From the
Previous studies showed a complex relationship between
nucleotide composition of a gene and the rate of the gene's
evolutionary variation. We have investigated mechanisms by constructing
M13 phagemids containing part of the Escherichia coli lacZ gene, in which an opal codon is flanked either by nine
adenine
The studies described in this paper were inspired by a report
(Wolfe et al., 1989) that the rate at which mammalian DNA
sequences undergo evolutionary variation is a complex function of the
guanine
Most in vitro studies have been carried out either at
rather extreme dNTP pool biases, or with unnatural DNA templates, or
both. The present studies were designed to ask: 1) whether replication
of a natural gene sequence, at dNTP concentrations approximating
natural pool asymmetries, is mutagenic; and 2) whether the immediate
base sequence context influences replicative error rates in ways that
would help explain the observed relationships between base composition
of a gene and its evolutionary variation.
These studies used the modified phage M13mp2SV, described by
Roberts and Kunkel (1988). This phage contains in its genome an SV40
DNA replication origin and a mutational target consisting of the first
45 codons for Escherichia coli
Other methods were also as described by
Roberts and Kunkel (1988) and by Roberts et al. (1991),
including preparation of HeLa cell extracts, preparation and
purification of double-strand replicative form DNAs, conditions for
SV40 origin-dependent DNA replication catalyzed by HeLa cell extracts,
DpnI digestion to eliminate unreplicated DNA from analyses,
electroporation of replicated DNA, plating on E. coli CSH50,
and scoring mutations on the basis of plaque color. The host strain for
electroporation, E. coli NR9162, was
mutS
dNTP pool measurements were
carried out essentially as described by North et al. (1980).
These analyses, when carried out on the concentrated HeLa cell extracts
used for the replication reactions, confirmedthat the dNTPs in these
extracts contributed negligibly toward the dNTP concentrations in each
reaction mixture. Also, similar analyses confirmed that dNTP
degradation in the replication reactions was negligible (less than 5%
of the starting values) over the course of the replication reactions.
For analysis of the M13mp2SV derivatives that had replicated in
vivo, COS7 cells were grown in Dulbecco's modified Eagle
medium (DMEM)(
Originally we contemplated a ``global'' approach to
analyzing the relationship between DNA base composition, dNTP pool
asymmetry, and mutagenesis. We planned to modify the G+C content
of the 135-base pair protein-coding part of the lacZ gene in
M13mp2SV, to values as high and low as possible without changing the
amino acid sequence of the encoded lacZ
However, a preliminary analysis
(Table I) indicated that this approach would not be feasible. M13mp2SV
DNA was replicated either in the presence of an equimolar dNTP mixture
(100 µ
M each dNTP) or an asymmetric mixture representing
the estimated dNTP concentrations in S-phase HeLa cell nuclei (60
µ
M dATP, 60 µ
M dTTP, 30 µ
M dCTP,
10 µ
M dGTP) (Leeds et al., 1985). In both cases
the replicated DNA samples showed significantly more mutants than
unreplicated controls, which were treated identically to the
experimental reaction mixtures, except for the omission of SV40
T-antigen during incubations with HeLa cell extract. However, we did
not see a significant difference in mutation frequencies between the
equimolar and asymmetric dNTP mixtures. It was apparent that an
extremely large number of plates would have to be counted, if we were
to learn whether the small difference we did observe was significant,
since detection of white or light blue plaques can be done only if
there are fewer than about 500 dark blue plaques/plate.
Accordingly,
we turned from the global to a more local approach, involving reversion
and pseudoreversion events within one codon. Reversion analysis in this
system involves scoring blue plaques against a white plaque background,
and this allows inspection of a far larger number of plaques per plate
than does the forward mutation assay. For our analysis we chose a
serine codon (residue 7) in a flexible part of the lacZ
Extensive sequence analysis of
mutants generated during in vitro replication of the
lacZ
Because we
are interested in sequence context as a determinant of replication
fidelity in the presence of biologically biased dNTP concentrations, we
wished to alter the base sequences flanking the local mutational
target, namely the opal codon introduced in place of the serine-7
codon. Accordingly, we designed two sets of flanking sequences, as
shown in Table II. In one construct,
(AT)
Data from two experiments,
summarized in Table III, reveal several noteworthy results. First, as
noted elsewhere ( cf. Roberts et al. (1991)),
replication in this in vitro system is quite accurate. The
DNAs replicated in equimolar dNTP mixtures showed error rates
comparable to those seen in the unreplicated controls
(``background'' in Experiment 2). Second, the biological
asymmetry in DNA precursor pools apparently does contribute toward the
natural mutation rate. In all three constructs the mutant fraction was
significantly higher when the DNA was replicated at
``biological'' dNTP concentrations, biased as described in
. This effect was particularly significant in the
(AT)
The third noteworthy result is the fact that
replication of the three constructs in vivo, rather than under
biologically biased pool conditions in vitro, also yielded
mutant fractions significantly above background. In fact, as shown in
the fourth line of Experiment 2, these values were even somewhat higher
than the corresponding values from the in vitro experiment
(third line). Of course, factors other than dNTP asymmetries may well
contribute toward the error rates seen during replication in living
cells. Mismatch repair, for example, could occur in vivo, but
this would tend to decrease the mutant fractions to values lower than those seen after incubation in vitro. In any event,
the results are consistent with the hypothesis that biological dNTP
pool asymmetries contribute toward the natural replication error rate.
Fourth, whether replicated in vivo in bacterial cells
(``background'') or mammalian cells, or in vitro in
equimolar or biologically biased dNTP pools, replication was
significantly more accurate when the opal codon was flanked by G
Fifth, although
a GC-rich sequence context seems to promote correct base pairing at the
insertion step, maintenance of high fidelity is highly dependent upon
proofreading of insertion errors that do occur. Note both from
Experiment 1 and from the fifth and sixth lines from Experiment 2 in
I the extraordinary sensitivity of the
(GC)
Essentially the same conclusion can be drawn from the ``dGTP
excess'' experiments (Experiment 2, last line). Presumably, the
mutations here were caused largely by the next nucleotide effect, which
involves pool-driven incorporation of nucleotides past the site of a
substitution error before that error can be repaired exonucleolytically
(Roberts et al., 1991). Again, if helix unwinding is slower
when the site of an error is flanked by guanine
To propose
an effect of flanking helix stability upon proofreading efficiency in
this system is to propose that proofreading in eukaryotic DNA
replication involves significant helix unwinding to place the primer
terminus in the 3` exonuclease site, as evidently occurs in prokaryotic
DNA replication (Beese et al., 1993). Whereas structural
studies on eukaryotic replication proteins make this a reasonable
expectation ( cf. Wang, 1991; Beckman and Loeb, 1994), it has
not been explicitly demonstrated. However, our results are consistent
with this model.
The influence of base sequence context upon
replication fidelity has long been apparent, simply from the existence
of hot spots for spontaneous mutagenesis. However, systematic analysis
of this phenomenon has begun only recently. Of particular interest is a
study of Bloom et al. (1994), who used pre-steady-state
kinetic analysis to analyze 3` exonucleolytic proofreading, and who
showed also the influence of helix stability at the primer terminus
upon replication accuracy. The system of Bloom et al. involves
proofreading of a nucleotide analog, in replication of synthetic DNA
templates by a purified DNA polymerase. By contrast, our system
involves replication of natural or near-natural DNA sequences by a
multiprotein replication apparatus using natural DNA precursors at
concentrations that can be adjusted to near-natural levels. Both kinds
of analyses should be mutually supportive as investigations of
spontaneous mutagenesis continue.
The preliminary results reported
here demonstrate, we believe, the utility of this approach to
understanding the effects of natural dNTP asymmetries upon replication
accuracy and spontaneous mutagenesis. The results suggest a variety of
informative approaches to be taken in subsequent investigations,
including sequence analysis of the revertants, analysis of different
sequence contexts ( e.g. AT upstream, GC downstream), and more
definitive analyses of the effective dNTP concentrations at eukaryotic
DNA replication sites.
0.5 µg of
M13mp2SV replicative form DNA was replicated in each assay for 6 h, as
described by Roberts and Kunkel (1988), at the specified dNTP
concentrations. Incorporation of radioactivity from
[
Aside from the sequence alterations shown, each construct is
identical to M13mp2SV. Each altered sequence extends from codon 4
through 10 of the coding sequence for the lacZ
The DNA constructs
described in Table II were replicated in vitro as described
for Table I, except that the incubation period was 3 h. All dark blue
and light blue plaques were scored as mutations, and the mutant
fraction is the ratio of mutant to total plaques counted. The actual
numbers of mutant and total plaques scored after each incubation are
shown in parentheses. ``Background'' denotes DNAs incubated
in the absence of SV40 T antigen, where no detectable replication
occurred. ``Replicated in vivo'' means replicated
in, and isolated from, COS7 cells. ND, not determined.
Much of the work described in this paper was carried
out in the laboratory of Dr. Thomas A. Kunkel, NIEHS, National
Institutes of Health during separate visits by each of the two authors
to that laboratory. We are grateful to Dr. Kunkel and to Drs. John D.
Roberts and David C. Thomas of that laboratory for hospitality,
instruction, and guidance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
thymine base pairs on each side, or by nine
guanine
cytosine pairs, or by its wild-type sequence context.
Reversions or pseudoreversions within the opal codon yield a lacZ
-peptide that can undergo
-complementation and yield a
blue plaque when plated with a chromogenic substrate. When these
constructs were replicated in HeLa cell extracts, in the presence of
equimolar deoxyribonucleoside triphosphate (dNTP) mixtures, reversion
was near background levels in both the AT-rich and GC-rich contexts. By
contrast, when the DNAs were replicated at dNTP concentrations
approximating those in HeLa cell nuclei, increases over background were
seen in all three contexts. Replication of the phagemids in vivo led to even higher mutation frequencies. Replication in the
presence of dGMP, added to inhibit proofreading, caused extraordinarily
high reversion frequencies in the GC-flanked opal codon. Apparently,
dNTP concentrations approximating intracellular concentrations are
mildly but significantly mutagenic, and pool asymmetries and base
sequence context both contribute to the natural fidelity of DNA
replication.
cytosine content of the sequence, with the highest rates
observed in sequences containing about 50% G+C. It seemed likely
that natural asymmetries in intracellular concentrations of
deoxyribonucleoside triphosphates (dNTPs) could be at least partly
responsible for the variations observed (Mathews and Ji, 1992).
Specifically, dGTP accounts for only 5-10% of the total pool of
the four common dNTPs in most mammalian cell lines that have been
studied. Thus, misinsertion opposite template dCMP residues might be
relatively frequent, causing increased mutation rates with increasing
G+C content. At the same time, replication immediately 5` to a
template dCMP residue might be more accurate if dGMP insertion at that
site were slow enough to increase the probability of excision of
misinserted nucleotides at the upstream site; this effect would
decrease the replication error rate as a function of increased G+C
content, as schematized below.It is well known that dNTP pool biases are mutagenic during DNA
replication both in vitro and in vivo ( cf. Kunkel (1992) and Kunz et al. (1994)). The relationship
between dNTP pool biases and replication fidelity has become of special
interest with the report that pool imbalances during reverse
transcription may be responsible for hypervariability of the human
immunodeficiency virus genome (Vartanian et al., 1994).
-galactosidase plus 115
nucleotides of upstream sequence. Expression of the 45 codons of
wild-type sequence generates a peptide that can undergo
-complementation when introduced into a host strain, E. coli CSH50, which expresses the remainder of the lacZ gene.
Complementation is scored by plating in the presence of
5-bromo-4-chloro-3-indoyl
-
D-galactoside, a chromogenic
substrate for
-galactosidase. A deep blue plaque is scored as
wild-type, while mutants yield white or light blue plaques. Constructs
for the reversion assays described here were prepared from M13mp2SV by
site-directed mutagenesis, using the methods of Kunkel et al. (1987).
, to minimize loss of replicational
heterozygotes due to mismatch repair. In the reversion assays, plates
were incubated for 15-18 h at 37 °C, followed by an
additional 48-h incubation at room temperature, to allow detection of
the maximum number of mutational events.
)
plus 10% fetal bovine serum to
about 40% confluence, then washed twice with Opti-MEM reduced serum
medium (Life Technologies, Inc.). For each 100-mm culture dish, 3
µg of replicative form I DNA and 10 µl of Transfectase reagent
(Life Technologies, Inc.) were diluted to 300 µl with Opti-MEM I
reduced serum medium, then mixed together. After standing 20 min at
room temperature for formation of lipid-DNA complexes, each mixture was
diluted to 3.0 ml with the same medium and added to cells treated as
described above. Cells were incubated for 10 h at 37 °C, and then
3.0 ml of DMEM plus 20% fetal bovine serum was added, and incubation
was continued for 24 h. At that point, medium was replaced with DMEM
containing 10% fetal bovine serum and incubation continued for 12 h
more. After trypsin treatment and centrifugation, each cell pellet was
washed with phosphate-buffered saline and resuspended in 200 µl of
50 m
M Tris-HCl, pH 7.5, 10 m
M EDTA, and 100 µg/ml
RNase A. Cells were lysed by adding an equal volume of 0.2
M NaOH and 1% SDS. Chromosomal DNA and cell debris were precipitated
by adding potassium acetate, pH 4.8, to a final concentration of 0.44
M, followed by centrifugation. Episomal DNA was purified
through Wizard mini-columns (Promega), and unreplicated DNA was
eliminated from each mixture by digestion with DpnI prior to
electroporation into E. coli NR9162 and subsequent analysis
for revertants.
-peptide. We
would then replicate these modified constructs in vitro, in
the presence of dNTP concentrations chosen to represent the approximate
levels within HeLa cell nuclei (Leeds et al., 1985) and
determine the extent to which the natural asymmetry in dNTP levels
influenced replication error frequencies. This latter analysis would
involve a forward mutation assay ( lacZ
lacZ
), where mutations anywhere in the
135-base pair target could be scored as a change in plaque color from
dark blue to white or light blue.
mutational target. The TCA encoding this serine was changed to an opal
codon (TGA), and revertants or pseudorevertants were scored as dark
blue or light blue plaque formers.
target in M13mp2 and its derivatives has revealed
few null (white plaque) mutations within this region ( cf. Kunkel and Alexander (1986)), suggesting that most mutations
occurring here allow some retention of wild-type protein function. This
means that: 1) we can alter the sequences flanking this codon and
expect relatively little effect on protein function, and 2) we can
expect most single-base substitution errors involving the engineered
opal codon to generate a wild or pseudo-wild phenotype and, hence, to
be scored as mutational events in a reversion analysis.
TCA(AT)
, the opal codon was flanked on each
side by nine adenine
thymine base pairs, which generated two
conservative changes in the six codons from the wild-type flanking
sequence. Design of the other construct,
(GC)
TCA(GC)
, required more changes in order to
flank the opal codon with nine guanine
cytosine pairs on each side.
However, the changes apparently had little effect on function of the
gene product, because many of the revertant plaques seen in analysis of
all three constructs had a deep blue color indistinguishable from that
given by the wild-type sequence.
TCA(AT)
construct, where the mutant
fractions in biological and equimolar dNTP mixtures differed by a
factor of 3.5.
C
base pairs than with either A
T base pairs or with the natural
nucleotides. This may reflect the stability of guanine
cytosine
base pairs, which could lower the tolerance for insertion of
incorrectly base paired nucleotides. This interpretation is consistent
with the relatively large difference in mutant fraction between
equimolar and biologically biased pools for the AT-flanked opal codon,
described above. Formation of a C
dGTP pair occurs during normal
replication of the 3`-ACT-5` trinucleotide in the antisense strand at
the opal codon. If mispairing in this site at low dGTP concentrations
occurs more readily in an AT-rich sequence context, then the results
described in the previous two paragraphs are readily understood. It
seems unlikely that variations in mismatch repair are involved, because
mismatch repair activities are thought to be low during replication in
HeLa cell extracts (Roberts et al., 1991).
TGA(GC)
target to inhibition of
proofreading, brought about by addition of a deoxyribonucleoside
monophosphate at high concentrations. Error rates increased in all
three constructs, but the severalfold increment in replication
accuracy caused by GC-rich flanking sequences when proofreading was not
inhibited was replaced by a decrement in replication accuracy,
by about an order of magnitude, when proofreading was inhibited.
cytosine pairs, the
sensitivity of the (GC)
TGA(GC)
construct to
mutagenesis under these conditions is easily understood.
Table: Forward mutation assay:
laclac
-
P]dCTP confirmed that replication was
undetectable in the controls incubated in the absence of SV40 T
antigen.
Table: DNA constructs used in the reversion assay
-peptide.
Table: Reversion and pseudoreversion
mutations generated during DNA replication
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.