From the Laboratory of Molecular Genetics, NIEHS,
National Institutes of Health, Research Triangle Park, North Carolina
27709-2233, the ¶ Department of Biochemistry, Tulane University
Health Sciences Center, New Orleans, Louisiana 70112-2699, and the
Department of Molecular Biophysics and Biochemistry, Yale
University, New Haven, Connecticut 06510-3219
Received for publication, August 23, 2000, and in revised form, December 11, 2000
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ABSTRACT |
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The DNA polymerases (gp43s) of the related
bacteriophages T4 and RB69 are B family (polymerase Bacteriophage RB69 is a relative of phage T4, with which it shares
many similarities in genetic organization (1, 2) and structures and
functions of the phage-encoded DNA replication proteins (3, 4).
Replication fidelity in T4 and presumably also in RB69 is determined
almost exclusively by the fidelities of the phage-encoded DNA
polymerase and its associated proofreading 3'-5' exonuclease (5). This
useful simplicity reflects the fact that T4 DNA replication appears to
be devoid of DNA mismatch repair; phage T4 is not subject to the action
of the several Escherichia coli mismatch repair systems (6)
and seems unable to repair mutational heteroduplexes on its own.
Screens for T4 mutator mutations have failed to uncover evidence for
the involvement of mismatch repair in mutagenesis, and the mutational
dose response to base analogues does not display the mismatch
repair-dependent lag seen in E. coli (5).
The DNA polymerases of phages T4 and RB69 (gp43, product of
phage gene 43) are members of the polymerase RB69 gp43 (903 residues) and T4 gp43 (898 residues) differ at ~40%
of their amino acids but are probably quite similar in tertiary
structure and function because they can substitute for each other to
conduct phage DNA replication in vivo (3). In particular,
RB69 gp43 has almost the same proficiency and fidelity as T4 gp43 when
replicating the T4 genome in vivo (3, 18). Thus, one can
readily use clones of specifically altered alleles of RB69 gene
43 to study the roles of its five structural domains (and
amino acids therein) in supporting T4 DNA replication. Although numerous T4 gp43 alterations can alter the fidelity of DNA replication (12), the ~40% divergence between the T4 and RB69 gp43s mandates a
focus on the RB69 enzyme when pursuing structure-function relationships because a crystallographic structure is available for RB69 gp43 but not
for T4 gp43. Such structural information is apt to be particularly
important when probing interactions between gp43 domains, notably of
the type to be described in this report, because conserved residues
exist in environments that are likely to be influenced by nonconserved
residues. Another advantage of the RB69-T4 complementation system is
that although recombination occurs vigorously between a phage and a
plasmid carrying the T4 gene 43, such recombination is rare
(e.g. < 10 Gp43 and many other DNA polymerases control fidelity through two
catalytic functions, a template-dependent 5'-3' nucleotidyl transferase activity (the Pol function) and a single-stranded DNA-dependent 3'-5' proofreading exonuclease activity (the
Exo function). Although some replicative DNA polymerases have separate Pol and Exo subunits, the catalytic centers for the two gp43 activities reside in separate structural modules of the same polypeptide (15). It
is clear that the biological role of the Exo function is to erase
errors committed by the Pol function, but it is much less clear how the
Pol function makes (or avoids) errors in the first place and how the
enzyme recruits the Exo function to reverse such errors. Some insights
have been obtained from biochemical studies of T4 gp43 and of the
polymerase I family (A family) T7 DNA polymerase, which, like gp43,
bears separate Pol and Exo modules in the same polypeptide
(19-21). Kinetic assays with these enzymes indicate that the fidelity
of the Pol function is achieved through two transactions that precede
proofreading and that occur at or near the Pol catalytic center (20).
The first step is accurate selection of an incoming dNTP at the single
dNTP-binding site, and the second step is slowed primer extension from
a mispaired base at the primer terminus. The base selection step
provides a large contribution to fidelity and appears to depend both on base pair geometry and on the hydrogen bonding potential of the incoming dNTP (22-25). The primer extension step depends particularly strongly on the hydrogen bonding potential of the 3'-terminal nucleotide (26) and may provide the signal for transferring the primer
terminus to the Exo catalytic site for proofreading (20). Both
structural and biochemical evidence suggest that the switch from primer
extension to proofreading involves a conformational transition in the
enzyme from a "closed" (or Pol) mode to an open (or Exo) mode. This
transition includes fraying the primer end to allow its appropriate
positioning relative to the Exo catalytic center (17, 27).
Recently, a cluster of amino acid residues at the juncture of the Palm
and Fingers domains of RB69 gp43 was implicated in dNTP binding (28).
One of these residues, Tyr567, was proposed to play a role
in interactions with the base component of the incoming dNTP during the
alignment of the nucleotide for nucleotidyl transfer. We show here that
substitutions at this residue can dramatically increase replication
errors while exhibiting only small effects on total DNA synthesis and
viable phage production. An RB69 gp43 with the Y567A substitution is
highly mutagenic in vivo while exhibiting normal
3'-exonuclease activity in vitro. Thus, the mutator activity
of Y567A-gp43 is not caused by a proofreading-exonuclease defect. An
RB69 Y567A-gp43 mutant that is also defective in the Exo function
(through the introduction of a D222A/D327A double substitution) does
not support viable phage production, although it does support 50-70%
of the normal amount of DNA synthesis. Combining Y567A with the
proofreading defect increases the mutation rate only modestly over the
increase caused by either component alone. However, the combined
increase appears to be sufficient to cause error catastrophe. Based on
an analysis of the in vivo mutational spectra produced by
Exo Materials
Strains--
Wild type T4 and the T4 gene 43 double-amber mutant amE4332 amE4322 (hereafter referred to
as 43am) with UAG codons at positions 202 and 386 were
described previously (18). Using two amber mutations minimizes
translational read-through in nonpermissive (supo) bacterial hosts (29).
Three T4 rII mutants (30) were used for reversion tests.
rIIUV131 carries a
Most E. coli strains have been described (18). E. coli strain B40 su+II (supE)
efficiently suppresses T4 amber mutations. E. coli strain BB
lacks amber-suppressing activity; it displays the characteristic r
plaque phenotype with T4 rI mutants and also with a few
other kinds of T4 r mutants but not with T4 rII
mutants. E. coli K12 strain QA1(
pRB.43 denotes a plasmid encoding RB69 gene 43 (or a mutant
version of this gene) in a T7 promoter vector in which gene
43 expression is repressed. Although leak-through expression
is weak, it produces sufficient RB69 gp43 to replicate T4
43am somewhat better than is achieved using the
amber-suppressor B40 su+II. Good replication is
achieved in T4 with only a small amount of gp43 (29, 32). Recombination
between the phage and plasmid-borne gene 43 homologues is
negligible (3, 18).
Media--
LB broth, M9 synthetic medium supplemented with
0.25% vitamin-free casamino acids and 1 µg/ml vitamin B1
(M9SB), Drake bottom agar, and Drake super-soft top agar have been
described (33, 34).
Other--
T4 polynucleotide kinase was from New England
Biolabs. [ Methods
Growth, Screening, and Assay Conditions--
Cells for genetic
experiments were grown in LB broth in a rotary shaker water bath at
37 °C. Plates were incubated overnight at 37 °C. T4
am43 stocks (with or without rII mutations) were grown on BB cells carrying the desired version of pRB.43. QA1 cells
were used to assay am43 rII+ revertants. B40
su+II cells were used to assay am43
stocks with or without rII mutations. BB cells carrying
pRB.43 (wild type) were used to screen for mutant plaques displaying
the rI phenotype.
Cloning--
Plasmid CW19R carries a wild type RB69 gene
43 (designated pRB.43 Pol+ Exo+)
under the control of the T7 Phage T4 DNA Synthesis and Progeny Production in Vivo--
For
the experiments to be described later in Fig. 1A, cultures
of E. coli BB cells carrying a pRB.43 plasmid were grown at 30 °C with vigorous aeration to 3 × 108 cells/ml
in M9SB medium containing ampicillin at 20 µg/ml. Then 0.2 ml of
culture was added to 0.1 ml of fresh medium containing 6 × 108 plaque-forming units of T4 43am phage
(multiplicity of infection = 10), and aeration was continued at
30 °C. After 22 min, [3H]thymidine was added (5 µCi/ml 3H at a specific activity of 10 µCi
3H/µg dT). After 15 min (37 min postinfection), further
[3H]thymidine labeling was slowed in an ice bath and
trichloroacetic acid-precipitable 3H counts were
determined. Only T4 DNA is synthesized after the first few moments of infection.
To measure burst sizes, E. coli BB was grown to 3 × 108 cells/ml at 30 °C in M9SB medium. At 0 min, 1-ml
samples were infected with 107 plaque-forming units of T4
43am or T4 wild type added in 10 µl of M9SB. At 12 min the
cultures were diluted 103-fold in M9SB and aerated at
30 °C, and infective centers were assayed. Under these conditions,
cell lysis begins at about 42 min. Cell lysis was completed by the
addition of chloroform to the diluted infected cultures at 60 min.
To measure T4-induced DNA synthesis in E. coli NapIV cells
carrying pRB.43 (shown later in Fig. 1B), the cells were
grown at 30 °C with vigorous aeration to 3 × 108
cells/ml in M9SB medium containing ampicillin at 20 µg/ml. 2 ml of
each culture were added to 1 ml of fresh medium containing 6 × 109 plaque-forming units of T4 43am phage
(multiplicity of infection = 10), and aeration was continued at
30 °C. After 5 min, [3H]thymidine was added (20 µCi/ml at a specific activity of 20 µCi/µg dT). Samples (0.1 ml)
were withdrawn at various times thereafter to determine trichloroacetic
acid-precipitable counts.
For alkaline gel analysis of newly synthesized T4 DNA (Fig.
1C), E. coli NapIV cells carrying the desired
pRB.43plasmid were grown and infected with T4 43am phage as
described above. [3H]thymidine (32 µCi/ml, 10 µCi/µg) was added to 8 ml of each infected cell at 15 min
postinfection. Radiolabeling was stopped at 30 min postinfection by
chilling the cultures in an ethanol/wet ice bath. The cells were
harvested by centrifuging (5000 × g, 5 min) in the
cold and resuspending in 250 µl of TE buffer (10 mM Tris-HCl; 1mM EDTA pH 8.0), and their DNA
was extracted by the Phase Lock Gel method (catalogue number p1-678901,
5 Prime Reversion Tests--
To measure rII+
revertant frequencies, stocks of T4 am43 rII mutants were
grown in E. coli BB cells carrying the desired pRB.43 allele. Revertants were scored by plating on QA1 cells, and the total
phage were scored by plating on B40 su+II cells.
Revertant frequencies are the median values for 21 stocks; for as few
as 5 stocks, 2-fold reproducibility is observed about 95% of the time
(6).
Forward Mutation Tests--
Mutations in several T4 r
(rapid lysis) genes result in large plaques with sharp edges.
rI mutants predominate among mutants detected by plating on
BB cells. The rI gene is a good candidate for a mutation
reporter gene (35, 36). It seems not to be involved in DNA metabolism,
it is of appropriate size (294 base pairs including the termination
codon) for repetitive sequencing, and it displays a mutant phenotype
for many missense mutations (as confirmed in this study). Phage stocks
for measuring rI mutant frequencies consisted of individual
T4 43am plaques recovered in their entirety from lawns of BB
cells carrying the desired pRB.43 allele. The plaques were resuspended
in 1 ml of LB broth plus a drop of chloroform and were plated on BB
cells bearing pRB.43 Pol+ Exo+ (pCW19R) to
yield about 600 plaques/plate, and the plates were then screened for
r mutants. rI frequencies (median values for 7-21 stocks) consist of the total r mutant frequency
multiplied by the fraction of rI mutants among all
r mutants (as determined by subsequent sequencing). The
correction factor (0.64) was the ratio of 287 rI mutants
among 449 sequenced r mutants distributed among the four
gene 43 genotypes.
Measuring Mutations beyond the Error-Catastrophe Barrier--
T4
43am phage infecting BB cells carrying pRB.43
PolY567A Exo Calculating Mutation Rates--
In the experiments reported
here, sufficiently large numbers of mutational events occurred per
T4-infected culture to justify using the expression µrI = f/ln(N/N0), where
µrI is the mutation rate of the rI gene per
replication, f is the observed mutation frequency,
N is the final population size, and N0 is the initial population size, or, for
N0 Sequencing rI Mutants--
Mutant plaques were resuspended in 40 µl of water, and the rI region was directly
amplified by PCR and sequenced. The upstream primer was
5'-GTTAAGGCCCTGCATCG-3' and the downstream primer was 5'-CCTAAGTATTCATCTGCCTTTG-3' for both PCR and sequencing. The PCR
consisted of 30 cycles of 1 min at 94 °C, 1 min at 55 °C, 1 min
at 72 °C, with a final extension time of 10 min at 72 °C using
Taq large fragment polymerase (Display System Biotech TAQFL from PGC Scientifics, Gaithersburg, MD). PCR products were purified with the PCR purification kit (Qiagen). Sequencing was performed with an ABI Prizm 377 automatic sequencer using the dRhodamine terminator cycle sequencing kit (PE Applied
Biosystem). Each mutation was identified by sequencing in both directions.
Determining Kinetic Parameters--
Steady-state kinetic
parameters for both misinsertion and mispair extension were determined
as described (28). For misinsertion assays, 13-mers were
5'-32P-labeled by standard procedures, annealed with the
complementary 20-mers, and used as substrates. Three sets of 13/20-mers
were designed for misinsertion assays, where the base in bold type is
the template base to pair with the correct or incorrect incoming dNTP:
P/T-1 for T/T misinsertion,
5'-CCGACCAGCCTTG-3'/3'-GGCTGGTCGGAACTGGGGGG-5'; P/T-2 for
T/G and G/G misinsertions,
5'-CCGACCAGCCTTG-3'/3'-GGCTGGTCGGAACGTTTTTT-5'; and P/T-3
for C/C misinsertion,
5'-CCGACCACGGAAC-3'/3'-GGCTGGTGCCTTGCAAAAAA-5'. For mispair
extension assays, the 13/20-mer primer terminus and its cognate
template base are in bold type: P/T-4,
5'-CCGACCACGGAAC-3'/3'-GGCTGGTGCCTTGCAAAAAA-5'; P/T-5,
5'-CCGACCACGGAAG-3'/3'-GGCTGGTGCCTTTCAAAAAA-5'; and P/T-6,
5'-CCGACCACGGAAC-3'/3'-GGCTGGTGCCTTTCAAAAAA-5'.
Effects of Substitutions at RB69 gp43 Tyr567--
The
tyrosine at position 567 of RB69 gp43 (Tyr564 in T4 gp43)
is highly conserved in Region III of B family DNA polymerases (7, 11).
Nevertheless, we found that replacing this residue with alanine or
several other amino acids affects replication functions only weakly. T4
am43 was used to infect E. coli BB cells carrying a plasmid expressing one or another allele of RB69 gene 43,
and both DNA synthesis and phage growth were monitored (Fig.
1, A and B). The
Exo
In contrast to alanine, serine, threonine, and valine substitutions at
Tyr567, alanine substitutions at conserved residues located
close to Tyr567 in the crystal structure sharply reduce DNA
synthesis and/or phage production, e.g. N564A (Fig. 1,
A and C) and others (28). Unexpectedly, the
conservative substitution Y567F failed to support DNA replication. In
addition, gp43 PolY567F Exo
The PolY567A Exo
When we used denaturing gel electrophoresis to examine the size
distributions of 3H-labeled DNA synthesized in
vivo, no notable differences were observed among the four
genotypes used in this study (Pol+, PolY567A,
Exo+, or Exo Reversion Tests--
Measuring reversion of specific T4
rII mutations can quickly reveal changes in mutation rates
along particular mutational pathways. To this end, we used
rII mutants that revert by +1 frameshifts in a run of five
A·T base pairs or by base pair substitutions at either a G·C base
pair or an ochre codon (TAA/ATT). The results are summarized in Table
I. When Tyr567 was replaced
by serine, threonine, or alanine, the result was weak mutator activity
for frameshift mutations in an A·T run and strong mutator activity
for BPSs at both a G·C and an A·T site. The polymerases with serine
or threonine substitutions appear to be slightly more prone to
frameshift mutator activity than the polymerase with an alanine
substitution. In contrast to these Pol effects, a defect in exonuclease
function strongly promotes all three mutational pathways. Note that the
values in Table I are frequencies and not rates; under these
experimental conditions, values for rates are roughly an order of
magnitude smaller than for frequencies, but relative rates are similar
to relative frequencies.
Forward Mutation Tests--
We used forward mutation in the T4
rI gene to determine the mutational spectrum generated by
the RB69 gp43 mutator mutants. In contrast to reversion tests, which
tend to display high sensitivity, forward mutation tests provide
generality and, when augmented by sequencing, provide detailed
information about mutability at specific sites. Forward mutation tests
can also reveal classes of mutations that are not detected in reversion tests.
For the polymerases that supported high levels of T4 DNA synthesis and
phage production, it was straightforward to measure r mutant
frequencies (discussed later) and to collect mutants of independent
origin for sequencing. For gp43 PolY567A Exo Mutational Classes--
Table III
lists the numbers of mutations of different kinds arising in
Pol+ Exo+, Pol+ Exo
The Pol+ Exo+ mutational distribution contains
a majority of BPSs, a characteristic of most collections of spontaneous
mutations in diverse wild type organisms studied in
vivo; the exceptions involve distributions in genes that harbor
extraordinarily strong frameshift mutation hot spots or organisms
experiencing outbursts of transposon mobility. The Pol+
Exo+ distribution contains roughly twice as many
transversions as transitions, whereas the Pol+
Exo
The PolY567A Exo+ distribution consists almost
exclusively of BPSs with a substantial bias in favor of transitions.
This distribution is consistent with the reversion tests (Table I).
The PolY567A Exo
When three different substitutions at Tyr567 in the
Exo+ background were compared in the forward mutation test,
their mutational patterns (Table IV) and
their spectra (not shown) were qualitatively similar, although
quantitative differences were discernable. In reversion tests, we
observed more frameshift mutations with PolY567S and
PolY567T than with PolY567A (Table I). This
tendency is repeated in the forward mutation tests (
In all of the distributions, G·C Mutational Spectra--
Mutational spectra reveal widely different
intrinsic mutabilities at different sites. Highly mutable sites are
often called hot spots, but this designation is arbitrary because sites
typically display a smooth gradient of mutabilities rather than
discrete steps, at least within the resolving power of almost all
spectra. Hot spots are of considerable interest because they identify
genetically unstable sequences. They may also interfere with the
analysis of error proclivities by diluting out other kinds of
mutations. Most spontaneous frameshift hot spots are now interpreted as
being the result of slippage within repeated bases or short sequences during DNA replication. Repeats of six or more base pairs form very
strong frameshift hot spots in phage T4 (39, 40) and account, for
instance, for half of all rII mutations (41); fortunately, the rI reporter gene lacks repeats of more than five base
pairs. Spontaneous base substitution hot spots, on the other hand,
remain largely unexplained. The many contacts between a DNA polymerase and both the primer template and the incoming dNTP (25) probably modulate error frequencies in still uncharacterized ways that can vary
with local DNA sequence up to a dozen bases away (42, 43).
Fig. 2 shows the mutational spectra
obtained with Pol+ Exo+, Pol+
Exo
The Pol+ Exo
The PolY567S Exo+ and PolY567T
Exo+ spectra are very similar to the PolY567A
Exo+ spectrum and are therefore not shown here. Their
ratios of transitions to transversions, which are somewhat higher than
those of the PolY567A Exo+ spectrum (Table IV),
are reflected in the sharply reduced numbers of G·C
At sites of multiple occurrences, the PolY567A
Exo Sequence Determinants of Genetic Instability--
Mutationally
warm and hot sites and regions constitute DNA sequences that constrain
polymerase fidelity and that therefore may provide insights into
fidelity mechanisms. The hot spot at rI position 247 is
imbedded in a generally hypermutable sequence, 5'-TGGCAC-3', that is
similar to another hypermutable sequence, 5'-TGGCAA-3', previously
described in the T4 rIIB gene (44). In both cases the
central G is a transition hot spot, whereas the adjacent C mutates
moderately often and the first G only slightly more than average. (The
complement of this sequence, TTGCCA, is located at position 26-31 but
does not contribute mutations to any of our spectra; however, a
transition at the first C of this sequence would produce a Ser
Further inspection of all six rI spectra reveals a strong
association between hypermutability and GG (or CC) dinucleotides. We
then examined four other spontaneous spectra available for T4 (all
produced by T4 gp43) (36, 45) and found an identical association. Both
the central GG/CC motif and sites to its left and right frequently
display increased mutability, and this motif accounts for almost all
sites of hypermutability observed in T4 in vivo. The
increased mutability of G·C-rich regions may in part reflect a
previously suggested role for the increased stability of G·C base
pairs compared with A·T base pairs. Such differential stability might
modulate the melting of an adjacent mispair prior to partitioning from
the Pol site to the Exo site (21, 46). Indeed, the contribution of GG
and CC regions to mutability is higher in the two Exo+
spectra than in the two Exo Mutation Rates--
An experimentally determined mutation
frequency f (such as total mutants per total phages) can be
converted into a mutation rate µ (such as mutations per chromosome
replication) provided that the topology of replication is known (such
as semiconservative DNA replication) and the growth parameters of the
population have been measured (most often the final population size
N). The conditions required to apply the expression µ = f/ln(µN) are well met for the gp43 constructs
studied here except for gp43 PolY567A Exo
The forward mutation rates appear in Table
VI. As in the reversion tests, all of the
mutant polymerases display strong mutator activity. The top three
values are expected to be reproducible to within 1.5-fold or less (and
the next two to within 2-fold or less) about 95% of the time (6), so
that all four single-mutator values are indistinguishable. Because the
forward mutation test averages over many base pairs, at some of which
BPSs will not produce a mutant phenotype, the relative increases for
the Pol site mutators are lower than in the reversion tests. (Relative µg values, not shown here, are not identical to relative
µrI values because the correction factor C varies
according to the fraction of BPSs in each spectrum.) The mutator
activity of the Pol+ Exo Kinetic Parameters in Vitro--
Using highly purified,
nuclease-free Exo
The kinetic parameters for mispair formation are presented in Table
VII. Although these results are
preliminary, the trends are unequivocal. The catalytic efficiency with
PolY567A Exo
The kinetic parameters for mispair extension are presented in Table
VIII. The catalytic efficiencies for extending a normal base pair are
similar (2.2 versus 3.1) for these two gp43s. Both a
transition mispair and a transversion mispair were extended very
inefficiently, in some cases beyond the limits of measurement. Perhaps
surprisingly, the PolY567A enzyme extended both the
transition and the transversion mispairs less efficiently than did the
Pol+ enzyme, with the Km contribution
outweighing the kcat contribution. A somewhat
similar result was reported for the Y766S variant of the Klenow
fragment of E. coli DNA polymerase I (49), where catalytic
efficiencies rather than discrimination factors revealed a misinsertion
mutator factor of about 130, although in this case most of the
difference was contributed by the kcat term. We
note in closing that a mispair might extend poorly either because of
its inappropriate geometry within the Pol site (25) or if it resided
for a long time in the inactivated Exo site.
This report describes the first detailed analysis in
vivo of determinants of the fidelity of DNA synthesis in a B
family DNA polymerase. One determinant is the proofreading exonuclease
(Exo) function, which was studied using a bimutational knockout of Exo activity. Another determinant is the Pol function, in which more subtle
modifications define a residue that turns out to be critical for
accurate base selection. The interpretation of the results is simpler
than in many other systems because phage T4 is not subject to the
action of the several E. coli mismatch repair systems (6)
and seems to be unable to repair mutational heteroduplexes on its own
(5). Therefore, comparing mutations arising in Pol+
Exo+ and Pol+ Exo The Proofreading Contribution to Fidelity--
Although T4
Exo
In E. coli, proofreading discriminates about 4-fold more
strongly against transversions than against transitions, whereas mismatch repair discriminates about 14-fold more against transitions than against transversions (51). Transition mismatches are more easily
extended and are less efficiently proofread than transversion mismatches by most polymerases (21, 25). Because RB69, like T4, almost
certainly lacks mismatch repair, we might expect to find a reversed
proofreading balance, and we do. The transition rate increases about
1370-fold (from 0.43 × 10
The absence of complex mutations and large additions and deletions from
the Pol+ Exo A Polymerase Contribution to Fidelity--
We chose to examine the
role in accuracy of RB69 gp43 residue Tyr567 because this
residue is unequivocally close to the Pol active site (10, 15) and
because kinetic parameters for the incorporation of correct bases were
almost unaffected in a PolY567A Exo
RB69 gp43 Tyr567 may be related functionally (even if not
strictly structurally) to either or both of two A family E. coli Klenow Pol site residues, Tyr766 (15) and
Phe762 (10), that are crucial for accuracy. Because
substitutions at gp43 Tyr567 increase BPS mutagenesis far
more than frameshift mutagenesis, Tyr567 is involved more
deeply in the fidelity of base selection than in preventing slippage
errors. This result is consistent with the properties of Klenow
fragment Y766S and Y766A, which are strong BPS mutators promoting
especially T·G and G·T mispairs in vitro but also
generating deletions of two or more bases (49, 52). The mutator
activities of RB69 gp43 Y567A, Y567S, and Y567T are similar, although
Y567S and Y567T are slightly more prone to transition and frameshift
mutagenesis than is Y567A (Tables IV and VI). Understanding the similar
effects of these three substitutions and the unanticipated near
lethality of the Y567F substitution must await further structural information.
Genomic Mutation Rates--
The genomic mutation rates of the
mutators are instructive. The rate for each of the single mutators is
about 3-4. T4 can survive with this high rate at least long enough to
grow into a population of roughly 109 phages because of
several relieving factors: more than half of the genome is comprised of
genes whose function is not required for survival under laboratory
conditions, the fraction of BPSs is at least 75% among the mutators
(and many of these are relatively innocuous), and selection against
deleterious mutations occurs during the growth of a stock. However, the
genomic rate for the double mutator is roughly 15, rendering it unable
to propagate. Despite the uncertainty in this last value, it is clearly
greater than 3, a value that does permit propagation. On the other
hand, a rate of 15 is far lower than expected if the component mutator activities in PolY567A Exo Pol-Exo Coupling--
Depictions of the accuracy of
replicative DNA synthesis usually attribute multiplicative effects to
fidelity factors for insertion, proofreading, and mismatch repair. In
E. coli, mutationally dissecting the latter two revealed
them to be coupled; specifically, a double knockout displayed mutator
strength not much greater than did a knockout of proofreading alone,
because the large number of input mutations that were not proofread
quickly saturated mismatch repair (53). In phage T4, both the
deleterious effects of Exo defects upon polymerase activity (47) and
the properties of numerous mutants that seem to affect partitioning
between the Pol and Exo site (13) imply coupling between the Exo and
Pol sites. In no polymerase, however, had the interaction between Pol
and Exo mutators been examined previously. After overcoming the
complications of error catastrophe, we observed that
PolY567A and Exo
In a structural sense, coupling means that the partitioning of mispairs
between the Pol and Exo sites is determined by more than simple melting
and diffusion, and in particular it means that local changes in
molecular architecture can affect partitioning strongly. Our results
might be explained in several ways: 1) Most DNA polymerases extend
mispaired primer termini far more slowly than correctly paired primer
termini (25), favoring partitioning to the Exo site. If gp43
Tyr567 replacements strongly promoted mismatch extension,
residence time in the Exo site could be diminished sufficiently to
render the PolY567A Exo+ enzyme functionally
Exo Future Directions--
It will be important to extend our analyses
to these same gp43 variants in vitro to gain insights into
how well in vitro analyses of polymerase fidelity accurately
reflect the situation in vivo. Such analyses will benefit
from reconstituting the replication complex as fully as possible, in
addition to studying unassisted gp43s. This will include extending our
kinetic analyses to the pre-steady state. It is also crucial to gain
structural information on gp43 in the closed configuration complexed
with a primer template and an incoming dNTP. The logical later
extension is a structural comparison of correct versus
incorrect dNTPs, extending eventually to a comparative structural
analysis of mutationally cold and mutationally hot sequences.
class)
enzymes that determine the fidelity of phage DNA replication. A T4
whose gene 43 has been mutationally inactivated can be
replicated by a cognate RB69 gp43 encoded by a recombinant plasmid in
T4-infected Escherichia coli. We used this phage-plasmid
complementation assay to obtain rapid and sensitive measurements of the
mutational specificities of mutator derivatives of the RB69 enzyme.
RB69 gp43s lacking proofreading function (Exo
enzymes)
and/or substituted with alanine, serine, or threonine at the conserved
polymerase function residue Tyr567
(PolY567(A/S/T) enzymes) were examined for their effects on
the reversion of specific mutations in the T4 rII gene and
on forward mutation in the T4 rI gene. The results reveal
that Tyr567 is a key determinant of the fidelity of base
selection and that the Pol and Exo functions are strongly coupled in
this B family enzyme. In vitro assays show that the
PolY567A Exo
enzyme generates mispairs more
frequently but extends them less efficiently than does a
Pol+ Exo
enzyme. Other replicative DNA
polymerases may control fidelity by strategies similar to those used by
RB69 gp43.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
class or B
family of DNA polymerases, which includes the replicative polymerases
,
, and
of eukaryotic cells and the polymerases of several of
their DNA viruses (7). Some archaeons also encode gp43-like B family
enzymes (8-10). As such, T4 gp43 and RB69 gp43 are attractive subjects
for studies of mechanisms of replication by this class of enzymes,
particularly because of the amenability of the phage system to combined
genetic and biochemical analyses (11-14). A recently determined
crystal structure of RB69 gp43 reveals five discrete domains termed N,
Exo, Palm, Fingers, and Thumb (15). This structure is in the "open"
configuration and provides a preliminary framework for understanding
the dynamics of DNA polymerase interactions with the DNA primer
template, with incoming dNTPs, and with other proteins of the DNA
replicase complex (16, 17). These multiple interactions are critical
for regulating the processivity and accuracy (fidelity) of replicative
DNA synthesis.
7 am+
progeny) when the plasmid carries the diverged RB69 gene 43 (3, 18). In addition, future structural information is more likely to
be derived from the RB69 enzyme than from the T4 enzyme (16, 17).
gp43 and from three different substitutions at the
gp43 Tyr567 site, together with some in vitro
kinetic properties of the double-mutator enzyme, we conclude that
Tyr567 is an important determinant of base selection by
RB69 gp43. The structural similarities between gp43 and other DNA
polymerases of the B family (8-10) may reveal the existence of
similarly positioned tyrosine residues at the dNTP binding sites of
these enzymes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 frameshift mutation that reverts
primarily by the addition of an A·T base pair within a run of five
A·T base pairs, although other frame-restoring alterations are
possible. rIIUV356 carries a base pair substitution
(BPS)1 that can revert by
transitions (and perhaps also transversions) at a G·C site.
rIIUV375 carries an ochre (UAA) mutation that can revert to
sense codons by both transitions and transversions. For the reversion
tests, each rII mutation was combined with 43am by recombination.
h)
su+I (supD) (hereafter referred to as
QA1) restricts the growth of nonamber T4 rII mutants.
E. coli strain BE NapIV (31), used for some DNA
labeling experiments, requires vitamin B1 supplementation.
-32P]ATP was from Amersham Pharmacia
Biotech. dNTPs were from Pharmacia/LKB. Oligonucleotides for kinetics
experiments were synthesized by the W. K. Keck Foundation
Biotechnology Resource Laboratory (Yale University). All other
chemicals were analytical grade. The PolY567A
Exo
derivative of RB69 gp43 was constructed and purified
as described previously (28).
10 promoter of cloning vector pSP72 (Promega) (11, 18). Mutant derivatives of pCW19-borne gene 43 were constructed by site-directed mutagenesis and
confirmed by sequencing and are designated pRB.43 followed by one of
the Pol Exo annotations described below. Plasmid pCW107 (pRB.43
PolY567A Exo+) expresses gp43
PolY567A Exo+, plasmid pSNG14-1 (pRB.43
PolY567S Exo+) expresses gp43 Y567S, plasmid
pSNG8-1 (pRB.43 PolY567T Exo+) expresses gp43
Y567T, plasmid pCW50R (pRB.43 Pol+ Exo
)
expresses exonuclease-deficient gp43 D222A/D327A, and plasmid pSNG3-1
(pRB.43 PolY567A Exo
expresses gp43 Y567A
D222A/D327A, where "CW" plasmids were constructed by C.-C. Wang and
"SNG" plasmids were constructed by S. Ng.
3 Prime, Inc., Boulder, CO). Samples of purified DNA (3 µl containing 70,000-110,000 cpm) were incubated for 5 min at
65 °C with 12 µl of a gel loading buffer containing 20 mM NaOH, 1 mM EDTA (pH 8.0), 10% Ficoll, 0.0215% bromcresol green. The samples were then subjected to
electrophoresis for 24 h at 25 V in a 0.6% agarose gel (Seakem
GTG) in a continuously recycling buffer containing 30 mM
NaOH, 1 mM EDTA (pH 8.0). The gel was stained with ethidium
bromide and destained, and the DNA lanes were visualized with a UV
transilluminator. Subsequently, each ~14-cm lane was sliced into 25 pieces of equal size (~5 mm) that were transferred to scintillation
vials and counted for 3H in Ultima Gold scintillation fluid
(Packard Instruments); this scintillator decreases quenching from
agarose in the samples.
produce no viable progeny,
probably because the double mutator has a mutation rate so high that
all progeny are mutationally inactivated. Therefore, we designed growth
conditions in which both the wild type (Pol+
Exo+) and double-mutator (PolY567A
Exo
) polymerases were provided, hoping to recover some
viable progeny in which the rI region had been replicated by
the mutator gp43. The gp43 mixture was achieved by infecting cells
bearing a plasmid expressing double-mutator RB69 gp43 (or, as a
control, wild type RB69 gp43) with T4 particles expressing wild type T4
gp43 from the cognate phage gene. BB cells carrying a plasmid
expressing either pRB.43 Pol+ Exo+ or pRB.43
PolY567A Exo
were grown to about
108/ml and were concentrated by centrifugation to
109/ml. T4 43+ was adjusted to
1010/ml. At t = 0, equal volumes of
prewarmed phage and cells were mixed at 37 °C on a rotary shaker
water bath. At t = 10 min the mixture was diluted
2-fold in warm LB broth. At t = 15 min, before the
appearance of significant numbers of intracellular phage under these
conditions, a sample was taken into LB broth with chloroform and
assayed to estimate the fraction of unadsorbed parental phages. At
t = 20 min the mixture was diluted an additional
50-fold in warm broth. At t = 40, infection was
terminated with chloroform, and the lysate was assayed for progeny
phages and further screened for r mutants on BB cells
bearing pRB.43 Pol+ Exo+. The contribution of
unadsorbed parental phages to viable progeny phages was negligible.
1/µrI, the
expression µrI = f/ln(NµrI)
(37, 38). The genomic mutation rate µg = µrIC(168,897 base pairs/genome)/(294 rI
base pairs), where C is the ratio of all mutations to
detected mutations (the reciprocal of the efficiency of mutation
detection). We assume that all non-BPS mutations are detected but that
only chain-terminating mutations are efficiently detected among BPSs, synonymous mutations and many missense mutations remaining undetected (37). If we let B = the fraction of mutations that are
BPSs and D = the correction factor for the fraction of
all BPSs detected, then C = (1
B) + DB. The Pol+ Exo+ rI spectrum
contained 47 BPSs of which 9 produced chain-terminating codons. Because
the T4 genome is about two-thirds A·T, about 0.073 of random
BPSs will produce a chain-terminating mutation (37). Therefore,
D = 9/(47 × 0.073) = 2.6. Although only
approximate, this value is lower than those encountered in most of a
variety of other systems (37), demonstrating that the rI
system detects many missense mutations.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
defect had little effect on DNA synthesis and phage
growth. The Y567A substitution slightly reduced the rate of DNA
synthesis in vivo (by ~20%), whereas phage growth was
hardly affected. The Y567S, Y567T, and Y567V substitutions reduced DNA
synthesis moderately (by 30-40%) and phage yield somewhat less (Fig.
1A).
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Fig. 1.
DNA synthesis and phage production in T4
infections supported by different RB69 gp43s. See the
"Experimental Procedures" for experimental details. A,
relative DNA synthesis is based on [3H]thymidine
incorporation between 22 and 37 min after infection with T4
43am, where the value of 1.0 for Pol+
Exo+ represents 6 × 104 dpm
3H/107 infected cells. Phage growth images are
spot tests in which 50,000 or 50 viable particles of either T4
43am or 43+ phages were spotted onto
lawns of cells carrying a plasmid producing the indicated RB69 gp43.
The values below the images are burst sizes relative to those for
Pol+ Exo+, whose burst sizes were 100 with
43am and 114 with 43+. B,
the rate of thymidine uptake is approximately linear over the interval
from 22 to 37 min and representative of most of the interval from 5 to
65 min. The cpm values are 3H counts/min/0.1-ml sample.
C, alkali-denatured DNAs synthesized by the four indicated
RB69 gp43s have indistinguishable size distributions.
exhibits very
weak activity in an M13 gap filling assay (results not shown). The poor
catalytic efficiency of Y567F-gp43 was confirmed both by repeating its
construction and by reverting the mutated gene 43 at the 567 site (results not shown). Tyr567 is unusual among highly
conserved RB69 gp43 residues tested to date (results not shown) in
tolerating nonconservative amino acid substitutions while not
tolerating a conservative substitution.
construct reduced DNA
synthesis by a moderate 40% but reduced phage growth sharply. Unlike
the other gp43 constructs listed in Fig. 1, RB69 PolY567A
Exo
also inhibited the growth of wild type T4. This
dominant lethality has two possible explanations, both probably
operating here. First, dominant lethality can be exhibited by RB69 gp43
mutants that are deficient in polymerase activity but retain the
capacity to repress the translation of the gene 43 mRNA
transcribed by the infecting wild type T4 particles (3, 11). This
PolY567A Exo
enzyme can indeed repress
heterologous translation (results not shown). Second, as we show later,
the PolY567A Exo
enzyme has such low fidelity
that most of the genomes it synthesizes carry numerous mutations.
) (Fig. 1C). In
particular, the PolY567A Exo
enzyme appears
to accumulate no more single-stranded DNA of reduced size than does the
Pol+ Exo+ enzyme. We therefore presume that DNA
is packaged in phage progeny with similar efficiency in all four infections.
Reversion analyses of mutator predilections of mutant RB69 DNA
polymerases
,
which failed to support the production of viable phage, we designed a
procedure in which T4 infection was supported competitively by T4 gp43
Pol+ Exo+ and RB69 gp43 PolY567A
Exo
(see "Experimental Procedures"). The results of
an infection supported by this mixture of gp43s appear in Table
II. The average number of viable progeny
per infected cell fell about 90-fold, whereas the frequency of
r mutants among the progeny rose by 60-fold. Thus, although
the ratio of DNA synthesis conducted by the two competing gp43s is
unknown, the DNA in the large majority of the mutated rI
regions must have been synthesized by the double-mutator gp43. We
believe that these rI regions are embedded in genomes that
were mostly synthesized by Pol+ Exo+
polymerase, the mutated rI regions then finding their way
into otherwise little-mutated genomes by recombination; T4 has a high frequency of recombination, about 1% per 150 base pairs. An
alternative but less attractive possibility is that the mutated
rI regions were introduced by brief intervals of synthesis
by the double-mutator enzyme. Because these r mutants arose
during a single round of infection, they are all presumed to be of
independent origin.
Effects of a double-mutator RB69 DNA polymerase on burst size and
mutant frequency
,
PolY567A Exo+, and PolY567A
Exo
backgrounds. Complex mutations (closely spaced
multiple BPSs and/or frameshift mutations) (5) were excluded from
further analysis here because they are rarely produced by our mutator polymerases.
Classes of rI mutations generated by different RB69 gp43s
spectrum displays the reverse ratio. Small additions
and small deletions (frameshift mutations) are equally frequent in the
Pol+ Exo+ spectrum, whereas small additions
predominate in the Pol+ Exo
spectrum.
Therefore, gp43 proofreading appears to operate roughly four times more
efficiently to repair transition mispairs than transversion mispairs
and three or four times more efficiently to repair +1 than
1
frameshift mutations. (Remember that T4 mutations are not subject to
mismatch repair.) These results are in good agreement with the
reversion tests (Table I).
distribution appears to be
quantitatively intermediate in character between the Pol+
Exo
and PolY567A Exo+
distributions when broad categories such as transitions, transversions, or frameshift mutations are examined (Table III). If the
PolY567A and Exo
mutator activities operated
independently, then the high frequencies of transitions produced by
PolY567A should have continued to predominate in the
Exo
background, and few or no frameshift mutations would
have been seen. It therefore appears that these two mutator activities
do not act independently of each other.
Mutational proclivities of RB69 DNA polymerases modified at
Tyr567
A·T transitions outnumbered
A·T
G·C transitions. G·C
T·A mutations predominated
among the transversions, whereas G·C
C·G transversions were
completely absent. The predominance of G·C
A·T transitions and
G·C
T·A transversions is consistent with the A·T-rich nature
of the T4 genome. The rarity of G·C
C·G transversions suggests
that these polymerases form C·C and G·G mispairs much less readily
or extend such mispairs less efficiently than they do G·A, C·T, and
other transversion-generating mispairs. Almost all frameshift mutations in these spectra arise within short repeats of single base pairs.
, PolY567A Exo+, and
PolY567A Exo
gp43s. As expected, frameshift
mutations cluster within AAAAA, TTTTT, AAAA, and TTTT, and most are
simple additions or deletions of single bases.
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Fig. 2.
Mutational spectra characteristic of
different RB69 gene 43 alleles. The sequence is
that of the wild type rI strand complementary to the coding
strand. P+, Pol+; E+,
Exo+; PM, PolY567A;
E , Exo
. Transitions are entered
above the wild type bases, and transversions are entered below.
Additions (+) appear above the underlined short repeats and
are almost all of single bases. Deletions (
) appear below the
underlined short repeats and are almost all of single bases. Complex
mutations and large additions and deletions are omitted because they
appeared almost exclusively in the Pol+ Exo+
spectrum and only once among the 268 mutations in the other spectra;
they will be described in a subsequent report. The four top displays
(above the horizontal line) are the first halves of the four different
spectra, and each second half appears below the dividing line and
position numbers.
spectrum exhibits no pronounced
hot spots but does display numerous sites of intermediate mutability.
The PolY567A Exo+ spectrum displays two hot
spots, each imbedded within a small region of generally increased
mutability. The hot spot at position 247 is specific for G·C
A·T transitions. It contains 23% of all the mutations in the
spectrum, whereas positions 248 and 250 each contain two G·C
A·T transitions. The hot spot at position 203 produces C·G
T·A transitions and C·G
A·T transversions about equally
often, and together they account for about 19% of all the mutations in
this spectrum.
T·A
transversions at position 203 and the mildly increased numbers of
transitions at positions 203 and 205.
spectrum much more often resembled the
PolY567A Exo+ than the Pol+
Exo
spectrum. However, the PolY567A
Exo
mutations at positions 2, 3, 77, 109, 110, 131-135,
and 154 are predicted by neither the Pol+ Exo
nor the PolY567A Exo+ spectrum. If the
PolY567A and Exo
mutators acted sequentially
and independently, then the PolY567A Exo
spectrum should resemble the predominant PolY567A
mutational input rather than the Pol+ mutational input seen
in the Pol+ Exo
spectrum. Thus, the
PolY567A Exo
spectrum tends to reinforce the
conclusion drawn from the mutation distribution (Table III) that the
PolY567A and the Exo
mutator activities do
not interact multiplicatively. The exceptions to a simple spectral
mixture noted above highlight the complexity of this interaction.
Val
replacement, which might well go undetected.) The hot spot at
rI position 203 is imbedded in yet another generally
hypermutable sequence, 5'-CCCGTG-3', where the third C is the most
mutable, sometimes producing both transitions and transversions, and
the T is a little less mutable and produces transitions. The
hypermutable region around position 203 begins with three G·C base
pairs, the only run of three G·C base pairs in rI. From
the rI sequence, the sum of CCC and GGG expected from a
random distribution of bases is 3.5, so perhaps the frequency of these
runs has been reduced by mutation pressure. The entire T4 genome has
A·T = 109278 and G·C = 59619 and is unequivocally depleted of such runs (expected = 1857, observed = 1239), and this deficit also appears in the observed infrequent use in T4 of the
codons CCX, XCC, GGX, and
XGG.
spectra at positions 202-203
and 247, although not at positions 3 and 109-110. However, our search
for associations between the flanking and nearby sequences and the
degree of hypermutability failed to reveal a mutability thermostat.
Thus, understanding variable GG/CC hypermutability in T4 is likely to
require systematic studies of the kinetics of misincorporation in
vitro as a function of nearby bases.
.
However, the growth conditions described above, which sufficed to
provide a mutational spectrum, also sufficed to estimate the PolY567A Exo
mutation rate. Because of the
high rates characteristic of these mutators, mutants sometimes contain
multiple mutations. If the mutations are randomly distributed among
rI genes, then most multiple mutations should lie many base
pairs away from each other in any particular mutant, and the
distributions of these distances should appear to be random. Such is
the case (Fig. 3). For randomly
distributed mutations, the Poisson distribution predicts that
M
f/(ef
1), where
M
2 rI mutations and M is the total number of
rI mutants. Thus, f can be estimated solely from
the numbers of multiple and single mutations among sequenced mutants
and can then be combined with an estimate of N to obtain the
mutation rates; this is the method of multiples. The numbers of single
and multiple mutations for the relevant RB69 gp43 variants are shown in
Table V. Several rI mutation
rates were then calculated in two ways. The method of the median was
simply the median rI mutation rate calculated using µ = f/ln(µN) and was used to obtain the
Pol+ Exo
and PolY567A
Exo+ rates. The method of multiples was used to obtain
values of f for all three gp43s. These f values
were then converted to rates using µ = f/ln(µN). (In the case of PolY567A
Exo
, we assumed that the average number of progeny phage
was the same, 182, for both infections described in Table II but that most of the phages in the mixed polymerase infection carried lethal mutations. This assumption is justified by the previously described vigor of the polymerase in vivo where nearly normal amount
of DNAs of normal sizes are synthesized by PolY567A
Exo
. Here, N = 182 instead of the total
number of particles in the stock. However, because the condition
N0
1/µ is not met here, we must
calculate the rate using µ = f/ln(N/N0). The
multiplicity of infection was about 10, but not all particles can
participate under our experimental conditions. We therefore used the
full estimated range N0 = 1-8.) Note, however,
that these multiples values are overestimates for at least two reasons.
One is that all of the synonymous mutations and some of the missense
mutations among the multiples have an r+
phenotype as singles and therefore engender false multiples in the
sense of our argument. Another is that some of the multiples may have
arisen by recombination between singles. These confounding issues can
be roughly factored out by determining the ratio of the multiples rate
to the median rate for the Pol+ Exo
and
PolY567A Exo+ polymerases, averaging them (to
obtain 5.32), and then dividing the multiples rate for
PolY567A Exo
(0.0391-0.652) by this average
value to obtain the corrected median value (0.00740-0.0122). Although
this value may have an experimental uncertainty of a few-fold even
beyond its stated range, it still turns out to be very useful.
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Fig. 3.
Distances between double mutations.
Whereas complex mutations are always close together, most pairs of
randomly arising single mutations will be far apart. No multiple
mutations were observed in the Pol+ Exo+
spectra, as expected from the low mutation frequency.
Distributions of multiple rI mutations produced by mutant RB69 gp43s
2/M ratio as described in
the text. The top two multiples/median ratios were averaged to obtain
the third value, which was then applied to the bottom multiples value
to obtain the bottom (underlined) median value.
polymerase
(510-fold) is the same as observed previously (490-fold) using an assay
that screens all T4 r mutants (18) and is similar to values
obtained with various T4 Pol+ Exo
mutants:
650-fold (47) and 760-fold (48) when screening for acridine resistance
and 310-fold when screening for all r mutants (18).
Forward mutation rates produced by mutant RB69 gp43s
gp43, we measured steady-state kinetic
constants for Pol+ and PolY567A for the
incorporation and extension of one correct base pair and various
mispairs. Using measured values of kcat and
Km(app), we calculated the steady-state
catalytic efficiency kcat/Km, a discrimination factor against mispair formation by a particular gp43,
and a mutator factor or antimutator factor for gp43
PolY567A versus gp43 Pol+.
for the correct G·C base pair
is about 0.2 of the efficiency with Pol+ Exo
.
This difference is somewhat smaller than the relative rate of total DNA
synthesis in vivo for these two genotypes (about 0.7; Fig.
1). However, the in vitro value of 0.2 varies depending on the sequence context of the measured site. In vitro, gp43
Pol+ Exo
discriminates strongly against all
tested mispairs but more strongly against transversion mispairs than
against transition mispairs (a result concordant with the higher
in vivo frequency of transitions than transversions seen in
Table III). Conversely, the PolY567A Exo
mutator factor is stronger for a transition mispair than for any of
three transversion mispairs. Discrimination against C·C and G·G are
particularly strong in gp43 Pol+ Exo
and
remain strong in gp43 PolY567A Exo
(with no
change at all in the case of C·C), a result concordant with the
absence of G·C
C·G transversions in all these spectra (Table
III). For all four mispairs, the mutator factor is influenced more
strongly by a change in kcat than in
Km. Thus, studies both in vivo (Table
III) and in vitro (Table VIII)
demonstrate that PolY567A is a strong BPS mutator with a
preference for generating transition mutations.
Steady-state kinetic parameters for insertion accuracy by Exo
Pol+ and Exo
PolY567A
Steady-state kinetic parameters for mispair extension by Exo
Pol+ and Exo
PolY567A
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
backgrounds
reveals both the kinds of mutations introduced by the Pol function in
the first place and the efficiencies with which each kind of mutation
is proofread.
mutations often impair polymerase activity (47), we
were fortunate to observe only a small effect of the D222A/D327A
combination on polymerase activity either in vivo (Fig. 1)
or in vitro (28). The Exo
form produces a
strong mutator effect, increasing both the rI mutant
frequency (Table III) and rate (Table VI) and the total r
frequency (18) about 500-fold. In reversion tests, the
Exo
state increased mutation frequencies by 500- to
2600-fold (Table I) and rates by similar factors. The standard DNA
microbial genomic mutation rate is 0.0034/replication (50). For phage
RB69, whose genome size is very close to that of T4 (34), this genomic
rate corresponds to an average rate per base pair of 2.0 × 10
8. If proofreading contributes a fidelity factor of
about 1/500 = 2 × 10
3 to this rate, then the
average fidelity of DNA synthesis itself must be 10
5/base
pair. Applying the standard genomic rate to E. coli and dividing by genome size gives an average rate/base pair of 7.3 × 10
10. Extending fidelity factors for base substitution
(51) to all kinds of mutations gives a synthesis fidelity of 0.9 × 10
5, a proofreading factor of 1.7 × 10
2, and a mismatch repair factor of 5 × 10
3/base pair. Thus, RB69 achieves its spontaneous
mutation rate starting with almost exactly the same accuracy of DNA
synthesis as achieved by E. coli but attains the remaining
balance in a proofreading step that is about 8-fold stronger than in
E. coli. E. coli uses additional powerful DNA mismatch
repair systems to achieve the standard rate. However, we should point
out that these computations do not take into account any coupling that
may occur between the several determinants of fidelity.
5 × 15/79 in
Pol+ Exo+ to 220 × 10
5 × 39/77 in Pol+ Exo
), whereas the transversion
rate increases only about 310-fold (calculated similarly). The
corresponding factors for frameshifts arising predominantly in runs are
roughly 1800-fold for +1 mutations and 530-fold for
1 mutations.
distribution may mean either
that these arise at intrinsically low frequencies and are not proofread
efficiently or that they are generated by an aberration of proofreading
in the first place. The latter, for instance, might occur by the
removal of a correct base followed by misaligned reannealing of the
primer terminus to a distant complementary template sequence.
mutant
(28). It therefore seemed likely that if Tyr567 interacts
with the incoming dNTP, it does so with the base rather than with the
phosphate or deoxyribose. As it turns out, modifications at this
residue produce either a robust polymerase with sharply reduced
fidelity (Y567(A/S/T)) or a moribund polymerase (Y567F) whose fidelity
could not be measured.
were
multiplicative, in which case the genomic mutation rate would be
roughly 1800.
interact only a little more
strongly than additively. First, the double-mutator spectrum is an
approximate mixture of the component spectra, although with important
exceptions (Fig. 2). Second, the double-mutator mutation rate is only a
little greater than the sum of the component rates (220 + 210 = 430 versus ~970), where the probable accuracy of the
values does not preclude simple additivity (Table VI). We have observed
a quantitatively similar interaction between these two mutator
activities when the purified polymerases are assayed in
vitro.2
. However, the PolY567A Exo+
and PolY567A Exo
mutational distributions and
spectra would then be very similar, which they are not, and direct
estimates of mispair extension would reveal a large increase, whereas
they reveal a substantial decrease (Table VIII). 2) Because
Tyr567 replacements reduce mismatch extension, the DNA
might simply dissociate from the enzyme, and the defective primer
terminus might then be unable to reassociate with a gp43 molecule in a productive manner. Thus, most mutations would be lost. 3) If the amino
acid replacements in the Exo domain lower the rate of return of the
primer to the Pol site, the outcome could be the same, the mutations
being lost. 4) The Exo defect might unexpectedly improve fidelity at
the modified Pol site. However, there is neither precedence nor
structural hint of such a possibility, and it seems unlikely.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Tom Kunkel and Kasia Bebenek for many helpful discussions during the course of the work and Youri Pavlov for a close, critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by United States Public Health Service Grants DK09070, GM18842, and GM54627.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Permanent address: Institute of Biochemistry and Biophysics, Polish Academy of Science, 02-106 Warsaw, Poland.
** To whom correspondence should be addressed: Laboratory of Molecular Genetics E3-01, National Institute of Environmental Health Sciences, Rm. E-344, 111 South Alexander Dr., Research Triangle Park, NC 27709. E-mail: drake@niehs.nih.gov.
Published, JBC Papers in Press, December 22, 2000, DOI 10.1074/jbc.M007707200
2 A. Bebenek, H. K. Dressman, and J. W. Drake, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: BPS, base pair substitution; PCR, polymerase chain reaction.
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REFERENCES |
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1. | Russell, R. L. (1967) Speciation among the T-Even Bacteriophages , University Microfilms Inc., Ann Arbor, MI |
2. |
Russell, R. L.,
and Huskey, R. J.
(1974)
Genetics
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989-1014 |
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