Active Site Tightness and Substrate Fit in DNA Replication

Annu. Rev. Biochem. 2002.
71:191–219
DOI: 10.1146/annurev.biochem.71.110601.135453
Copyright © 2002 by Annual Reviews. All rights reserved
First published as a Review in Advance on March 4, 2002
ACTIVE SITE TIGHTNESS AND SUBSTRATE FIT

IN DNA REPLICATION
Eric T. Kool
Department of Chemistry, Stanford University, Stanford, California 94305; e-mail:
kool@stanford.edu
Annu. Rev. Biochem. 2002.71:191-219. Downloaded from www.annualreviews.org
by University of Central Florida on 01/26/14. For personal use only.
Key Words fidelity, hydrogen bond, hydrophobic effects, base stacking,

solvation, sterics
f Abstract Various physicochemical factors influence DNA replication fidelity.
Since it is now known that Watson-Crick hydrogen bonds are not necessary for
efficient and selective replication of a base pair by DNA polymerase enzymes, a
number of alternative physical factors have been examined to explain the efficiency
of these enzymes. Among these factors are minor groove hydrogen bonding, base
stacking, solvation, and steric effects. We discuss the concept of active site tightness
in DNA polymerases, and consider how it might influence steric (size and shape)
effects of nucleotide selection in synthesis of a base pair. A high level of active site
tightness is expected to lead to higher fidelity relative to proteins with looser active
sites. We review the current data on what parts and dimensions of active sites are
most affected by size and shape, based on data with modified nucleotides that have
been examined as polymerase substrates. We also discuss recent data on nucleotide
analogs displaying higher fidelity than the natural ones. The published data are
discussed with a view toward testing this sterically based hypothesis and unifying
existing observations into a narrowly defined range of effects.
CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
NUCLEOSIDE ANALOGS AS PROBES OF DNA POLYMERASES . . . . . . . . 193
Simple Modifications of Natural Bases . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Nonpolar Shape Mimics of Natural Bases . . . . . . . . . . . . . . . . . . . . . . . . 194
Nonpolar DNA Bases Designed De Novo . . . . . . . . . . . . . . . . . . . . . . . . 195
Size-Altered DNA Bases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Sterically Augmented Sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
THE VARIED FIDELITY OF DNA POLYMERASES . . . . . . . . . . . . . . . . . 198
Wild-Type Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
Mutations That Affect Fidelity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
EVIDENCE AGAINST A PURE HYDROGEN BONDING MODEL OF FIDELITY. 200
The A Rule and Noninstructional Lesions . . . . . . . . . . . . . . . . . . . . . . . . 201
Nonpolar Nucleoside Analogs Are Replicated Efficiently . . . . . . . . . . . . . . . 202
0066-4154/02/0707-0191$14.00 191
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Replication of Nonpurine, Nonpyrimidine Molecular Shapes. . . . . . . . . . . . . 202

THE STERIC MODEL OF DNA REPLICATION FIDELITY . . . . . . . . . . . . . 203
Definition of the Active Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Consensus Base Pair Shape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Size Exclusion in the Active Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Solvation Effects on Nucleobase Size and Shape . . . . . . . . . . . . . . . . . . . . 208
Limitations of the Steric Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
THE CONCEPT OF ACTIVE SITE TIGHTNESS . . . . . . . . . . . . . . . . . . . . 210
The Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
Testable Predictions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
EVIDENCE FOR ACTIVE SITE TIGHTNESS AS A FACTOR IN FIDELITY . . 211
Variations in Selectivity Without Hydrogen Bonds. . . . . . . . . . . . . . . . . . . 211

High-Fidelity Nucleoside Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
Effects of Polymerase Mutations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
OTHER SIGNIFICANT ACTIVE SITE INTERACTIONS . . . . . . . . . . . . . . . 213

Base Stacking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Watson-Crick Hydrogen Bonds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
Triphosphate and Sugar Recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
Minor Groove Hydrogen Bonds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
CONCLUSIONS AND FUTURE PROSPECTS . . . . . . . . . . . . . . . . . . . . . . 215
INTRODUCTION
Many noncovalent interactions between the deoxynucleoside triphosphate

(dNTP), the DNA template, and the polymerase are necessary to support
successful DNA replication. Even the simplest and smallest DNA polymerases
are complex machines that utilize several kinetically distinct steps to incorporate
a nucleotide into a growing DNA strand. Polymerases are in most cases highly
accurate at choosing a correct nucleotide to pair with a partner in a template they
are copying, and the rate-limiting step for incorporating “correct” and “incorrect”
nucleotides is usually different. However, a large amount of recent work has
made it clear that polymerases have several different functions in the cell,
including leading strand and lagging strand synthesis, primer synthesis, gap
filling, extension past lesions, and repair synthesis, and thus their properties can
vary widely. One of the important ways that these enzymes vary is in their
fidelity. The hypothesis of this chapter is that fidelity can be strongly affected by
steric effects. Steric effects in the polymerase active site depend on (a) the sizes
and shapes (and shape complementarity) of the template base and the base on the
incoming nucleotide; and (b) the closeness and rigidity of the fit of the enzyme
around its substrate as it is synthesizing a base pair. We examine what is known
about the various noncovalent effects that can influence nucleotide choice, and
what existing data have to say about the hypotheses of steric exclusion and active
site tightness.
ACTIVE SITE TIGHTNESS IN DNA REPLICATION 193
Figure 1 Examples of various methylated nucleobases derived from thymine and

guanine.
NUCLEOSIDE ANALOGS AS PROBES OF

DNA POLYMERASES
Simple Modifications of Natural Bases

Nucleosides have been modified in many ways over several decades. In this
review we focus chiefly on those modifications that retain activity in polymerase
enzymes and especially those that change their size and shape, so as to probe
steric limits of the active site.
One of the simplest ways to change the size and shape of nucleotides is to add
simple substituents, and one of the smallest substitutions available is the methyl
group. Methyl groups are approximately 2.4 Å in length (about 1.5 Å longer than
a hydrogen alone). Chemists have substituted methyl groups at multiple positions
about all four nucleobases, and the effects of these substitutions have in a few
cases been examined with DNA polymerases. If we examine a simple pyrimidine
base such as thymine, for example, there are four possible positions for a methyl
group to be placed (O2, N3, O4, and C6) (the top row of Figure 1 shows the first
three of these), and all four have been synthesized at one time or another. Of
these, O2, N3, and O4 methyl groups have been examined for their effects on
DNA replication (1– 4). Note that a complete examination of DNA replication
effects has scarcely been done for any of the modified bases discussed here, since
“complete” can be very involved indeed. In some cases there has been a
qualitative gel-based or radioactivity-based assay for incorporation or bypass,
performed in vitro. This is most often done with the modified base in either the
template strand or in a nucleoside triphosphate, but not always both (an important
distinction). More useful for the present kind of analysis are cases where
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quantitative kinetics have been performed for incorporation in vitro with at least
one purified polymerase.
The DNA base guanine can be methylated at C8, N7, O6, N3, N2, and N1.
Those examined for their effects on DNA replication are N7 and O6 (5–9). The
bases cytosine and adenine can be methylated at several positions, and although
many of these methylated derivatives have been synthesized, they have not been
studied very often with DNA polymerases. Of special interest here would be N4-
or O2-methyl-cytosine, or N1-, N3-, or N4-methyl-substituted adenine. Of these,
only N3-methylA has been studied appreciably (10, 11).
The existing studies of methylated bases have found them to be very poor
polymerase substrates. Of course, even relatively inefficient bypass of these
lesions may be biologically relevant to mutagenesis. However, in analysis of

active site interactions it is difficult to draw useful distinctions between different
influences, such as steric effects versus hydrogen bonding effects, because

methylated bases generally differ in both respects.
Nonpolar Shape Mimics of Natural Bases

The analogs mentioned above retain most of their hydrogen bonding groups and
have methyl groups added. When methyl groups are added at positions where
hydrogen bonding is unaffected, they can be quite useful as probes of steric
effects. However, such positions are few. For example, for thymine, of the four
positions of interest, all may affect hydrogen bonding directly. O4 and N3
substitutions directly affect hydrogen bonding, and O2 affects it indirectly by
changing N3 from a hydrogen bond donor to an acceptor. Substitutions of methyl
at C6 on pyrimidines lead to possible confusion by altering the syn/anti prefer-
ence of the base. Thus discussion of C6 substitution is not particularly useful here
either.
When methyl groups or other substituents are placed on DNA bases, then, two
factors are changed rather than one in the experiment: The size and shape of the
DNA base is altered (the desirable effect here), and the hydrogen bonding ability
is altered (which here would be an undesirable change that might have masking
effects on the steric influences).
As a result of these problems, we introduced a set of molecules that lack the
hydrogen bonding functional groups of natural DNA/RNA bases but mimic their
size and shape as closely as possible (12, 13) (Figure 2). Because of the lack of
hydrogen bonding groups, these isosteric molecules are nonpolar. Two of the
isosteres are nearly perfect shape mimics [the G analog H and the T analog F (14,
15)], with nearly indistinguishable sizes and shapes, and the other two are good
although imperfect in their size/shape mimicry [the A analog Z (16) and the
as-yet-unstudied C analog, abbreviated D].
If these nonpolar isosteres have the same size and shape as natural nucleo-
sides, what can they tell us about steric effects on DNA replication that the
natural ones cannot? The answer is that they represent four different molecular
shapes that are not affected by differences in hydrogen bonding strength and
Figure 2 The structures of the “bases” of nonpolar nucleoside isosteres F, D, Z, and H.

They are isosteres for the natural DNA bases T, C, A, and G, respectively. These are the
closest size and shape mimics of the natural bases that lack Watson-Crick hydrogen
bonding groups.
complementarity, nor by strong differences in solvation. Thus, one expects that

if size and shape alone have influences on replication, then they will exhibit some
of these effects even when hydrogen bonding is not present. The data to date are
discussed below.
Nonpolar DNA Bases Designed De Novo

Perhaps the best way to test size and shape effects in replication is to make
potential substrate molecules that have varied sizes and shapes but have no
hydrogen bonding groups. This allows one to eliminate the strong effects of
solvation and desolvation on pairing of polar molecules, and also allows the
elimination of the strong steric effects that the solvating water molecules can
have (17).
To date, upwards of two dozen different nonpolar DNA bases have been
synthesized in more than one laboratory, representing a wide variety of shapes
and sizes (reviewed in 18, 19) (Figure 3). Many of these have also been examined
quantitatively as substrates for various DNA polymerases. Of particular interest
are the phenyl derivatives (20), the isocarbostyril derivatives (21), the pyrene
variant (22, 23), and the benzimidazole (24) and indole analogs (25). Some of the
results with these molecules in template strands and as nucleoside triphosphates
are described below.
Size-Altered DNA Bases

Another interesting way to test size effects in the active site, and tightness effects
of the enzyme itself, is to retain all hydrogen bonding functional groups but
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Figure 3 Examples of base pairs designed de novo that are replicated with
moderate to high efficiency by DNA polymerases. See text for references.
simply increase the size of the DNA base. Although this has not yet been tested
quantitatively with DNA polymerase enzymes, this exact strategy was described
decades ago for testing the active sites of ATP-dependent enzymes. Leonard and
Figure 4 The structures of Leonard’s extended nucleobases, lin-benzoadenine and

lin-benzoguanine (26). They are longer than the natural bases by the width of a
benzene ring (2.4 Å), and have been used to study the active sites of ATP-dependent
enzymes.
coworkers described extended analogs of the nucleobases A and G (Figure 4), in

which a benzene ring was added in the middle of the base (26). This has the effect
of increasing size by 2.4 Å, but without changing the hydrogen bonding potential.
It remains to be seen whether strategies such as this would be useful in probing
the DNA polymerase enzymes rather than ATP-dependent enzymes, but it would
seem a potentially interesting and useful approach (27). Presumably, an extended
version of dATP would act as a good substrate for insertion opposite T only if
there is considerable steric room in the active site, or if there is sufficient
flexibility to adapt to the added size. A base pair between, for example, extended
A and standard T would be ⬃25% wider than the standard pair. Conversely, if
the active site is very tight, then this base pair would be a very poor substrate. By
comparing different quantitative efficiencies for processing the pair, the tightness
of different enzymes could be measured.
Another interesting prediction is that if a molecule such as expanded A were
in a template strand in an enzyme that had sufficient looseness, the expanded
A 䡠 T pair might be handled with higher fidelity than the natural pair. The
looseness might allow this pair to just fit, thus acting like a normal pair in a
tighter (higher fidelity) active site. The added size should then help prevent
mismatches by steric clashes with inappropriately sized or shaped bases C, T,
or G.
Sterically Augmented Sugars

The above mechanism for increasing fidelity may have been observed recently
for an interesting set of molecules that have sterically augmented sugars (28, 29)
(Figure 5). It was shown that methyl or ethyl groups substituted at C4⬘ of
deoxynucleoside triphosphates are well tolerated by the Klenow fragment (Kf)
polymerase (the large proteolytic fragment of E. coli DNA polymerase I) and,
remarkably, that this added size apparently increased fidelity. The data are
discussed below. One possible explanation is that this alkyl substitution may
have the effect of tightening the active site as described above, or that it simply
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Figure 5 The sterically augmented thymidine nucleosides of Marx et al. (28). The
smallest two examples are replicated with higher fidelity than natural thymidine by the Kf
polymerase.
allows the base pair to fill the entire space available in the active site. Thus such
molecules may also be useful for probing enzymes of different fidelity, testing
the hypothesis of active site tightness.
THE VARIED FIDELITY OF DNA POLYMERASES
Wild-Type Enzymes
The fidelity of native DNA polymerases can vary widely, depending on their
biological function and on the organism from which they are derived (reviewed
in 30 –32). Virally derived enzymes may often be under selection pressure to
have relatively low fidelity to increase mutation rate in the virus, and the same
may often be true of bacterial enzymes. In eukaryotic systems, where mutation
rates are lower, the replicative enzymes have very high fidelity. However,
eukaryotes have a number of different polymerases in the cell, each with a
specific function, and some have very low fidelity indeed.
Table 1 lists approximate fidelities of a number of different DNA polymerases
from widely varied sources. The fidelity measurements are listed as fidelity for
initial nucleotide insertion, which is the main number with most relevance here,
rather than overall fidelity of incorporation, which depends on the presence or
absence of 3⬘ editing activity. Overall fidelity in vitro can be affected by a
number of polymerase activities beyond simple selection of a nucleotide, which
is the first step. The other activities that can strongly affect fidelity include
fidelity of extension and proofreading by a 3⬘ exonuclease domain (if present)
(33). Of course, in vivo still other activities increase fidelity, including most
importantly the various DNA repair mechanisms (34). Finally, additional sub-
units of multicomponent enzymes can also affect overall fidelity; one example is
the sliding clamp subunits of replicative enzymes, such as proliferating cell
nuclear antigen (PCNA) associated with polymerase delta (pol ␦) (35, 36).
TABLE 1 A survey of nucleotide insertion fidelities for various DNA polymerases
Polymerase Error ratea Reference
Phage/viral
T4 exo- 10⫺2 101
⫺5
AMV-RT 10 102
⫺5
MMLV-RT 10 102
⫺4
HIV-RT 10 103
Bacterial
Pol I (Kf exo-) 10⫺3 to 10⫺4 104,105
⫺4
Pol II 10 106
10⫺4 to 10⫺5
Pol III 107

⫺4
Pol IV 10 108
Pol V 10⫺3 108
⫺1
RB69 exo- 10 101
Taq exo- 10⫺4 109
Eukaryotic
Pol ␣ 10⫺4 110
Pol ␤ 10⫺3 110
⫺4 ⫺5
Pol ␦ 10 to 10 37
⫺4
Pol ␥ 10 110
Pol ␩ 10⫺2 38
Pol ␫ 10 0
39
Pol ␬ 10⫺2 to 10⫺3 111
a
Values are approximate, since fidelity depends on context, conditions, and methods. Values represent initial
insertion fidelity without 3⬘ exonucleolytic proofreading. For multisubunit enzymes, values are for core polymerase.
Of this group of widely varied DNA polymerases, the fidelity of initial

insertion ranges from the highly accurate enzyme pol ␦, giving a value of 10⫺5
(meaning one error in 105 insertions is made) (37) to a low of approximately
1/10, meaning a remarkable one in ten error rate, for pol ␩ (eta) (38). Even more
remarkable is pol ␫ (iota), which in vitro actually favors misinsertion of G
opposite T rather than A opposite T (39).
This wide range of fidelities begs the question: What is it about these enzymes
that makes them differ in fidelity? Are there a number of widely varying factors
that change, or are there some simpler factors that can alter fidelity? It would not
be surprising if, in the long run, more than one physical factor is found to affect
fidelity of initial insertion. However, it also seems quite possible that the factor
of active site tightness may be a simple answer that plays a large (and perhaps the
largest) role in most variations in fidelity.
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Mutations That Affect Fidelity

Many mutations have been studied in several different DNA polymerases, and these
mutations also provide useful insight into the origins of high and low fidelity.
Because this topic has been reviewed in detail elsewhere (40, 41), only a few
examples are mentioned here. Some of the more extensively studied enzymes in this
regard include HIV-RT, pol I, and pol ␤ (beta). Interestingly and importantly, it has
been demonstrated that various mutations for these enzymes not only can lower
fidelity but in some cases can increase fidelity. In HIV-RT, for example, the Y115A
mutation involves loss of a tyrosine that stacks directly on the incoming nucleotide
in the active site. This loss results in a fourfold decrease in fidelity (42). However,
several mutations in this enzyme are now known to increase fidelity (up to 14-fold),
including M184V, E89G, D76V, and R78A (42, 43). Interestingly, V. N. Pandey and
coworkers suggest that mutations that increase fidelity, such as Q151N, may have a
“more stringent, less flexible” binding pocket, and suggest that mutations that remove
flexible residues may result in higher fidelity (44).
Loeb has studied many mutations of pol I, and has noted that the active site
is highly mutable with retention of activity. That laboratory has observed
several active site mutations that affect fidelity (45). In a different enzyme,
polymerase ␤, effects at several locations in the active site have been noted.
In this enzyme, residue R283 interacts very closely with the minor groove of
the template strand, and the R283A mutation lowers fidelity (46). This has
been explained by steric effects, in that the arginine side chain likely
sterically prevents mispairs from fitting into the active site, whereas the
smaller alanine methyl group would create much more steric room for
mispairs to be accommodated (32). Also in pol ␤, residues 294 and 295 act
to help position the template strand, and it is known that the N294Q mutation
increases fidelity by up to 19-fold while lowering overall activity (47).
Conversely, N294A results in fidelity that is lower than in the wild type. This
also may be interpreted as a steric effect; the larger glutamine side chain may
cause the active site to be tighter and thus less forgiving of the additional size
or misplaced groups of a mismatched pair.
Significantly, mutations do not need to be very near a polymerase active site
in order to affect fidelity. For example, the Y265C mutation in polymerase ␤ is
not in the active site but lowers fidelity markedly (48). Longer-distance influ-
ences such as this may occur by several mechanisms, but one possible way is to
alter active site motions or closeness of fit (tightness).
EVIDENCE AGAINST A PURE HYDROGEN BONDING

MODEL OF FIDELITY
It is now becoming clear that Watson-Crick hydrogen bonds alone do not explain
many examples of efficient or selective replication by different DNA poly-
merases. Although textbooks often have cited only the influence of these bonds,
a few investigators have considered the effects of other influences such as active
site geometry (49 –51). Below are outlined a few examples where other factors,
including size and shape, stacking ability, solvation, and minor groove interac-
tions, all play significant roles.
The A Rule and Noninstructional Lesions

The hydrolytic loss of a DNA base results in an aldehydic abasic lesion. The
effect of this structure on replication has been widely studied with polymerases,
as have more chemically stable models of abasic lesions, such as a common
tetrahydrofuran derivative. It has long been observed that most DNA poly-
merases preferentially insert adenine opposite these lesions, doing so with low to
moderate efficiency (52, 53). Similarly, many polymerases also preferentially
insert A after the end of a template strand (54). These A-rule phenomena are
sometimes referred to as a default mechanism of polymerases. However, it seems
more likely that they are the result of selective interactions of adenine in the
active site (see below). Regardless of the mechanism, A-rule activities are clear
examples of moderate efficiency and selectivity in the absence of Watson-Crick
hydrogen bonds, and in the absence of any obvious chemical information
encoded in the template strand. Once again, the physical origins of this activity
are worth examining.
The simplest explanation for selective insertion of A at abasic sites and at ends
of templates is probably its superior stacking ability. The relative stacking ability
of the four natural bases is A ⬎ G ⬎ C,T (55, 56), and this is (not coincidentally)
also their order of insertion preference at abasic sites (57). The active nucleotide,
dATP, is likely bound and processed in the active site because it retains most of
the noncovalent interactions that are needed for activity: The triphosphate can
bind the enzyme, the deoxyribose sugar can bind, the edges of the base can bind,
the flat nucleobase surface can stack on the primer/template duplex end, and there
is no steric clash that counteracts binding. Indeed, recent experiments with
unnatural nucleoside analogs are consistent with this picture; a larger-than-
normal nucleotide with pyrene replacing the DNA base is an extremely good and
highly selective substrate at abasic sites, with efficiencies essentially the same as
a wild-type base pair (58). Once again, the nucleotide cannot form Watson-Crick
hydrogen bonds, but it stacks quite strongly, which compensates for this absence.
Its larger size is not an issue because its partner is missing, making more than
adequate room.
Another possible influence on the selective insertion of adenine at “nonin-
structional” lesions is its relatively weak solvation. DNA base adducts that lack
hydrogen bonding potential are often classified as noninstructional in analogy to
abasic sites because of their lack of hydrogen bonding groups (59), and because
they selectively direct the insertion of A opposite themselves. A good example
is ethenodC, which is a poor polymerase substrate in a template strand (60).
When a natural DNA nucleobase is inserted opposite such a nonpolar surface, the
polar edge of the nucleobase must lose its strongly bound waters of solvation, and
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gain little in return except stacking. Adenine has the advantage over the other
three bases, likely because it is solvated more weakly (61) and it stacks more
efficiently (56).
Nonpolar Nucleoside Analogs Are Replicated Efficiently

A second, and even more convincing, replication activity that occurs in the
absence of Watson-Crick hydrogen bonds was observed with nucleoside analogs
that lack such hydrogen bonding ability. Nonpolar nucleoside isosteres,
described above, were found in some cases to be surprisingly good polymerase
substrates. For example, difluorotoluene (F, a shape analog of T) in a template
strand of DNA is processed efficiently and selectively by enzymes such as KF,

bacteriophage T7 DNA polymerase, and HIV-RT (20, 63, 64). Adenine is
inserted with nearly wild-type fidelity and efficiency with these enzymes.
Conversely, the nucleoside triphosphate analog dFTP is also a good polymerase
substrate, and once again is handled with selectivity as high as the natural version
(TTP) (24, 64).
It should be noted, however, that varying polymerases differ in these
activities; for example, polymerases ␣ and ␤ do not apparently process
difluorotoluene as a substrate (65). The reason for this difference is unclear,
but it is an important observation to address in generalized models for
replication. It has been hypothesized that polymerases ␣ and ␤ do not process
the non-hydrogen-bonding analog because they possess extremely tight active
sites (see below), and/or they require minor groove interactions in the
insertion active site (also addressed below).
Replication of Nonpurine, Nonpyrimidine Molecular

Shapes
More recently a number of base pairs have appeared that are efficiently replicated
in the absence of Watson-Crick hydrogen bonds. The steric model of replication
fidelity (see below) does not require that individual nucleobases take on the shape
of the natural ones; rather, it requires that the combination of two bases in a pair
being synthesized does not cause any steric clashes between the two or with the
enzyme surrounding them (17, 66, 67). Thus, for example, if one base in a
template strand is larger than a natural purine, its partner must be smaller than a
pyrimidine. If the shape of the template base is unusual, then its partner must
avoid steric clashes with that shape. Such a pair may well be a good polymerase
substrate if the two are also flat, aromatic structures that stack well.
In the past four years or so several examples of bases and base pairs have
appeared in this category, and there is a good deal of data on their ability to be
accepted by polymerase enzymes. Bases tested range in size from nitropyrrole,
with a single five-membered ring (68), to pyrene, with four six-membered rings
(58). In addition, the smallest nucleoside tested as a polymerase substrate is the
abasic site (tetrahydrofuran model), which has been tested both in template
strands (58) and as an abasic nucleoside triphosphate derivative (T. J. Matray &
E. T. Kool, unpublished).
Studies with this varied class of molecules make it clear that insertion of a
nucleotide can occur efficiently and selectively in the absence of Watson-Crick
hydrogen bonding effects. Thus other effects, such as stacking and size/shape
effects, are likely showing their influences. However, an additional set of lessons
has been learned with these molecules: first, that successful insertion of the
nucleotide into the primer strand does not guarantee its further elongation
through the active site; second (and sometimes related), that effects of hydrogen
bonding in the minor groove must not be ignored for this elongation; and third,
that polymerases vary widely in their ability to handle nonnatural nucleosides.
Some of these factors are discussed below.

THE STERIC MODEL OF DNA REPLICATION FIDELITY
Definition of the Active Site

Before discussing size and shape effects in the active site of DNA polymerases,
we must first define exactly what the active site is. In its working mode, the
enzyme makes contact with several base pairs of DNA and binds a template
strand of DNA, a primer strand of DNA, and a nucleoside triphosphate. Which
of these are to be included in the active site? For the present discussion we are
concerned only with the insertion step of DNA synthesis. More specifically, we
are considering only what forces allow a particular nucleoside triphosphate to
bind and become covalently bonded to the primer strand (and what forces, or lack
of forces, prevent the wrong dNTPs from forming this bond). When this
nucleotide is being chosen, the enzyme is already bound to a primer/template
duplex. Thus we will consider the active site for nucleotide binding and insertion
to be the following (Figure 6): The floor of the active site is defined by the flat
side of the primer 3⬘ end base. The far wall is the template base already in place.
The ceiling of the active site will be defined by the enzyme as it closes down over
the base of the incoming nucleotide. Thus the shape of the pocket is a slot having
the height (thickness) of an aromatic pi-system (⬃3.4 Å). The widths of the two
(left and right) sides are also defined by the enzyme, on the major and minor
groove sides of the base pair being made.
Because all successful nucleotides also have a sugar and a triphosphate
moiety, we leave these moieties out of the discussion for the moment. Thus,
the focus here is narrow, with discussion of only the part of the nucleotide-
binding pocket that binds the incoming nucleobase (the only part that varies
with varying sequence). We refer to this as the nucleobase binding pocket
(Figure 6).
With this narrow definition of the active site (the nucleobase binding pocket),
there is only one small but important part that changes as the chemical informa-
204 KOOL
Figure 6 Schematic diagram illustrating the dimensions and determinants of the

binding pocket for the base of the incoming nucleotide during DNA replication. The
depth and fit against the far wall depend on the size and shape of the template base.
The plasticity of this fit depends on the tightness of the enzyme along the H5-H8 axis.
The fit against the front and back walls depends on the tightness of fit of the protein
against the minor and major grooves, respectively. The ceiling of the pocket is
defined (in HIV-RT) by stacking of Tyr115 on the incoming nucleobase. Top,
correctly matched pair. Middle, wobble pair. Bottom, pairing of methylated (sterically
too long) base with a natural partner.
tion (e.g. base sequence) in the template strand varies: the far wall changes depth
and shape as the template base changes size and shape. In addition, if the
template base changes in number or arrangement of polar groups, these effects
are also felt in the far wall of the nucleobase binding pocket.
Consensus Base Pair Shape

The floor and ceiling of this nucleobase binding pocket are expected to be fixed
in the size and shape needed to handle the four nucleotides, since they all have
essentially the same thickness (the thickness of an aromatic ring is ⬃3.4 Å). The
depth and shape of the far wall varies, however, because of differing sizes and
shapes of the template base.
However, the analysis of the side (minor and major groove) walls of the
nucleobase binding pocket is less clear. If DNA polymerases handled only one
base pair, or if all four pairs (A 䡠 T, T 䡠 A, G 䡠 C, C 䡠 G) were exactly the same in
overall size and shape, then these two side walls could be fixed to fit tightly about
one specific shape. However, the four base pairs vary in their size and shape in
the major and minor groove dimensions. Figure 7 shows approximate space-
filling shapes of the four pairs to illustrate this. Figure 8A shows them over-
lapped, making clear that there is considerable variation of the four pairs, but
only in the minor and major groove dimensions. Near the center of the minor
groove there is variation largely because of the differences in minor groove
substitution of G and A. In the major groove there is variation because of the
differences in shape of purines and pyrimidines, and because of the presence or
absence of the thymine methyl group.
It is important to note that in one dimension— overall length—there is
virtually no variation from pair to pair. The extreme ends of the pairs consist
of H8 of purines and H5 of pyrimidines. Thus a polymerase could make close
contact on the ends of the base pair and on the sugars holding the bases at this
distance. The size exclusion hypothesis relies on this to tightly define a
“correct” base pair dimension, and the hypothesis of active site tightness asks
how rigid these far walls of the enzyme are.
The variations in minor and major groove sides of the natural base pairs
imply that polymerases must have enough space in the minor and major
groove to accept all four combinations. There are two ways this may happen:
either the nucleobase binding pocket is quite flexible only in these two walls,
and thus adapts size to match the changes, or more likely, the pocket is large
enough on these two sides that it accommodates all four base pair shapes.
Either way, one may consider the aggregate of all four natural base pairs in
designing the limits of sizes that are likely to be tolerated in the nucleobase
binding pocket, and in any pair of bases under consideration for replication.
Figure 8B illustrates the consensus base pair pocket, taking on the largest
dimensions of all four natural base pairs. The size exclusion hypothesis (see
below) adopts this size and shape of base pair pocket as a limit for successful
base pair processing in the nucleotide insertion step. The hypothesis of active
site tightness (also below) addresses to what degree the walls as drawn in this
consensus pocket are able to be pushed outward—that is, how flexible
they are.
206
KOOL
Figure 7 Schematic diagram illustrating the space-filling shapes of the four base pairs in isomorphous
orientation.
Figure 8 Schematic diagram

showing the overlay of the four base
pair shapes from Figure 7. (A)
Direct overlay, showing variability
(marked by arrows) at sides of
major groove and in center of minor
groove. R represents deoxyribose
and phosphodiester backbone. (B)
Overlay showing the consensus
largest dimensions along the outer
surface. A polymerase accommo-
dates at least this consensus shape in

its active site in order to process all
four pairs approximately equally

well. Pairs that violate the walls of
this shape may have steric clashes
with the active site, and the ener-
getic cost of such clashes depends
on the degree of deformability of
this pocket. Highly selective
enzymes are expected to be less
deformable and/or closer fitting than
enzymes with low fidelity.
Size Exclusion in the Active Site

The size exclusion hypothesis starts with the premise that to be successfully
processed (i.e. a complementary nucleotide is inserted opposite a template base),
a base pair must fit into the consensus base pair shape. In analyzing fidelity we
are most interested in a situation with a given template base, and we ask which
nucleotide from solution will be inserted opposite it. In this case, roughly half of
the pocket for the consensus base pair is already filled by the template nucleo-
base, and the binding pocket remaining for the incoming nucleobase is defined
clearly. Thus we are concerned here only with the shape and size of this incoming
nucleobase, and whether it will fit into this binding pocket opposite the template
208 KOOL
nucleobase without clashing with the defined walls of the active site. If it is too
long, it will be rejected (unless some flexibility can accommodate it, see below).
If it is too thick, it will also be rejected (of course, aromatic systems all have
similar thicknesses, but if the base were not flat, or one or more of the double
bonds were reduced, this would both increase thickness and reduce stacking
ability). Finally, it must fit into the dimensions and shape defined by the minor
and major groove sides, as illustrated by the consensus base pair shape (Figure
8B). If the incoming nucleobase does not fit because of steric clashes, then either
it will not bind in the pocket or if it does partially insert itself it will not
allow the triphosphate moiety to be aligned correctly for phosphodiester bond
formation.
Of course, this pocket can fully take its shape only after the enzyme undergoes
a conformation change to close and tightly define the active site (69, 70). This is
likely only for correctly shaped pairs; thus, it may well be the steric clashes
mentioned above that prevent the active site from forming, rather than the
reverse, where the formed active site prevents the steric clashes.
The steric fit model as described above lists only energetically unfavorable
reasons why incorrect nucleotides are not processed successfully. However, we
have not yet described the energetically favorable interactions that encourage the
correct nucleotide to bind, leading to phosphodiester bond formation. These
favorable interactions are for the most part clear. Some of them are essentially
constant for all nucleotides: the triphosphate and sugar contribute to binding of
the nucleotide. The other energetically favorable factors vary with different
nucleotides. The most important of these are the stacking ability of the incoming
nucleobase and to a lesser extent the hydrogen bond complementarity (if any)
with the template base. Also important is the solvation of the template and
incoming bases prior to base pair formation (see below), which also influences
size and shape. Thus the more general steric model takes into account the
stacking ability and the solvation of the incoming nucleobase.
Solvation Effects on Nucleobase Size and Shape

The water molecules solvating polar DNA bases are expected to have real and
measurable influences on their physical size and shape. This steric effect of
solvation has been discussed in detail elsewhere (17), so it is only summarized
here. Experimental measures of steric size of organic groups have clearly shown
that polar functional groups like amino and hydroxyl groups are effectively larger
in solvents that strongly hydrogen bond than they are in nonpolar solvents (71).
This is because such polar groups are essentially always closely solvated
(hydrogen bonded) by at least one, and often more, solvent molecules. Although
these exchange rapidly with other solvent molecules, they are essentially never
lost unless an energetically equivalent interaction can be made in exchange.
For example, water molecules are closely associated with the polar Watson-
Crick edges of DNA bases prior to pairing, and will not be lost until a
complementary base pair can be formed. Overall, the change in enthalpy
(counting numbers of hydrogen bonds) for forming an isolated pair is small to

zero, whereas if waters were lost completely without the formation of compen-
sating hydrogen bonds, the enthalpy change would be large and unfavorable (61,
72).
We have pointed out that, based on this solvation, the natural polar DNA bases
behave as if they are larger than we typically draw them. If there is no way to
exchange away the waters, then they will have real steric effects on replication.
Thus we have argued that it is these waters that can help to sterically prevent
misinsertion of a wrong nucleobase opposite a template base.
These solvation effects are especially useful in helping to rationalize why
small hydrogen bonding nucleobases are usually not incorporated opposite other
small nucleobases. The simple steric model (without solvation effects) would not
easily explain why small nucleobases such as thymine are not easily misinserted
opposite another template thymine, for example; after all, there should be steric
room for the two to fit into the consensus base pair pocket. The solvation-based
explanation, then, is that the solvating waters make T too large to fit opposite
another T (probably also solvated), and since one cannot make new bonds to
compensate for loss of waters, they are not lost, and the nucleobases remain
effectively too large to accommodate each other. One piece of evidence support-
ing this notion is that poorly solvated nucleobases having the same shape and size
as thymine are, in fact, inserted efficiently opposite one another (24).
Limitations of the Steric Model

Although steric effects such as those discussed above clearly do have important
effects in DNA replication, it remains to be seen whether they can explain all
effects seen during replication. It seems more likely that Watson-Crick hydrogen
bonding does, in fact, play some role in the selectivity of replication. For
example, although some of the non-hydrogen-bonded replicable base pairs
studied so far show considerable efficiency in replication, they do not display
fidelity levels as high as those of the natural pairs. This lower fidelity may be
partly due to the possibility that Watson-Crick hydrogen bonds do increase the
level of selectivity overall. This can be examined in more detail in the future by
studying replication fidelity for various non-hydrogen-bonding base pairs having
small variations in shape. Such studies will provide information on how much
selectivity is available from shape differences alone; the remainder may well be
due to hydrogen bond selectivity.
Other factors in replication efficiency and fidelity are not addressed by the
steric model. The first is specific polar (hydrogen bonded) interactions between
the enzyme side chains and the minor groove. Although they appear to be
unimportant during initial insertion of a nucleotide for bacterial and viral
enzymes studied to date, they may well be a factor in some eukaryotic proteins.
This is discussed in some recent articles (73–76) and also below. A second factor
that is not addressed by the steric model is the relative stacking abilities of
incoming nucleotides. This is also mentioned below. Finally, there is one effect
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that has a close relationship to the steric model: the possibility that the active site
does not behave in completely rigid fashion. Some interesting questions remain:
How flexible is the active site, and how does this flexibility affect the fidelity of
DNA replication?
THE CONCEPT OF ACTIVE SITE TIGHTNESS
A few recent literature reports have raised the issue of the tightness of polymer-
ase active sites (77– 80). Here we expand on this concept, defining it in more
detail and outlining its relationship to the sizes and shapes of the DNA bases in
an incipient pair.
The Hypothesis
We define active site tightness here as a measure of the environment around the
walls of the incoming nucleobase pocket. “Tightness” measures two parameters:
(a) closeness of fit of the walls of the pocket; and (b) flexibility of the
enzyme/DNA defining the pocket to move outward to adapt to sterically larger
nucleotide sizes. As discussed above, clearly the side chains of DNA poly-
merases cannot tightly surround DNA bases in the central minor groove or
around the major groove surfaces, since they change in size and shape for the
four natural base pairs (see Figure 8A). Thus, in these parts of the pocket, either
there is not a close fit in the first place or there is a significant degree of flexibility
already available for all polymerases. Here flexibility means that side chains can
move away from the base or base pair with little energetic penalty at the
transition state for base pair formation.
However, it is quite possible that the binding pocket for the incoming
nucleobase is more tightly defined in the length dimension. Here length is defined
roughly by C1⬘-C1⬘ distance; this also correlates with the H8-H5 distance
(marked in Figure 8A) in a purine-pyrimidine pair. We argued above that
polymerases may well make very close contact with these positions, since their
size, distance, and shape do not vary within the four natural pairs.
In this section we argue further that it is in the best evolutionary interest of a
high-fidelity polymerase to hold these distances rigidly, by bolstering the groups
in contact with these positions, thus defining base pair length rigidly. In this way,
any mutagenic changes to the polar Watson-Crick pairing surface (e.g., methyl-
ation) will cause steric problems that push the bases outward, clashing with the
rigid walls of the protein. The more rigid this surface is, the steeper the energetic
penalty for incorporating a given offending incorrect or damaged nucleotide.
Conversely, there are certainly examples where lower fidelity is evolutionarily
desirable; for example, in viral or bacterial polymerases where higher mutation
rates lend survival advantages, or in enzymes that process damaged bases. Here
again, it seems quite possible that evolving a lower rigidity in the nucleotide
binding pocket in general, and in the H8-H5 axis in particular, would be one
simple way to lower fidelity overall. In such a case, a template base or incoming
nucleotide might have a small lesion that changes size/shape significantly; in a
more flexible active site there would be a lower energetic penalty for binding and
subsequent phosphodiester bond formation.
Another example would be mispairing; for example, formation of the common
G 䡠 T mismatch. When G 䡠 T pairs are formed they adopt the wobble geometry,
which clearly has difficulties fitting in the consensus base pair shape, defined
above. The offset geometry of the pair means a steric clash in the major or minor
groove side of the pair, unless the enzyme has added flexibility to adapt to this
by moving side chains with little energetic penalty.
Testable Predictions
The concept of active site tightness provides some testable predictions that may
be worthy of examination in future work. The most obvious prediction is that the
DNA binding pocket may be found to be more rigid in enzymes that display high
fidelity, and more flexible in enzymes that act with low fidelity. Rigidity is not
a simple factor to quantify, but some NMR methods may provide insight into
these proteins. In addition, combined quantum mechanics/molecular mechanics
methods and other molecular simulation methods may prove useful in analyzing
changes in active site volume as a function of time as the protein goes through
vibrations. Second, the active site volume may be inherently larger (in the steady
state) for lower-fidelity enzymes than for the ones with highest fidelity. An
examination of the available high-resolution co-crystal structures of polymerases
with DNA substrates (81– 89) could be enlightening in that regard.
A third prediction is that the natural base fidelity of various polymerases
should correlate inversely with their ability to process base pairs that have small
perturbations to their size. That is, one may develop a simple set of base pair
analogs that have slightly larger sizes than the natural pairs, but otherwise change
little about their structure. One predicts that such pairs would be processed more
efficiently by lower-fidelity enzymes than by high-fidelity enzymes. Examples of
this approach are mentioned below.
EVIDENCE FOR ACTIVE SITE TIGHTNESS AS A

FACTOR IN FIDELITY
Variations in Selectivity Without Hydrogen Bonds

Non-hydrogen-bonding nucleoside analogs have provided some evidence con-
sistent with the idea that polymerase fidelity may depend on the tightness of the
active site. These analogs can serve as slightly larger-than-normal probes of
active site size because they lack hydrogen bonding potential. Hydrogen bonded
pairing of two DNA bases allows them to approach each other more closely than
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the sum of their van der Waals radii (90). In Watson-Crick base pairs, this
hydrogen bonding contraction amounts to approximately 0.5 Å. Thus, a pair
between A and T has a length (along the C8-H5 axis) approximately 0.5 Å
shorter than the analogous non-hydrogen-bonded pair between analog F and A.
Similarly, pairs involving non-hydrogen-bonding analog Z (a mimic of A) will
also be larger than the standard one by ⬃0.5–1.0 Å (91).
A comparison of fidelity and selectivity of polymerase processing of these
analogs shows that a higher-fidelity enzyme (T7 DNA pol exo-) handles these
analogs with higher selectivity than a lower-fidelity enzyme (Kf exo-) (24, 65).
(An exo- polymerase has a 3⬘ exonuclease-free mutation.) Although this is not
direct evidence for a tighter active site in T7 DNA pol than in Kf, it is at least
consistent with that notion.

High-Fidelity Nucleotide Analogs

A very important recent development also lends credibility to the hypothesis
of active site tightness. Summerer & Marx prepared nucleotide analogs in
which the C4⬘ carbon on the backbone was substituted with methyl, ethyl, or
isopropyl groups instead of the standard hydrogen (28) (Figure 5). This, of
course, increases the size of the deoxyribose sugar, and consequently, the
diameter of the double helix containing these analogs would be increased by
as much as ⬃1.5 Å in the methyl case. If a polymerase active site were
relatively tight, then a C4⬘ methyl nucleotide should be handled with low
efficiency. If it were relatively loose, then a polymerase might accept such an
analog with little penalty in efficiency.
Such a size-augmenting substitution might be expected to affect fidelity as
well. In either a tight or a loose active site, one might expect that a larger-than-
normal nucleotide would be tolerated better opposite its natural partner (for
example, if 4⬘-methyl dTTP were paired opposite deoxyribosyladenine) than in
a mismatched case, where added steric problems compound the situation. For
example, if, as expected, a G 䡠 T mismatch causes a steric problem when fitting
into an active site, then adding to the size of the dTTP nucleotide will be expected
to increase the severity of the problem.
Remarkably, this is what Summerer & Marx observed. Their experiments
showed that C4⬘-methyl- and -ethyl-substituted thymidines were accepted
with little penalty in efficiency by the Kf enzyme (28). However, larger
substituents such as isopropyl led to a low efficiency, consistent with the
possibility that the DNA became too large for the active site. Most impor-
tantly, the Kf enzyme processes the methyl analog with higher fidelity than
it processes normally substituted thymidine. This may be some of the best
existing evidence for the hypothesis of active site tightness. However, it will
be important to see whether such analogs show the predicted behavior with
enzymes of varying fidelity. One predicts that a very high-fidelity enzyme
(having a very tight active site) will process such expanded-size analogs quite
poorly, whereas low-fidelity enzymes will accept them much more readily.
Effects of Polymerase Mutations

It has been noted that mutations both near to and remote from the active site of
a polymerase can have significant effects on fidelity. Quite possibly these effects
are the result of static and/or dynamic changes in active site size and flexibility.
Again, such a result is not proof that active site tightness is affected, but it is
consistent with what one expects if this were the case. In the future, comparisons
of different mutants of the same enzyme will provide a useful test of the
hypothesis. For example, the R283A and N294Q mutations of pol ␤ (46, 47) are
hypothesized to add some extra room in the active site pocket, and so such
mutations (especially the latter) are expected to allow for more efficient incor-
poration of larger-than-normal base pairs. If the active site tightness varies

because of either nearby or remote mutations, then one expects that backbone-
size-altered analogs such as those of Marx will reflect this difference, and other
more direct measures of tightness and flexibility, such as NMR or molecular
dynamics methods, will as well.
OTHER SIGNIFICANT ACTIVE SITE INTERACTIONS
It is important to note that the hypothesis of active site tightness does not
preclude the possibility that other noncovalent interactions in the polymerase
active site may also influence nucleotide binding and discrimination at the
transition state for phosphodiester bond formation. A number of other interac-
tions are possible, and some if not all of the following certainly influence either
efficiency or fidelity of nucleotide incorporation. They can act either without
regard to, or in concert with, the steric effects of base shape complementarity and
active site tightness.
It is also noteworthy that the above steric effects, to the extent that they play
an important role, act largely in an energetically negative way to select against
incorrect base pair formation. But clearly the binding of a nucleotide requires that
there are energetically favorable interactions in the active site that encourage
complexation; among these are base stacking, Watson-Crick hydrogen bonds,
and triphosphate and sugar recognition, discussed below. We note here merely
that these interactions are largely favorable regardless of what nucleotide is
chosen, and so we rely on differential steric effects to explain most selectivity.
Base Stacking
It is certainly true that base stacking plays an important role in the binding of the
incoming nucleotide at the transition state for formation of a new base pair.
Indeed, much evidence suggests that stacking is the most important noncovalent
interaction that stabilizes the DNA helix (92). One simple piece of evidence that
stacking alone can influence dNTP binding is the observation that the order of
selectivity A ⬎ G ⬎ C,T for polymerase insertion at abasic sites correlates with
214 KOOL
the order of base stacking proficiency (56, 93, 94). Consistent with this is the
observation that a larger pyrene nucleotide, which stacks considerably better than
adenine, is found to be a much better substrate for polymerase insertion at abasic
sites (58).
It is also noteworthy that nucleotides are essentially not at all inserted by
polymerase when they lack a base. We prepared the 5⬘-triphosphate derivative of
an abasic deoxyribose (the common tetrahydrofuran analog), and we studied the
efficiency of its insertion opposite various template bases by the Kf enzyme (T. J.
Matray & E. T. Kool, unpublished data). The results showed that it was an
extremely poor substrate regardless of the template; we estimate that it is at least
six orders of magnitude less efficient than a natural nucleotide opposite its
complement. This suggests a value of as much as 3–5 kcal/mol of lost interac-

tions, much of which is stacking.
Watson-Crick Hydrogen Bonds

It is also true that Watson-Crick hydrogen bonds add a measure of favorable free
energy of binding of the correct dNTP in the active site. The highly polar aqueous
environment lessens the impact of this interaction in two ways: first, by providing
a high dielectric that lessens the favorable electrostatic component that is the
major part of hydrogen bonding; and second, water molecules provide direct
competition for the hydrogen bonds between bases by forming favorable hydro-
gen bonds of their own to the bases prior to nucleotide insertion. Despite these
effects, however, hydrogen bonds may add an estimated 0.25–1.0 kcal/mol each
of stabilization in a base pair, depending on context (67, 95, 96). It is also quite
possible that they increase specificity; however, it must be noted that mismatched
pairs also commonly form one to two hydrogen bonds. Thus, it seems likely that
one of the major ways that hydrogen bonding increases specificity is by helping
to enforce steric effects. Mismatched pairs that are hydrogen bonded adopt a
geometry (such as the wobble geometry) that will not fit in the active site. In
addition, the solvating water molecules have steric effects of their own (see
above).
Triphosphate and Sugar Recognition

Polymerase enzymes also recognize the triphosphate by electrostatic and hydro-
gen bonding interactions, and likely also make favorable interactions with the
deoxyribose sugar. This has no direct effect on selectivity of replication, since all
nucleotides have the same triphosphate and sugar, but these interactions are
necessary for phosphodiester bond formation, and no doubt help the dNTP bind
favorably.
Minor Groove Hydrogen Bonds

It has been recognized for some time that polymerases make several hydrogen
bonds with DNA, between amino acid donor groups and acceptors in the floor of
the minor groove (97, 98). It is not yet clear how important these may be for the
actual insertion step for a given base pair formation. Mechanistic studies have
demonstrated that these minor groove interactions can have large effects on the
DNA synthesis as a given base pair passes through the active site during further
extension of the duplex. But it does not necessarily follow that such a minor
groove interaction is important for the incoming nucleotide. Indeed, studies of Kf
and of T7 DNA pol show no significant effect on the incoming dNTP (65, 99).
Structural studies suggest that some polymerases may make a hydrogen bond
with the minor groove acceptor of the incoming nucleotide, but that is not
necessarily energetically or mechanistically important. Regardless, it is also true
that polymerases seem to vary considerably in their minor groove interactions, so
more studies are needed on this point.

CONCLUSIONS AND FUTURE PROSPECTS
Among all the noncovalent interactions in the active site, we conclude that steric
effects are the most likely candidate for supplying the lion’s share of fidelity
during DNA synthesis. These steric effects depend on the complementarity of
sizes and shapes of the two bases being paired, and on the tightness of the
polymerase active site under study. Watson-Crick hydrogen bonding groups may
also contribute to fidelity, but may do so in part because of the steric effects of
waters bound to them.
The hypothesis of active site tightness predicts that high-fidelity polymerases
surround their substrates more closely and/or with greater rigidity, and that
low-fidelity polymerases exhibit more flexibility and may not have as close a
contact with the substrate. Analysis of base pair consensus predicts that the
enzymes make looser contact with the major and minor groove portions of a base
pair, since those parts are already variable in their size and shape, and suggests
that flexibility near the minor groove hydrogen bond acceptors and along the
H8-H6 axis may decrease fidelity. Finally, recent studies with nucleotide analogs
having altered sizes have lent important support to the hypothesis of active site
tightness, by showing that analogs having slightly expanded size can be pro-
cessed with higher fidelity in an enzyme that is ordinarily a relatively low-fidelity
polymerase.
Clearly there needs to be more study of how base size and shape, and active
site tightness, affect replication fidelity. At least two or three broad research
directions are worth pursuing to examine these issues, including the testing of
new nucleotide analogs of varying shape and size, the further examination of
polymerase mutants, and direct spectroscopic measurements of proteins.
As described above, a growing number of nucleotide analogs are now
available that can be used to test the size/shape/tightness effects in polymer-
ase active sites. Yet more analogs are needed and will be developed shortly.
216 KOOL
Importantly, they need to be studied with polymerases using quantitative kinetic

methods and with a set of low-fidelity and high-fidelity enzymes. Analogs need to be
tested that extend the size of the natural ones by small, medium, and large amounts,
to test a broad range of active site tightness and fidelity.
A second valuable approach for continuing study of the issue of active site
tightness is the testing of preexisting and new polymerase mutants that strongly
affect fidelity. In the case where enzymes have already been crystallized with
DNA and incoming nucleotide, one might make rational mutations that increase
active site space. One predicts that this will decrease fidelity. Conversely,
mutations that increase steric closeness of the DNA in the active site of a
low-fidelity enzyme might be expected to increase fidelity. In addition to such
rational tests, one might also randomly make mutations and select for changes in
fidelity using published screening methods (100). This will be especially useful
in cases of mutations that are remote to the active site but might affect active site
tightness in an unforeseen way.
Finally, it would be of added interest to more directly measure the tightness
of the active sites of polymerase enzymes. First, the amount of active site space
can be carefully analyzed for existing and future co-crystal structures of poly-
merases with their substrates. Second, molecular dynamics simulations of poly-
merases could be carried out to measure the extent to which the active site
surrounds a set of base pair substrates (matched and mismatched), which should
measure the closeness of fit, and the extent to which the active site volume varies
over time, which should provide a measure of flexibility. Comparison of a
low-fidelity and a high-fidelity enzyme should be illustrative in this regard.
Third, careful analysis of NMR data, of thermal disorder X-ray diffraction of
polymerase crystals, and of data using advanced infrared spectroscopic methods
may also provide useful information on motions and flexibility in the active sites
of high- and low-fidelity enzymes.
ACKNOWLEDGMENTS
This work has been supported by the U.S. National Institutes of Health
(GM52956), and by a grant from the U.S. Army Research Office. I also thank my
coworkers for their contributions to this work; their names are found in the cited
references from our laboratory.
The Annual Review of Biochemistry is online at http://biochem.annualreviews.org
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Annual Review of Biochemistry
Volume 71, 2002
CONTENTS
FRONTISPIECE–Norman Davidson xii
MY CAREER IN MOLECULAR BIOLOGY, Norman Davidson xiii
FRONTISPIECE–Thressa Campbell Stadtman xxvi

DISCOVERIES OF VITAMIN B12 AND SELENIUM ENZYMES,
Thressa Campbell Stadtman 1

ERROR-PRONE REPAIR DNA POLYMERASES IN PROKARYOTES AND
EUKARYOTES, Myron F. Goodman 17
LONG-DISTANCE ELECTRON TRANSFER THROUGH DNA, Bernd Giese 51
THE BACTERIAL RECA PROTEIN AND THE RECOMBINATIONAL DNA REPAIR
OF STALLED REPLICATION FORKS, Shelley L. Lusetti and Michael M. Cox 71
V(D)J RECOMBINATION: RAG PROTEINS, REPAIR FACTORS, AND
REGULATION, Martin Gellert 101
EUKARYOTIC DNA POLYMERASES, Ulrich Hübscher, Giovanni Maga, and
Silvio Spadari 133
EUKARYOTIC RIBONUCLEASE P: A PLURALITY OF RIBONUCLEOPROTEIN
ENZYMES, Shaohua Xiao, Felicia Scott, Carol A. Fierke, and
David R. Engelke 165
ACTIVE SITE TIGHTNESS AND SUBSTRATE FIT IN DNA REPLICATION,
Eric T. Kool 191
GREAT METALLOCLUSTERS IN ENZYMOLOGY, Douglas C. Rees 221
ATP-DEPENDENT NUCLEOSOME REMODELING, Peter B. Becker and
Wolfram Hörz 247
BIOLOGICAL ROLES OF PROTEASES IN PARASITIC PROTOZOA,
Michael Klemba and Daniel E. Goldberg 275
METABOLISM AND THE CONTROL OF CIRCADIAN RHYTHMS, Jared Rutter,
Martin Reick, and Steven L. McKnight 307
DNA REPLICATION IN EUKARYOTIC CELLS, Stephen P. Bell and
Anindya Dutta 333
THE LA PROTEIN, Sandra L. Wolin and Tommy Cedervall 375
LIPOPROTEIN RECEPTORS IN THE NERVOUS SYSTEM, Joachim Herz and
Hans H. Bock 405
v
vi CONTENTS
ORDER OUT OF CHAOS: ASSEMBLY OF LIGAND BINDING SITES IN HEPARAN

SULFATE, Jeffrey D. Esko and Scott B. Selleck 435
NEURONAL CA2⫹/CALMODULIN-DEPENDENT PROTEIN KINASE II: THE ROLE
OF STRUCTURE AND AUTOREGULATION IN CELLULAR FUNCTION,
Andy Hudmon and Howard Schulman 473
BIOCHEMISTRY OF NA,K-ATPASE, Jack H. Kaplan 511
MAMMALIAN ABC TRANSPORTERS IN HEALTH AND DISEASE, P. Borst
and R. Oude Elferink 537
HOMOGENEOUS GLYCOPEPTIDES AND GLYCOPROTEINS FOR BIOLOGICAL
INVESTIGATION, Michael J. Grogan, Matthew R. Pratt,
Lisa A. Marcaurelle, and Carolyn R. Bertozzi 593

LIPOPOLYSACCHARIDE ENDOTOXINS, Christian R. H. Raetz and
Chris Whitfield 635

FORMATION OF UNUSUAL SUGARS: MECHANISTIC STUDIES AND
BIOSYNTHETIC APPLICATIONS, Xuemei M. He and Hung-wen Liu 701
NUCLEAR ACTIN AND ACTIN-RELATED PROTEINS IN CHROMATIN
REMODELING, Ivan A. Olave, Samara L. Reck-Peterson, and
Gerald R. Crabtree 755
MECHANISMS OF FAST PROTEIN FOLDING, Jeffrey K. Myers and
Terrence G. Oas 783
RNA EDITING BY ADENOSINE DEAMINASES THAT ACT ON RNA,
Brenda L. Bass 817
CATALYTIC PROFICIENCY: THE UNUSUAL CASE OF OMP DECARBOXYLASE,
Brian G. Miller and Richard Wolfenden 847
CATALYTIC STRATEGIES OF THE HEPATITIS DELTA VIRUS RIBOZYMES,
I-hung Shih and Michael D. Been 887
INDEXES
Author Index 919
Subject Index 995
Cumulative Index of Contributing Authors, Volumes 67–71 1035
Cumulative Index of Chapter Titles, Volumes 67–71 1039
ERRATA
An online log of corrections to Annual Review of Biochemistry chapters
may be found at http://biochem.annualreviews.org/errata.shtml

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Annu. Rev. Biochem. 2002.

ACTIVE SITE TIGHTNESS AND SUBSTRATE FIT

Key Words fidelity, hydrogen bond, hydrophobic effects, base stacking,

Replication of Nonpurine, Nonpyrimidine Molecular Shapes. . . . . . . . . . . . . 202

Variations in Selectivity Without Hydrogen Bonds. . . . . . . . . . . . . . . . . . . 211

OTHER SIGNIFICANT ACTIVE SITE INTERACTIONS . . . . . . . . . . . . . . . 213

Many noncovalent interactions between the deoxynucleoside triphosphate

Figure 1 Examples of various methylated nucleobases derived from thymine and

NUCLEOSIDE ANALOGS AS PROBES OF

Simple Modifications of Natural Bases

lesions may be biologically relevant to mutagenesis. However, in analysis of

influences, such as steric effects versus hydrogen bonding effects, because

Nonpolar Shape Mimics of Natural Bases

Figure 2 The structures of the “bases” of nonpolar nucleoside isosteres F, D, Z, and H.

complementarity, nor by strong differences in solvation. Thus, one expects that

Nonpolar DNA Bases Designed De Novo

Size-Altered DNA Bases

Figure 4 The structures of Leonard’s extended nucleobases, lin-benzoadenine and

coworkers described extended analogs of the nucleobases A and G (Figure 4), in

Sterically Augmented Sugars

THE VARIED FIDELITY OF DNA POLYMERASES

TABLE 1 A survey of nucleotide insertion fidelities for various DNA polymerases

Polymerase Error ratea Reference

Pol III 107

Of this group of widely varied DNA polymerases, the fidelity of initial

Mutations That Affect Fidelity

EVIDENCE AGAINST A PURE HYDROGEN BONDING

The A Rule and Noninstructional Lesions

Nonpolar Nucleoside Analogs Are Replicated Efficiently

strand of DNA is processed efficiently and selectively by enzymes such as KF,

Replication of Nonpurine, Nonpyrimidine Molecular

Some of these factors are discussed below.

THE STERIC MODEL OF DNA REPLICATION FIDELITY

Definition of the Active Site

Figure 6 Schematic diagram illustrating the dimensions and determinants of the

Consensus Base Pair Shape

Figure 8 Schematic diagram

dates at least this consensus shape in

four pairs approximately equally

Size Exclusion in the Active Site

Solvation Effects on Nucleobase Size and Shape

(counting numbers of hydrogen bonds) for forming an isolated pair is small to

Limitations of the Steric Model

THE CONCEPT OF ACTIVE SITE TIGHTNESS

EVIDENCE FOR ACTIVE SITE TIGHTNESS AS A

Variations in Selectivity Without Hydrogen Bonds

consistent with that notion.

High-Fidelity Nucleotide Analogs

Effects of Polymerase Mutations

poration of larger-than-normal base pairs. If the active site tightness varies

OTHER SIGNIFICANT ACTIVE SITE INTERACTIONS

complement. This suggests a value of as much as 3–5 kcal/mol of lost interac-

Watson-Crick Hydrogen Bonds

Triphosphate and Sugar Recognition

Minor Groove Hydrogen Bonds

more studies are needed on this point.

CONCLUSIONS AND FUTURE PROSPECTS

Importantly, they need to be studied with polymerases using quantitative kinetic

The Annual Review of Biochemistry is online at http://biochem.annualreviews.org

9. Haracska L, Prakash S, Prakash L. 2000.

1985. Mutat. Res. 150:77– 84 Enzymol. 262:217–32

71. Hirsch JA. 1967. Table of Conforma-

Kunkel TA. 1990. J. Biol. Chem. 265:

Tahmassebi DC, Kool ET. 2000. J. Am.