From the Laboratory of Molecular Genetics, NIEHS,
National Institutes of Health, Research Triangle Park, North
Carolina 27709 and § Department of Biochemistry and
Molecular Genetics, University of Colorado Health Sciences Center,
Denver, Colorado 80262
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
The in vitro fidelity of
Escherichia coli DNA polymerase III holoenzyme (HE) is
characterized by an unusual propensity for generating ( The error rate of a DNA polymerase depends not only on the
efficiency with which it discriminates against incorrect nucleotides during the insertion step but also on its efficiency in continuing synthesis from the mismatched primer-terminus that it created by
misinsertion. This is most obvious for enzymes containing an associated
proofreading activity. Proofreading and mismatch extension compete, and
a slow extension rate is likely to lead to very few mismatches being
able to escape removal by the exonuclease. Thus, the ability to extend
mismatches is an important factor in determining polymerase fidelity.
The accuracy of the duplication of the genetic material in the
bacterium Escherichia coli depends largely on the fidelity of the DNA polymerase III holoenzyme
(HE),1 which is responsible
for its chromosome replication (1). The HE is a large dimeric complex
composed of 10 distinct subunits that is capable of simultaneously
synthesizing the leading and lagging strands of the replication forks
(2-4). In vitro fidelity studies of purified HE using
either single-stranded phage templates (5, 6) or oligonucleotide
substrates (7) have revealed that HE is quite accurate for base
substitution errors. Interestingly, gap-filling synthesis of M13 DNA
(6) using a forward mutational assay capable of detecting various types
of synthesis errors revealed HE to be relatively inaccurate for
( Studies of ( A second mechanism, the "misincorporation model" proposed by Kunkel
and Soni (14), states that frameshift mutations can be initiated by
misinsertion of a nucleotide. If the misinserted nucleotide is
complementary to the next template base, then its 1-base forward
misalignment can form a frameshift intermediate containing a correctly
base-paired terminus (and an unpaired extra base in the template
strand). As in the direct slippage model, this frameshift intermediate
would be fixed into a ( A third possible model for ( Based on a detailed analysis of in vitro mutation spectra
produced by E. coli DNA polymerase III holoenzyme (both
wild-type and MutD5), we had suggested that most of the observed
( In this study, we have further investigated the mismatch extension
capability of the wild-type and MutD5 holoenzyme by using a fidelity
assay specifically designed to measure the relative probabilities by
which a polymerase chooses each of the three possible pathways upon
encountering a mismatched primer-terminus: (i) direct extension
(yielding a base substitution), (ii) misalignment extension (yielding a
Materials and Chemicals--
Bacteriophage M13mp2 and its mutant
derivatives containing a T Construction of M13mp2 Substrates with a 3' Terminal
Mismatch--
M13mp2G103 RF DNA containing unique sites for
restriction endonucleases BamHI and KpnI (15) was
doubly digested with the two enzymes to produce 4061- and 3135-bp-long
fragments. These fragments were separated by electrophoresis on a 0.8%
agarose gel, and the 3135-bp fragment was purified from the gel by
electroelution. The fragment was desalted and concentrated using a
Microcon-50 microconcentrator (Amicon Inc., Beverly, MA). To form the
terminally mismatched heteroduplexes, the purified fragment was
hybridized to single-stranded circular viral mp2 DNA that was either
wild-type (G102, T103) or altered at position 102 (A102, T103) (see
Fig. 1), as follows. The fragment was diluted to 5 µg/ml with water and incubated at 70 °C for 5 min to denature the strands.
Single-stranded circular viral DNA was added (5 µg/ml, final
concentration) just before removal from the 70 °C water bath, and
the mixture was placed on ice. After 5 min, SSC was added to a final
2× concentration (300 mM NaCl and 30 mM sodium
citrate), and the mixture was incubated for 5 min at 60 °C, after
which it was placed on ice. The resulting partial duplex DNA was
desalted and concentrated by a Microcon-50 microconcentrator. Using a
1:1 ratio of fragment to viral DNA, about one-half of the
single-stranded DNA was converted to partially heteroduplex molecules
containing the terminal T·C mismatch at position 103 (Fig. 2,
lane 1).
In Vitro DNA Synthesis Reactions--
Mismatch extension
experiments were performed in a 25 µl volume at 30 °C in 1.5-ml
Eppendorf tubes containing 45 fmol of partially heteroduplex substrate,
30 mM HEPES, pH 7.5, 16 mM Tris, pH 7.5, 6 mM dithiothreitol, 10 mM MgCl2, 100 µg/ml bovine serum albumin, 500 µM ATP, 1 µg of
single-stranded binding protein, and either 50 or 1000 µM
each of the four dNTPs. Reactions were initiated by adding 135 units of
MutD5 HE or 120 units of wild-type HE (1 unit = 1 pmol of
nucleotide incorporated per minute). At defined time points (from
15 s to 5 min), the reaction mixtures were quenched by adding 2 µl of 200 mM EDTA. Extension reactions by the Klenow fragment (0.5 unit/reaction; 1 unit = 10 nmol of nucleotide
incorporated per 30 min) were carried out for 30 min in the absence of
single-stranded binding protein and ATP. The reactions were extracted
twice with an equal volume of phenol:chloroform:isoamyl alcohol
(25:24:1) and separated by electrophoresis on a 0.8% agarose gel (70 V, 4 h). Full-length reaction products (RF II band) were cut out from the gel, and the DNA was extracted using a QIAquick Gel Extraction Kit (Qiagen Inc., Chatsworth, CA). The extracted DNA was desalted and
concentrated into a 10-µl final volume using a Microcon-50 microconcentrator.
Transfection and Plating--
2 µl of gel-purified RF II DNA
were used to transfect 50 µl of competent cells prepared from
E. coli strain MC1061 by electroporation with a Bio-Rad Gene
Pulser (Bio-Rad Laboratories, Inc.) set at 2.01 kV, 400 W, 25 µF.
Immediately after electroporation, 1 ml of SOC medium was added.
Plating was performed by adding the transfected cells to 3 ml of melted
soft agar (42 °C) containing 2.5 µg of 5-bromo-4-chloro-3-indolyl- To examine how E. coli DNA polymerase III HE processes
terminal mismatches in vitro, we used a fidelity assay that
permits distinction between the different pathways available to the
polymerase upon encountering a primer-template containing a 3' terminal
mismatch. The mismatched substrates used in this study are described in Fig. 1. They are based on the M13mp2
lacZ 1)-frameshift
mutations. Here we have examined the capability of HE isolated from
both a wild-type and a proofreading-impaired mutD5 strain
to polymerize from M13mp2 DNA primer-templates containing a terminal
T(template)·C mismatch. These substrates contained either
an A or a G as the next (5') template base. The assay allows distinction between: (i) direct extension of the terminal C (producing a base substitution), (ii) exonucleolytic removal of the C, or (iii),
for the G-containing template, extension after misalignment of the C on
the next template G (producing a (
1)-frameshift). On the A-containing
substrate, both HEs did not extend the terminal C (<1%); instead,
they exonucleolytically removed it (>99%). In contrast, on the
G-containing substrate, the MutD5 HE yielded 61% (
1)-frameshifts and
6% base substitutions. The wild-type HE mostly excised the mispaired C
from this substrate before extension (98%), but among the 2% mutants,
(
1)-frameshifts exceeded base substitutions by 20 to 1. The
preference of polymerase III HE for misalignment extension over direct
mismatch extension provides a basis for explaining the in
vitro (
1)-frameshift specificity of polymerase III HE.
INTRODUCTION
Top
Abstract
Introduction
References
1)-frameshift mutations. In fact, (
1)-frameshifts were the major
class of errors generated by HE in this assay (6).
1)-frameshift mutations have lead to the proposal of
several general mechanisms for their formation. One model proposed by
Streisinger et al. (8) postulates the occurrence of slippage
of the nascent DNA strand in homopolymeric sequences (the direct
slippage model). Within this model, the frameshift mutation frequency
increases with the length of the run, because larger runs produce more
(and more stable) misaligned intermediates. This model has been
suggested to contribute to spontaneous frameshift mutations in
vivo (9-12) as well as frameshifts produced in vitro by a variety of DNA polymerases (13).
1)-frameshift mutation by further extension.
The misincorporation mechanism has been shown to operate during
in vitro DNA polymerization by the Klenow polymerase (15)
and human immunodeficiency virus type I reverse transcriptase (16).
1)-frameshifts generated by polymerase
III HE in vitro results from recent work by Bloom et
al. (7), who observed elevated misincorporation by polymerase III HE in cases where the misincorporated base was complementary to the
next template base. The authors proposed a transient misalignment before incorporation, in which the incoming (incorrect) dNTP is aligned
on the next template base (dNTP-mediated misalignment). Continued DNA
synthesis from the misaligned intermediate (after incorporation of the
misaligned dNTP) would also generate a (
1)-frameshift.
1)-frameshift mutations produced by this enzyme occur via the
misincorporation plus slippage mechanism (6). This conclusion was based
on the following observations: (i) most of the (
1)-frameshifts
occurred at nonreiterated template positions, (ii) the (
1)-frameshift mutations were proofread with the same efficiency as the base substitution mutations, and (iii) (
1)-frameshift errors significantly increased under conditions of biased dNTP pools, specifically at
template positions where the 5' neighbor is complementary to the dNTP
provided in excess (6). We further suggested that the high level of
(
1)-frameshift mutations created by this enzyme reflects a general
inability to directly extend terminal mismatches and a greatly enhanced
extension efficiency if the terminal mismatch can be misaligned on the
next template base.
1-frameshift), or (iii) exonucleolytic proofreading (yielding no
mutation). We show that for a mispaired primer-template in which
misalignment is not possible, HE preferentially proofreads the
mispaired base (>99%). However, for a primer-template in which the
terminal primer base is complementary to the next template base, the
polymerase extends from the slipped intermediate to yield a
(
1)-frameshift mutation in a significant fraction of the cases. These
data support models in which the in vitro frameshift
specificity of HE results from its relatively low efficiency in direct
mispair extension compared with its efficiency in misalignment and
extension of the misaligned intermediate.
EXPERIMENTAL PROCEDURES
G substitution at position 103 (mp2G103)
or a G
A substitution at position 102 (mp2A102) in the
lacZ
gene were obtained from Dr. Katarzyna Bebenek
(National Institute of Environmental Health Sciences, Research Triangle
Park, NC). Strains MC1061, which was used to prepare competent cells,
and CSH50, which was used as an
-complementation host to score
plaque color, were stocks of our laboratory. The holoenzymes from a
wild-type and a mutD5 strain were purified as described
previously (6). Klenow polymerase (exo
) and restriction
endonucleases BamHI and KpnI were from New
England Biolabs (Beverly, MA). Ultrapure dNTP, ATP, and E. coli single-stranded binding protein were purchased from Pharmacia
Biotech, Inc.
-D-galactopyranoside, 0.24 mg
of isopropyl-1-thio-
-D-thiogalactoside, and 0.25 ml of a
mid-log culture of indicator strain CSH50. This mixture was poured onto
minimal agar plates. The plates were inverted and incubated for 18-24
h at 37 °C, followed by an additional 24-h incubation at room
temperature. Plaques were counted and classified according to their
dark blue, light blue, or colorless phenotype. The frequencies of
mispair extension and misalignment extension were calculated by
dividing the frequency of light blue plaques or colorless plaques,
respectively, by 0.55. The latter represents the average 55%
expression probability of a mutation contained in the (
)-strand of a
full-length RF II molecule (17, 18). The use of a mismatch
repair-proficient strain, such as MC1061, is preferred in this assay
because it minimizes the occurrence of mixed (blue/colorless) plaques
that make it difficult to score the light blue phenotype (17, 18).
RESULTS
-complementation system and use differential plaque
colors to distinguish the various reaction products. They represent a
modification from those originally described by Kunkel and co-workers
(17, 19-21), which have been used to assess terminal mispair
utilization by a number of different polymerases. Instead of using
duplex DNA containing a small gap, we have constructed half-duplex
M13mp2 molecules consisting of a 3135-bp duplex region and a 4061-bp
single-stranded region. The 3135-bp primer contains the terminal
mismatch. Upon synthesis by polymerase III HE, the full-length RF II
products can be readily separated from the starting molecules using a
0.8% agarose gel (see Fig. 2), thus
allowing us to recover and separately analyze the full-length extension
products. This feature is important for assaying rapid and highly
processive polymerases such as HE, because it permits the analysis of
extension products at very short reaction times, even when not all
substrates are bound or extended.
View larger version (19K):
[in a new window]
Fig. 1.
Construction of terminally mismatched
substrates. M13mp2G103 RF DNA was digested with restriction
endonucleases BamHI and KpnI to yield fragments
of 3135 and 4061 bp. The 3135-bp fragment was purified and used as a
primer. The primer was annealed to either wild-type single-stranded DNA
(G at position 102) or modified single-stranded DNA (A at position 102)
to produce heteroduplex molecules containing a T·C mispair at
position 103 of lacZ with either G or A as the next template
base (see "Experimental Procedures" for details).
View larger version (26K):
[in a new window]
Fig. 2.
DNA synthesis by polymerase III holoenzyme
from a mispaired primer-terminus. Lane 1, the starting
substrate with terminal T·C mispair (top band). The
lower two bands represent the single-stranded template and
the 3135-bp primer fragment from which the substrate was constructed.
Lane 2, the product of the extension reaction with 135 units
of MutD5 HE (a 5-min reaction in the presence of 1000 µM
of each of the four dNTPs) yielding a full-length RF II product.
Reactions and electrophoresis were carried out as described under
"Experimental Procedures."
Two substrates were prepared, both of which contained a T·C mismatch
at the 3' end of the primer strand (position 103 of the lacZ sequence) (Fig. 1). In one substrate, the next
template nucleotide to be copied is a G, in the other substrate, it is an A. In both cases, the template strand is phenotypically wild-type (dark blue plaque), because the G
A change at position 102 is silent
(17). However, the mismatched C in the primer strand is derived from a
T
G base substitution mutant at position 103, which produces a light
blue plaque. There are three possible outcomes for these two substrates
when processed by HE. First, HE may simply extend the mispaired
terminus C, creating a RF II heteroduplex molecule containing a base
substitution mutation in the (
)-strand. Upon transfection, this
molecule will yield a light blue plaque. (Actually, due to the action
of mismatch repair on the internal mismatch, upon transfection into the
competent cells, the probability of producing a light blue plaque is
about 55%; the remaining 45% carry the (+)-strand genotype and will
be dark blue. The 55% (
)-strand expression efficiency has been
determined experimentally for a large series of base·base and
frameshift mismatches (17, 18)). Second, in the case of the
G-containing substrate, the terminal mismatched C may first misalign on
the next template base G, followed by the extension of this misaligned
structure. Upon transfection, this will yield (again at 55%
efficiency) a mp2 frameshift mutant with a colorless plaque phenotype.
Third, instead of extending the terminal mismatch, HE may remove the
mispaired C by exonucleolytic proofreading. Upon extension, this would
produce a wild-type dark blue plaque. Thus, a simple enumeration of
light blue, colorless, and dark blue plaques reveals the fate of the
terminal mismatch upon polymerization.
Extension reactions were performed for each DNA substrate with
wild-type or MutD5 HE at two different dNTP concentrations: 50 and 1000 µM. The reaction products were separated by agarose gel
electrophoresis (see Fig. 2), and full-length RF II molecules were
purified and analyzed by transfection. Time-course experiments showed
that the RF II product had already appeared after 15 s of reaction
time, as expected for a rapid and highly processive enzyme such as HE
(data not shown). As a control, we also performed extension reactions
with the Klenow fragment (exo; see "Experimental
Procedures").
The data in Fig. 3 show that with the substrate containing the T·C mispair followed by a template A (right panel), most of plaques scored (>99%) in reactions with either the wild-type or MutD5 HE were dark blue, indicating that proofreading of the terminal base was the predominant outcome. The failure to detect light blue or colorless plaques even for the MutD5 HE, which possesses only limited (~5%) exonuclease activity (5), indicates that HE is very inefficient in extending directly from the mispaired terminus.
|
Extension by MutD5 HE of the substrate containing the T·C mispair
with the 5' neighboring template G (Fig. 3, left panel) yielded high numbers of colorless and dark blue plaques and
significantly fewer light blue plaques. Sequencing of randomly selected
colorless and light blue plaques (10 each) showed that all colorless
mutants had lost template T at the site of mispair, and that all light blue mutants carried the expected T to G base change at position 103. The proportion of colorless and light blue plaques was lower when the
dNTP concentration was decreased from 1000 to 50 µM, reflecting the stimulatory effect of high dNTP concentrations on the
two forward reactions as they compete with the proofreading step (6).
The data with the G-containing substrate demonstrate that MutD5 HE is
highly proficient in forming and extending a (1)-frameshift
intermediate (when permitted by the sequence context) as compared with
direct extension of the mismatch, which is inefficient. The wild-type
enzyme at either dNTP concentration produces a majority of dark blue
plaques, indicating that proofreading is the predominating pathway.
Nevertheless, among the mutants, colorless plaques outnumber the light
blue plaques, indicating that misalignment extension is strongly
preferred over direct extension in this case also. Note that the Klenow
fragment greatly prefers direct extension over misalignment extension,
as seen from the excess of light blue plaques versus
colorless plaques (Fig. 3).
In Table I, we present the calculated
probabilities for the G-containing substrate for each of the three
pathways. At 1000 µM dNTP, the MutD5 HE chooses the
misalignment extension pathway in 61% of the cases. The direct
extension pathway is chosen in only 6% of the cases, while 33% of the
mismatches are proofread. At 50 µM dNTP, the proportion
of proofread molecules increases to 80.8%, whereas 17.8% are
processed by misalignment extension, and only 1.5% are directly
extended. Thus, the ratio between direct extension and extension from a
misaligned intermediate at either dNTP concentration is about 1:10. The
wild-type HE, which has a strong exonucleolytic proofreading activity
(6), prefers to excise the mispaired C before polymerization. However,
at 1000 µM dNTP, about 2% of the primer-templates are
processed by HE to yield the (1)-frameshift mutation, whereas only
0.1% is directly extended. Reactions sampled at different time points
between 15 s and 5 min yielded the same ratios between direct
extension, misalignment extension, or proofreading (data not shown).
Because smaller HE subassemblies (core, polymerase III', polymerase
III*) cannot synthesize the required long stretch (~4000 bp) of DNA within a short time (and in the presence of single-stranded binding protein), there can be little doubt that the observed products are made
by HE. The results obtained with HE are in contrast to those obtained
with the Klenow polymerase: this enzyme efficiently extended the
mispaired C (95%) and only rarely chose the misalignment pathway
(5%).
|
![]() |
DISCUSSION |
---|
The data obtained in this study reveal properties of E. coli DNA polymerase III HE that appear to be unique among the
polymerases tested to date. First, when presented with a preformed
terminal mismatch, direct extension of the mismatch by the enzyme to
yield the expected base substitution is extremely infrequent. For
example, for the A-containing substrate (Fig. 1), only about 0.2% is
extended to yield the expected base substitution (Fig. 3), with the
vast majority (>99.8%) being removed by proofreading. This is true even for the proofreading-impaired HE purified from the
mutD5 mutator strain, in which only 5% or less of the
exonuclease activity remains (6). Secondly, when presented with a
terminal mismatch in a sequence context in which the terminal base in
the primer strand is complementary to the next template base (the
G-containing substrate; see Fig. 1), extension is greatly facilitated
but now takes place from the misaligned intermediate, yielding a
(1)-frameshift instead of the base substitution mutation. In the case
of the proofreading-impaired enzyme, this second pathway significantly exceeds the exonuclease pathway (61 versus 33%; see Table
I). For the wild-type enzyme, proofreading still predominates; however, most significantly, misalignment extension exceeds direct extension by
20:1 (Table I). This strong bias favoring misalignment extension over
direct extension is unique to polymerase III, because other enzymes for
which misalignment extension has been documented, such as Klenow
fragment (15, 21), avian myeloblastosis virus and Moloney murine
leukemia virus reverse transcriptases (19), mammalian polymerase
(19), and yeast polymerase I (20), generally produce the frameshift
mutation less efficiently than the base substitution. To illustrate the
extreme tendency of polymerase III HE in this respect, we compared the
enzyme side by side with the Klenow fragment. This enzyme also
performed misalignment extension, but at only 5% of the rate of the
direct extension (Table I).
The present data on mismatch extension by HE are relevant for
explaining the unusual spectrum of in vitro errors produced by HE in a forward mutagenesis assay using the lacI gene as
a mutational target (6). In these spectra, wild-type and MutD5 HE
produced a majority of (1)-frameshift mutations at nonreiterated sequences. We proposed (6) that these frameshifts originated as base
misinsertion errors that failed to be extended by the enzyme but were
processed efficiently by extension from the slipped intermediate in
sequence contexts where the next template base is complementary to the
misinserted base. The present data demonstrating the clear preference
for misalignment extension over direct extension when HE is faced with
a (preformed) 3' terminal mismatch are fully consistent with this
model. Although a preformed terminal mismatch may not resemble a
mismatch created during ongoing DNA synthesis in all respects, the
behavior displayed by HE is striking and must reflect some property of
HE that is likely relevant during ongoing DNA synthesis as well.
An important alternative context in which to consider the tendency of
HE to create (1)-frameshifts is provided by the "dNTP-stabilized misalignment" model proposed by Bloom et al. (7). These
investigators noted increased misincorporation by HE on an
oligonucleotide template under conditions in which the misincorporating
nucleotide was complementary to the next template base. The findings
were interpreted to indicate that the polymerase is able to read ahead
and use the information provided by the next template position to
direct (mis)incorporation. First of all, this model provides an
alternative mode of misincorporation by HE compared with direct
base·base misinsertion. If correct, both base·base mismatching and
misincorporation by dNTP-stabilized misalignment may provide the
terminally mismatched substrates that HE may extend from either the
aligned or misaligned state. Secondly, provided that the
primer-terminus remains in the misaligned state after phosphodiester
bond formation, the reaction product of a misincorporation via
dNTP-stabilized misalignment is the same as the intermediate that we
suggested to arise from a standard misincorporation and subsequent
forward misalignment. This feature of the dNTP-stabilized misalignment
model for misincorporation makes it a potentially attractive pathway
for the generation of the (
1)-frameshifts observed in
vitro (6). However, if the forward extension rate is slow relative
to the interconversion of the aligned and misaligned states, then any
distinction with regard to the initial mode of misincorporation would
disappear. Finally, it is likely that the preferred usage of misaligned
intermediates by HE and misincorporation by dNTP-stabilized
misalignment are manifestations of the same, unusual enzymological
property of HE that allows the enzyme to generate and/or use misaligned
intermediates with relatively high efficiency. This property, which is
obviously relevant for the fidelity of the enzyme, is most intriguing
and deserves further investigation.
In addition to providing insight into the mechanism of frameshift mutagenesis by HE, our present study also provides information about the contribution of proofreading to in vitro fidelity. The data from Table I indicate that for the MutD5 enzyme, 33 and 81% of the terminal mismatches are proofread at 1000 and 50 µM dNTP, respectively; for the wild-type enzyme, these numbers are 97.8 and 99.6%, respectively. Converted into fidelity factors (the fold reduction in mutant fraction due to proofreading), the proofreading activity of MutD5 HE contributes 1.5-fold [100/(100-33)] and 5.3-fold [100/(100-81)] to the accuracy of extension synthesis at the two dNTP concentrations, respectively, whereas for the wild-type enzyme, these factors are 45- and 250-fold. Thus, the efficiency of the MutD5 proofreading activity is reduced between 30- and 47-fold compared with the wild-type enzyme. These data are consistent with direct measurements of the exonuclease deficiency associated with the MutD5 enzyme taken in the absence of dNTPs that indicated that the exonuclease activity was decreased 26- to 47-fold (6). Thus, it appears that in the present experiments, which use a preformed terminal mismatch, proofreading correlates directly with the strength of the exonuclease. Interestingly, this correlation does not appear to hold for the proofreading contribution during ongoing in vitro DNA synthesis, in which we measured only a 4- to 6-fold difference in the error rates (for both base substitutions and frameshifts) between the wild-type and the MutD5 enzyme. This may be due to a different binding mode of the enzyme in the two assays. Possibly, upon initial binding to a primer, HE preferentially binds with the primer in the exonuclease subunit, as has been shown to be the case for T7 DNA polymerase (22). In contrast, during ongoing DNA synthesis, the terminal nucleotide is in the polymerase active site, and a kinetic barrier may exist for transfer to the exonuclease site.
Whereas this study provides new insights into the fidelity behavior of
the replicative complex of E. coli, numerous questions still
remain. Poor extension from terminal mismatches can be readily seen as
a valuable attribute for a high fidelity enzyme because it will greatly
increase the potential for exonucleolytic removal, but it is not clear
why a high tendency to transform a terminal mismatch into a frameshift
intermediate is particularly useful. This tendency seems to be a
property intrinsic to the subunit, because a similar frameshift
predominance was observed in fidelity experiments performed with the
isolated
subunit (23). Apparently, the numerous other subunits
present in the HE do not play a significant role in preventing these
frameshifts. However, because high levels of frameshift mutagenesis are
not observed in vivo (even before the action of mismatch
repair) (24), the question of how they are prevented inside the cell is
interesting. We suggest that this additional mode of error prevention
is somehow related to the functioning of HE at the in vivo
replication fork. Further experiments will be needed to address this
important question.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. T. Kunkel and K. Bebenek of National Institute of Environmental Health Sciences for kindly providing the mutant M13mp2 phages used in this study. We thank Drs. K. Bebenek and W. Osheroff for critically reviewing the manuscript.
![]() |
FOOTNOTES |
---|
* 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.
¶ To whom correspondence should be addressed: Laboratory of Molecular Genetics, E3-01, National Institute of Environmental Health Sciences, P. O. Box 12233, 111 TW Alexander Dr., Research Triangle Park, NC 27709. Tel.: 919-541-4250; Fax: 919-541-7613; E-mail: schaaper{at}niehs.nih.gov.
The abbreviations used are: HE, holoenzyme; dNTP, deoxynucleotide triphosphate; bp, base pair; RF, replicative form.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|