactions against each of the four DNA bases showed that all
ten compounds were enzymatically paired with adenine and
thymine in varied amounts but not significantly with guanine
or cytosine (see the Supporting Information). Thus, we
proceeded to measure the steady-state kinetic efficiencies
when these nonpolar thymine analogues are paired with A or
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T, wherein pairing with A defines absolute efficiency and
pairing with T, the most efficient mismatch, defines selectivity
or fidelity.
Data for analogues in the monochlorinated series 1–3
showed that they were generally more efficient in a template
strand than as dNTPderivatives (Figure 3A,B). The 2- and 4-
substituted compounds were enzymatically paired opposite
adenine preferentially, with efficiencies that were moderate
to high. The 2-chloro compound 1 showed greater selectivity
for adenine than did the 4-chloro case 3. The selectivity of 1
was moderate, with a 34-fold to 470-fold preference for
adenine over the most efficient mismatch (thymine). In
contrast to this, the 3-substituted compound 2 was paired very
poorly with adenine, 3–4 orders of magnitude below thymi-
dine, and the selectivity underwent a switch in the template
strand with a small preference for being paired with Tover A.
The second series (Figure 3C,D) compared enzymatic
processing of doubly chlorinated toluenes (4–6). Of the three,
only the 2,4-dichloro compound 6 showed selectivity for
adenine (selectivity was high, 64- to 810-fold), as previously
reported.[13c] In contrast to this, the other two compounds (4
and 5) showed selectivity for thymine over adenine. Thus
these two 3-substituted compounds demonstrated a selectivity
switch as had the other 3-substituted compound 3, above.
The kinetics data (shown in Figure 3, Figure 4, and
tabulated in the Supporting Information) as a whole showed
that the analogues were enzymatically paired with widely
varying efficiencies and selectivities. As template-base ana-
logues, the enzyme incorporated dATPopposite the com-
pounds with efficiencies that varied by three orders of
magnitude. The most efficient analogues were compounds 6,
7, and 10, which gave efficiencies within error limits of each
other and were only 2–3-fold less efficient than natural
thymidine as a template. As incoming nucleotide (dNTP)
derivatives, the compounds varied in efficiency by an even
larger 3500-fold for insertion opposite a template adenine.
The most efficient was compound 10, which was tenfold below
natural dTTPin efficiency. Thus a general inspection of the
data shows that shape effects are clearly large in magnitude
with this enzyme and that a number of the new analogues may
be even more efficient as thymidine mimics than any
previously reported.[13c,14]
Figure 3. Shape effects on DNA replication, as measured with thymidine analogues having the same size but altered shapes. A) and B) Data for
monochlorinated analogues 1–3. C) and D) Data for dichlorinated analogues 4–6. Kinetic efficiencies with the Kf enzyme are plotted on a log10
scale; black columns show data for replication opposite deoxyadenosine (as expected for a thymidine analogue), and striped columns show data
for replication against thymidine (the most efficient mismatch). Data are shown both for analogues as incoming nucleotides (A,C) and as
template bases (B,D).
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Figure 4. Effects of a gradually altering shape on DNA replication as measured with a series of thymidine analogues with one substituent of
varied size (over an 0.8 Š bond length range, Figure 2). Kinetic efficiencies with the Kf enzyme are plotted on a log scale. A) and B) Data for
varied size at the 2-position (analogues 3, 7, 6, and 8). C) and D) Data for varied size at the 4-position (analogues 1, 9, 6, 10). Black lines show
data for replication against deoxyadenosine and gray dashed lines show data for mismatched replication opposite the most efficient mismatch
(thymidine). Data are shown both for analogues as incoming nucleotides and as template bases. r=bond length.
The above data suggests strongly that the 2- and 4-
positions were important in the determination of thymidine-
like pairing behavior. Thus two series of compounds were
evaluated to investigate the effects of gradual shape changes
at these positions individually, by slowly increasing size
(Figure 4A–D). The data for the 2-position size variation
are plotted in Figure 4A,B and those for 4-position variation
are given in Figure 4C,D.
The incoming nucleotide analogues had gradually varied
shapes (Figure 4A,C), and the results showed that the
substituent size preference was strong at both the 2- and 4-
positions, with a 110-fold and 210-fold difference over the size
range. The maximum efficiency at the 4-position occurred
with bromo substitution, and this size preference appeared to
be slightly larger than that at the 2-position, wherein the
maximum appeared upon chloro substitution. The effects in
the template strand (Figure 4B,D) were somewhat similar in
that the 4-position had a larger size preference (chloro/
bromo) as compared with the 2-position (fluoro/chloro).
However, there was one substantial difference, the 4-position
selectivity overall was quite modest with a high efficiency
regardless of substituent size (Figure 4D).
Data were also plotted for the most efficient mismatch
(replication opposite thymidine; Figure 4, gray dashed lines),
which allowed for evaluation of the effects of gradually
altered nucleobase shape on pairing selectivity with this
enzyme. As a whole, the analogue with highest fidelity in a
template strand was the 2-chloro-4-bromo compound 10; as
incoming nucleotides, fidelities were the same for compound
10 and for the 2-fluoro-4-chloro compound 7. The overall
fidelity of compound 10 as an incoming nucleotide analogue
was approximately threefold lower than natural thymidine
(230-fold preference for adenine versus 790-fold). In the
template strand, however, compound 10 was processed with
higher fidelity than thymidine (1900-fold versus 1100-fold)
although identity of the most common mismatch was differ-
ent.
Taken together, the results demonstrate that the Klenow
DNA polymerase is highly sensitive to even small differences
in nucleobase shape. Overall, the shape analogues in this
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Communications
series displayed a remarkable 3500-fold range in enzymatic
low selectivity and little difference among the analogues seen
by Berdis, as all the molecules would present essentially the
same steric environment if they were oriented syn. Engels,
Kuchta, and co-workers studied closely related 5- and 6-
substituted benzimidazole nucleotides, and they observed
that nearly all of them gave pairing selectivity for guanine.[12]
Once again this could be readily explained if those molecules
flipped to the syn conformation in the active site, driven by
the expected large steric clash of the 5- and 6-substituents
with the template base. Importantly, the 9-nitrogen of
benzimidazole, if in syn orientation, would be analogous to
N3 of cytosine. Thus in this alternative conformation, one
would expect the best steric complementarity (and also H-
bonding complementarity) to guanine, which was the
observed replication result. The energetic cost of flipping to
the syn conformation might help explain the generally low
activity of the benzimidazole analogues that were studied.
In conclusion, we have shown that E. coli DNA polymer-
ase I (KF exoꢀ) can distinguish easily between nucleobases
that have the same sizes but different shapes. This enzymeꢀs
sensitivity to shape is surpisingly high, with subangstrom
changes being readily distinguished. Furthermore, we have
shown that the steric requirements of the 2-, 3-, and 4-
positions of thymine are all different. The 2- and 4-position
steric protrusions play crucial roles in defining the adenine-
encoding behavior of thymine, whereas steric substitutions at
the 3-position eliminate thymine-like behavior. Taken
together, our results suggest that nucleobase shape plays a
more prominent role in base-pairing efficiency and selectivity
than other factors in this enzyme. The results add insight into
the basic mechanisms of DNA replication, and into the
origins of mutations that arise during this process. More work
is needed, however, to evaluate how other enzymes respond
to differences in nucleobase shape and size.
efficiency despite the fact that they are all closely related
chlorinated toluenes. We showed previously that this same
enzyme is responsive to nucleobase analogues with variable
size but that maintain thymine-like shape.[13c] The present
results are distinct in that they shed light on the importance of
the location of steric substitutions rather than on the size of
the nucleobase as a whole. Changing a hydrogen substituent
to a chlorine (in dNTPanalogues) at positions 2 or 4 has a
strong positive effect (> 100-fold) on the efficiency of
insertion opposite adenine (compare 1 with 6, and 3 with 6).
In marked contrast, making the same steric change at the 3-
position has a negative effect on insertion opposite adenine
(by a factor of 2–5; compare 1 with 4 and 3 with 5).
We hypothesize that these local steric preferences are
defined by the shape of the base opposite these analogues,[22]
and that the magnitude of the shape preferences is enforced
by the active-site tightness of the enzyme around the incipient
base pair.[23,13c] The kinetic optimization that occurred here
with varying shape may reflect the optimal filling of space in
the active site; local steric protrusions that clash with adenine
(such as at the thymine 3-position) or with the enzyme at
other positions would be energetically costly at the closed
transition state for phosphodiester bond formation. On the
other hand, groups that are smaller than needed to fill the
space leave a void in the protein, which is also energetically
costly.[24]
We considered whether other factors apart from sterics,
such as polarity or electrostatic effects, might explain these
enzymatic results. For example, nucleobase dipoles might
conceivably influence base pairing or active site binding.[25]
However, comparison of members of the analogue series 1–3
and 4–6 reveals widely varying replication efficiencies with
essentially constant dipole strengths. In addition, local
electrostatic charges might conceivably contribute. However,
examination of data for the increasing size series (3!7!6!8
and 1!9!6!10), showed that the maximum efficiencies
occur with the largest halogens (bromine and chlorine),
whereas the strongest negative charge is associated with
fluorine. Moreover, local electrostatic charges associated with
chlorine would remain virtually the same in compounds 1–4
and in 4–6, but the replication efficiencies vary by orders of
magnitude. Thus we conclude that it is the size and location of
space-filling substituents, rather than polar factors, that best
explains these results.
Received: December 2, 2005
Keywords: base pairing · DNA replication · enzymes ·
.
steric effects
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