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J. Biol. Chem., Vol. 276, Issue 33, 31053-31058, August 17, 2001
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§,
,,**, and
From the Department of Biology and
Rosenstiel Basic Medical Sciences Research Center, Brandeis
University, Waltham, Massachusetts 02254-9110 and the
¶ Department of Biochemistry and Howard Hughes Medical
Institute, Duke University Medical Center, Durham, North Carolina
27710
Received for publication, June 13, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Previous biochemical analysis of
Escherichia coli methyl-directed mismatch repair implicates
three redundant single-strand DNA-specific exonucleases (RecJ, ExoI,
and ExoVII) and at least one additional unknown exonuclease in the
excision reaction (Cooper, D. L., Lahue, R. S., and Modrich,
P. (1993) J. Biol. Chem. 268, 11823-11829). We show
here that ExoX also participates in methyl-directed mismatch repair.
Analysis of the reaction with crude extracts and purified components
demonstrated that ExoX can mediate repair directed from a strand signal
3' of a mismatch. Whereas extracts of all possible single, double, and
triple exonuclease mutants displayed significant residual mismatch
repair, extracts deficient in RecJ, ExoI, ExoVII, and ExoX exonucleases
were devoid of normal repair activity. The RecJ The correction of DNA polymerase misincorporation errors plays an
important role in the maintenance of genetic integrity. DNA
biosynthetic errors in Escherichia coli are corrected by the methyl-directed mismatch repair
(MMR)1 system, which removes
incorrect nucleotides by a strand-specific excision reaction that is
directed to daughter DNA strands by virtue of the transient absence of
d(GATC) methylation within newly synthesized DNA (1). This system
processes both base-base and small insertion/deletion mismatches
(2-4). Inactivation of the genes encoding the nonessential MutH, MutL,
MutS, or UvrD components confers a large increase (100-500-fold) in
mutation rate (1, 5).
Biochemical analysis of the repair of artificial mismatch-containing
substrates has defined 10 activities in a methyl-directed excision
repair reaction reconstituted with purified components (6, 7). Repair
is initiated by the binding of MutS to the mismatch (2, 3, 8), with
MutL binding to the heteroduplex in a MutS- and
ATP-dependent manner (9-11). Assembly of this ternary complex is sufficient to activate the d(GATC) endonuclease activity of
MutH, which incises the unmethylated strand of a hemimethylated d(GATC)
sequence (12), as well as the unwinding activity of DNA helicase II
(uvrD/mutU gene product), which enters the helix at the
incised d(GATC) sequence and unwinds toward the mismatch (13). That
portion of the unwound, incised strand is subject to degradation by one
of several single-strand DNA (ssDNA) exonucleases (7, 14), and
DNA removed in this manner is resynthesized by DNA polymerase III
holoenzyme in the presence of single-strand DNA-binding protein.
Finally, DNA ligase restores covalent continuity to the repaired strand
(6).
In vitro excision can be directed by an incised d(GATC) site
that is located either 5' or 3' to the mismatch on the unmethylated strand, and the nature of the exonuclease requirement depends on the
relative orientation of the two DNA sites (7). If the initial incision
is made 5' to the mismatch, excision requires either ExoVII or RecJ
exonuclease, both of which are capable of 5' The phenotypes of strains mutant in RecJ, ExoI, and/or ExoVII
exonucleases have implicated these genes and their products in DNA
repair and recombination (18-20). In addition, ExoI and ExoVII also
appear to have overlapping roles in preventing frameshift and
quasi-palindrome templated mutations, two classes of mutations associated with strand slippage during DNA replication (21, 22).
Despite the biochemical evidence implicating ExoI, RecJ, and ExoVII in
mismatch repair (7), triple mutants deficient in the three activities
fail to show enhanced mutability in assays that score for spontaneous
base substitutions, a hallmark of MMR deficiency (21, 23). However, it
seems unlikely that DNA helicase II alone would be sufficient to
support methyl-directed excision, especially when one considers that
excision tracts can be a thousand nucleotides or more (14, 24).
Inasmuch as ExoI, RecJ, and ExoVII have been implicated in a UV-induced
recombination pathway that also depends on MutH, MutL, and MutS
mismatch repair activities (25), and because biochemical
experiments have suggested involvement of at least one additional
exonuclease in the methyl-directed reaction, we have sought the
identity of other exonucleases that might support methyl-directed excision.
A novel E. coli 3' We demonstrate here that ExoX also supports methyl-directed mismatch
repair in vitro. The results show that either RecJ or ExoVII
is sufficient to meet the exonuclease requirement when excision
is initiated 5' to the mispair, whereas either ExoI, ExoVII, or ExoX is
sufficient to meet the exonuclease requirement when excision is
initiated 3' to the mismatch. Simultaneous inactivation of ExoX, ExoI,
RecJ, and ExoVII abolishes normal mismatch repair in vitro
but confers only a modest increase in mutation rate. We provide
evidence elsewhere (27) that these exonucleases participate in
methyl-directed repair in vivo, and we demonstrate that the relatively low mutability of the quadruple exonuclease mutant is due to
under-recovery of mutants as a consequence of lethal events triggered
by the occurrence of mismatches in this genetic background.
Bacterial Strains, Media, and Antibiotics--
Isogenic strains
listed in Table I, which were derived from BT199, were constructed by
P1 phage-mediated transduction (28). Strains were grown in LB medium
(1% tryptone, 0.5% yeast extract, and 1% NaCl) or on solid LB medium
containing 1.4% agar. Medium for P1 lysates and transductions
consisted of LB medium supplemented with 10 mM calcium
chloride and 0.2% glucose. Transductants were selected on LB agar
containing 10 mM sodium citrate supplemented with
ampicillin, tetracycline, kanamycin, or chloramphenicol at concentrations of 100, 15, 25, and 15 µg/ml, respectively. LB media
for mutation assays contained rifampicin (Rif; 100 µg/ml). Growth was
at 37 °C, unless otherwise indicated.
Mutation Assays--
Mutation assay scoring for Rif
resistance was performed as described previously (21). Mutation rates
were calculated by the method of the median (29) using the following
formula: mutation rate = M/N, where
M is the calculated number of mutation events, and
N is the mean number of viable cells in the culture.
M is solved by interpolation from experimental determination
of r0, the median number of mutant cells
determined among the cultures for each set of assays, by the formula
r0 = M(1.24 + lnM). The 95% confidence intervals were calculated as described previously (30).
For experiments involving the temperature-sensitive allele of
recJ, culture growth and selection for Rif-resistant
colonies were maintained at 30 °C or 42 °C, as indicated.
In Vitro Mismatch Repair Assays--
E. coliextracts were
prepared as described previously (7) Briefly, ammonium sulfate pellets
were resuspended in 25 mM HEPES (potassium salt, pH 7.6),
0.1 mM EDTA, 2 mM dithiothreitol, and 50 mM KCl and dialyzed against this buffer until the
conductivity of the sample was equivalent to that of 25 mM
HEPES buffer containing 150 mM KCl. Mismatch repair in
cell-free extracts was performed at 37 °C in 20 mM
Tris-HCl buffered reactions (pH 7.6) containing 90 mM KCl
(final concentration including the contribution from the extract) as
described previously (7), except that reactions (20 µl) contained 24 fmol of heteroduplex and ~0.1 mg of extract protein. When indicated,
reactions were performed in a 20 mM Tris-HCl buffer (pH 8)
with a reduced salt concentration (55 mM KCl).
Exonuclease I (U. S. Biochemical Corp.) used for extract
complementation was dialyzed against 20 mM Tris-HCl (pH
7.6), 150 mM KCl, 0.5 mM EDTA, and 5 mM 2-mercaptoethanol for 6 h at 4 °C (with two
buffer changes) to remove glycerol, a potent inhibitor of the repair
reaction in extracts, and stored on ice. The dialyzed exonuclease was
diluted to 10 µg/ml in 20 mM KPO4 (pH 7.4),
50 mM KCl, 1 mM EDTA, 2 mM
dithiothreitol, 100 µg/ml bovine serum albumin, and 1 µl was
added to repair reactions.
Repair in a purified system was performed at 37 °C, essentially as
described previously (6, 7). Reactions contained (per 20 µl) 24 fmol
of heteroduplex DNA, 35 ng of MutS, 30 ng of MutL, 0.26 ng of MutH, 3 ng of DNA helicase II, 150 ng of single-stranded DNA-binding protein
(SSB), 20 ng of E. coli DNA ligase, 100 ng of DNA
polymerase III holoenzyme (generously provided by Dr. Mike O'Donnell,
Rockefeller University), and exonucleases as indicated.
E. coli Deficient in ExoI, ExoVII, ExoX, and RecJ Exonucleases
Display a Modest Mutator Phenotype--
Previous work implicated three
ssDNA-specific exonucleases (ExoI, RecJ, and ExoVII) in methyl-directed
mismatch repair in vitro and indicated that at least one
additional activity also participates in the pathway (7). To examine
the possible role of exonuclease X in this regard, an isogenic set of
mutant strains was constructed with null alleles of ExoX and the three
ssDNA-specific exonucleases that are known to participate in mismatch
correction (Table I). This panel of
mutants constituted a set of strains defective in all possible
combinations of one, two, three, or four null alleles of the
single-strand exonucleases cited above. The rate of spontaneous
mutation was determined for these strains in an assay for Rif
resistance, which scores for base substitution errors. The ExoX single
mutant as well as all triple exonuclease mutant combinations (Table
II) showed no significant increase in
mutation rate compared with the Exo+ control strain. The
quadruple mutant deficient in all four exonucleases exhibited a
moderate increase (7-fold) in mutation rate, suggesting a potential
defect in DNA repair. A quadruple exonuclease mutant with a
temperature-sensitive allele of recJ, recJ146
(20), exhibited a temperature-dependent increase in
mutation rate (Table III), confirming
that it is exonuclease deficiency that is responsible for the mutator
phenotype. The elevation of mutation rate in the quadruple null
exonuclease mutant is considerably less than that in strains defective
in MutS or UvrD, key components of the methyl-directed repair system of
E. coli, which exhibited a 30-100-fold increase in mutation
rate relative to the isogenic wild type control (Table II). The
mutation rates in MutS Orientation-dependent Exonuclease Requirements for in
Vitro Mismatch Repair--
Circular G-T heteroduplexes containing a
single asymmetrically located, hemimethylated d(GATC) site have been
used previously to study the exonucleases involved in the excision step
of methyl-directed mismatch repair in vitro (7, 14). DNAs of
the type shown in Fig. 1 have been used
to study the mismatch-dependent excision reaction that
removes the portion of the unmethylated strand spanning the shorter
path between the two DNA sites irrespective of placement of the
unmethylated d(GATC) sequence 3' or 5' to the mispair. Using such 3'
and 5' heteroduplexes, we have examined mismatch repair in cell-free
extracts derived from wild type E. coli and an otherwise
isogenic quadruple mutant deficient in ExoVII, RecJ, ExoI, and ExoX, as
well as in all possible single, double, and triple mutants (Table
IV).
Extracts of wild type BT199 support efficient repair of both 5' and 3'
heteroduplexes. As shown in Table IV, deficiency of a single
exonuclease has only a modest effect on repair rates. Genetic
inactivation of both ExoVII and RecJ (STL4556) abolished repair on the
5' heteroduplex, confirming a previous observation (7). Both RecJ and
ExoVII have 5'
With one exception, the repair proficiencies of triple mutants that
retain only one of the four activities were consistent with these
polarity assignments. Thus, STL4542 (ExoVII
The nature of the unanticipated 5' repair activity in STL4150 was
addressed in several ways. Introduction of a wild type
ExoI+ allele into STL4150 yielded a strain with an in
vitro repair specificity identical to that of STL4556
(ExoVII
The nature of the anomalous 5' heteroduplex rectification observed in
STL4150 (ExoVII Reconstituted Mismatch Repair--
ExoVII and RecJ have been
previously shown to be required for 5' heteroduplex repair in a
purified system, whereas ExoI was implicated in correction of 3'
heteroduplexes (7). The repair specificity of ExoX has now been
assessed in the reconstituted system. As shown in Fig.
4, ExoX supports repair of the 3'
heteroduplex in the purified system (lane 4) but displays
little if any activity on the 5' heteroduplex (lane 8).
Previous work with the purified system suggested that the action
of ExoVII was restricted to repair of 5' heteroduplexes (7). However,
the extract experiments described above indicated that ExoVII also
contributes to rectification of 3' heteroduplexes. Consequently, we
have re-examined this question in the purified system. As shown
in Fig. 4 (lane 7), ExoVII is highly active in 5'
heteroduplex correction, but this activity also supports a low but
significant level of repair on the 3' heteroduplex substrate
(lane 3). These results are consistent with the extract
results described above. In the absence of 5' exonucleases, a striking
feature of the reactions involving the 5' substrate is the
presence of an apparent repair band of 3.3 kbp, with
under-representation or absence of the expected 3.1-kbp repair band and
the appearance of anomalous species between the 3.3-kbp species and
full-length linear heteroduplex. Reconstitution experiments (data not
shown) demonstrated that this effect is dependent on MutS, MutL, MutH,
helicase II, SSB, and polymerase III holoenzyme. We think it
likely that these anomalous repair bands are due to helicase II strand
displacement from the MutH-incised d(GATC) site coupled with extension
by polymerase III of the exposed terminus. We postulate that the
anomalous 5' repair observed in extracts of STL4150
(ExoVII Previous work has implicated E. coli ExoX, as well as
the ssDNA exonucleases ExoI, ExoVII, and RecJ, in the repair of
UV-damaged DNA (21, 26). We have now demonstrated that these ssDNA
exonucleases also function redundantly during mismatch correction.
Inactivation of ExoI, ExoVII, ExoX, and RecJ conferred a moderate
mutator phenotype, and the corresponding cell-free extracts were devoid
of normal mismatch repair activity in vitro on model heteroduplexes.
The modest increase in mutability observed in the absence of these four
activities is less than that observed for other blocks of the
MMR pathway, including mutS, mutL, mutH, or uvrD
mutant strains. There are two possible explanations for this paradox. The simple heteroduplexes used for biochemical assay may not be good
models for natural substrates. Consequently, the four exonucleases studied here may process some but not all mismatches in the cell. The
alternate possibility is that all four exonucleases contribute to most,
if not all, methyl-directed repair events Depending on the relative position of the mispair to the incision site,
different exonucleases are recruited for the excision step of MMR.
ExoI, ExoX, and ExoVII are all capable of participating in the 3'
repair pathway. Both ExoI and ExoX are strict 3' These findings imply redundancy of exonuclease involvement in this
mutation avoidance pathway and the level of repair supported by
any one of these exonuclease activities is apparently sufficient to
maintain a low level of spontaneous mutability. Mismatch repair proficiency is exhibited in the absence of all but one of these four
exonucleases, further demonstrating that mismatch excision in
vivo is truly bidirectional and can be accomplished from a single
direction if necessary.
Although recJ and xseA (ExoVII large subunit)
orthologues can be found in almost all bacterial genomes sequenced to
date, the other exonucleases are apparently more restricted in their distribution. This means that, at least in bacteria, a different assortment of exonucleases is likely to be called into play for the
excision step of MMR in different species. The redundancy of
exonucleases in E. coli may be due to the specialized roles these enzymes play in other aspects of DNA metabolism. For instance, RecJ mediates recombination via the RecF pathway (20) and may process
stalled replication forks (33). RecJ and ExoI also assist recombination
via the RecBCD pathway (21, 34-36). ExoI and ExoVII play important
roles in the avoidance of misalignment errors during replication, such
as frameshifts (21) and quasi-palindrome templated mutations (22).
ExoVII ExoI ExoX strain
displayed a 7-fold increase in mutation rate, a significant increase,
but less than that observed for other blocks of the mismatch repair
pathway. This elevation is epistatic to deficiency for MutS, suggesting
an effect via the mismatch repair pathway. Our other work (Burdett, V.,
Baitinger, C., Viswanathan, M., Lovett, S. T., and Modrich,
P. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 6765-6770) suggests that mutants are under-recovered in the exonuclease-deficient strain due to loss of viability that is triggered
by mismatched base pairs in this genetic background. The availability
of any one exonuclease is enough to support full mismatch correction,
as evident from the normal mutation rates of all triple mutants.
Because three of these exonucleases possess a strict polarity of
digestion, this suggests that mismatch repair can occur exclusively
from a 3' or a 5' direction to the mismatch, if necessary.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3' hydrolysis of ssDNA
(15, 16), whereas exonuclease I, which is capable of 3'5' hydrolytic
activity (17), is sufficient to meet the exonuclease requirement when
d(GATC) incision occurs 3' to the mispair. Extracts prepared from a
RecJ ExoVII double mutant are defective in
repair directed by a 5' d(GATC) sequence. However, extracts of
ExoI-deficient strains retain repair activity on heteroduplexes where
repair is directed by a d(GATC) site located 3' to the mismatch,
suggesting that at least one additional activity can meet the 3'
exonuclease requirement (7).
5' exonuclease, exonuclease X, has
recently been identified (26). This activity hydrolyzes both single- and double-strand DNA, but its affinity for single-strand DNA is
approximately 10 times greater than that for duplex DNA. Unlike RecJ,
ExoI, and ExoVII, which are processive exonucleases, ExoX is
distributive, hydrolyzing only one or a few nucleotides before releasing from its substrate. The fact that ExoX is distributive does
not affect its potency as a nuclease; in fact, its affinity for
single-strand DNA and its specific activity rival those of RecJ and ExoI.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or UvrD derivatives
of the ExoVII RecJ ExoI
ExoX strain approximated those observed with the
MutS or UvrD strains, suggesting that the
bulk of the mutator effect associated with exonuclease deficiency is
caused by an inefficient mismatch repair pathway.
E. coli K-12 strains
Mutation rate to rifampicin resistance for exonuclease-deficient
strains
Temperature sensitivity of exonuclease-associated mutator phenotype
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Fig. 1.
Circular heteroduplex DNAs. The circular
DNA substrates used in this study contained a G-T mismatch and a single
hemimethylated d(GATC) sequence (2) separated by 1024 base pairs
(shorter path). Heteroduplexes are designated according to the d(GATC)
to mismatch orientation along the shorter path on the unmodified
strand. In 3' heteroduplexes, the unmethylated d(GATC) sequence lies 3'
to the mismatch, whereas this sequence is located 5' to the mismatch in
5' heteroduplexes. The G-T mispair is located within overlapping sites
for XhoI and HindIII, and repair is scored by
cleavage with one of these activities as well as Bsp106I
(2).
In vitro repair with extracts from exonuclease-deficient strains
3' exonuclease activity (15, 16), and both have been
previously shown to be functionally redundant for 5' heteroduplex
repair (7). Deficiency of ExoI (STL2694), ExoX (STL4534), or both
3'5' hydrolytic activities (STL4538) was without effect on repair of
the 5' G-T heteroduplex. ExoI deficiency (STL2694) reduced correction
of the 3' heteroduplex by 50%, and deficiency of both ExoI and ExoX
reduced repair by 63% (STL4538). Residual 3' repair in this double
mutant is attributable to the 3'5' activity of ExoVII (15) because
introduction of an ExoVII null mutation into the ExoI
ExoX background abolished repair activity on the 3'
heteroduplex (STL4541). These findings indicate that either ExoVII or
RecJ is sufficient to meet the exonuclease requirement for repair of 5'
heteroduplexes in E. coli extracts, whereas ExoI, ExoX, or
ExoVII can each contribute in a redundant fashion to repair of 3' heteroduplexes.
RecJ ExoX ExoI+) supported
efficient repair (57% of wild type) of the 3' heteroduplex but
displayed only trace activity on the 5' substrate, STL4541 (ExoVII RecJ+ ExoI
ExoX) displayed about 50% of wild type repair activity
on the 5' heteroduplex but was inactive on the 3' substrate, and
STL4539 (ExoVII+ RecJ ExoI
ExoX) supported significant repair with both heteroduplex
orientations. Surprisingly, extracts of STL4150 (ExoVII
RecJ ExoI ExoX+) supported
rectification of both 3' (15% of wild type) and 5' (80% of wild type)
heteroduplexes, despite the fact that ExoX is known to hydrolyze DNA
with strict 3'5' polarity, and extracts of STL4556
(ExoVII RecJ ExoI+
ExoX+) are devoid of repair activity on the 5'
heteroduplex. We also noted that whereas STL4150 extracts displayed
about 15% of the wild type level of activity on the 3' heteroduplex,
activity was increased to 30% of the wild type level by a slight
change in assay conditions (Table III parenthetic values; see
"Experimental Procedures"). Extracts of VB2 (ExoVII
RecJ ExoI ExoX) showed less
than 2% of the wild type levels of repair of 3' substrate under the
standard assay conditions; however, like STL4150, these extracts also
supported apparent repair of 5' heteroduplex (60% of wild type).
Repair of both 3' and 5' heteroduplexes in extracts of STL4150
(ExoVII RecJ ExoI
ExoX+) and VB1-VB4 (ExoVII RecJ
ExoI ExoX) was strand-specific (data not shown).
RecJ ExoI+
ExoX+) (data not shown), implying that the 5' repair
observed in STL4150 extracts is not a consequence of genetic activation
of an unknown 5'3' exonuclease in this genetic background. Indeed,
as shown in Figs. 2 and
3, the 5' heteroduplex repair observed in
STL4150 (ExoVII RecJ ExoI
ExoX+) and VB1-VB4 (ExoVII RecJ
ExoI ExoX) extracts was suppressed upon
mixing with extract derived from STL4542 (ExoVII
RecJ ExoI+ ExoX) or upon the
addition of purified exonuclease I, although in both cases 3'
heteroduplex repair was enhanced as expected. These results mimic the
repair activities obtained with STL4556 (ExoVII
RecJ ExoI+ ExoX+). Furthermore,
as shown below, ExoX supports repair of only the 3' heteroduplex in the
reconstituted mismatch repair system. We therefore attribute the
apparent repair of the 5' heteroduplex in STL4150 extracts to molecular
events that are suppressed in the presence of ExoI, despite the strict
3'5' hydrolytic polarity of this activity (17).
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Fig. 2.
Methyl-directed mismatch repair in
vitro. Mismatch repair assays were performed as
described under "Experimental Procedures" for 45 min in reactions
containing 150-180 µg of protein (for complementation, 75-90 µg
of each extract was used). Repair products were scored by cleavage with
Bsp106I and either XhoI (3' heteroduplex) or
HindIII (5' heteroduplex) that yield 3.1- and 3.3-kbp
products from repaired DNA (Fig. 1). The 3' and 5' heteroduplexes were
identical except for the state of methylation of the single d(GATC)
site: the 3' substrate was methylated on the complementary strand, and
the 5' heteroduplex was methylated on the viral strand (2). Genotypes
of strains used are summarized in Table I, and repair bands are
indicated by arrows. Repair activity is expressed as
fmol/h/0.1 mg protein.
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Fig. 3.
Exonuclease I restores 3' repair and
suppresses apparent 5' repair in extracts of STL4150
(ExoI ExoVII RecJ
ExoX+) and VB2 (ExoVII RecJ
ExoX). Mismatch repair assays ("Experimental
Procedures") contained extract from STL4150 (lanes 1-4)
or VB2 (lanes 5-8). Reactions corresponding to lanes
1, 2, 5, and 6 utilized 3' heteroduplex, and those for
lanes 3, 4, 7, and 8 contained 5' heteroduplex.
Reactions for odd-numbered lanes were unsupplemented,
whereas reactions for even-numbered lanes were supplemented
with 10 ng of exonuclease I.
RecJ ExoI
ExoX+) and VB2 (ExoVII RecJ
ExoI ExoX) extracts is unclear. As
described below, a related phenomenon was also observed in the purified
system. As shown in Fig. 1, in vitro repair is scored by
cleavage with a restriction enzyme diagnostic for correction as well as
Bsp106I. Repaired molecules thus yield two fragments of 3.1 and 3.3 kbp that are recovered with 1:1 molar stoichiometry.
Significant deviation from molar equivalence of these two species was
observed for repair of the 5' substrate in STL4150
(ExoVII RecJ ExoI
ExoX+) and VB1-VB4 (ExoVII RecJ
ExoI ExoX) extracts, with the smaller
3.1-kbp product consistently under-represented, indicating that these
repair events are to some degree anomalous. The ability of ExoI
to suppress these events in extracts raises the possibility of
preferential interaction between this activity and other components of
the mismatch repair system. The possibility of interaction of
exonucleases with each other or with other components of the repair
system is also suggested by comparison of the levels of 3' heteroduplex
repair observed in STL4555 (ExoVII RecJ+
ExoI ExoX+) and STL2729 (ExoVII+
RecJ ExoI ExoX+) with that
observed in STL4150 (ExoVII RecJ
ExoI ExoX+). The level of 3' heteroduplex
repair activity observed in extracts containing both RecJ and ExoX or
both ExoVII and ExoX was significantly less than that observed with
STL4150 extracts that contain only ExoX; moreover, in contrast to 3'
repair observed in STL4150 extracts, the activity in the former strains
failed to respond to alteration of reaction conditions (Table IV).
RecJ ExoI
ExoX+) or VB2 (ExoVII RecJ
ExoI Exo), discussed above, has a similar
explanation in that there is no suitable exonuclease is present that
can degrade a displaced 5' single strand.
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Fig. 4.
Heteroduplex repair in the purified system in
the presence of ExoVII or ExoX. Repair of 3' and 5' heteroduplexes
in the reconstituted repair system ("Experimental Procedures") was
performed in the presence of the indicated exonuclease. Lanes
1-4 show results with a 3' heteroduplex, and lanes
5-8 show results with a 5' substrate. Lanes 1 and
5, complete reaction in the absence of exonuclease incubated
for 60 min. Lanes 2 and 6, complete reaction in
the absence of exonuclease terminated at zero time. Lanes 3 and 7, 60-min reaction with the addition of exonuclease VII
(10 ng). Lanes 4 and 8, 60-min reaction with the
addition of exonuclease X (12.5 ng). The anomalous repair bands
(arrows) evident in lanes 5 and 8 are
exonuclease-independent. As described in the text, they are probably
derived from a strand displacement product produced by 5' 3'
extension by DNA polymerase III holoenzyme from the MutH-incised
d(GATC) sequence.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
the relatively low
mutability of the exonuclease-deficient strain may be due to
under-recovery of mutants due to lethal events triggered by the
occurrence of mismatches in this genetic background. We present evidence elsewhere (27) that the latter explanation is likely to be
correct. Quadruple mutants deficient in the four exonucleases are
cold-sensitive for growth, undergo filamentation, and are extremely
sensitive to 2-aminopurine, a base analogue that promotes mispairing
and triggers the methyl-directed system (31). These effects are
suppressed by the introduction of null mutations in mutS, mutL,
mutH, or uvrD, genes that mediate the earlier steps in
the MMR pathway. The exact nature of this lethality is not known, but
presumably accumulation of ssDNA displaced during MMR is
deleterious to the cell.
5' exonucleases (26,
32), and repair of the 3' substrate in extracts deficient in these two
exonucleases is reduced by 50%. No repair activity above background
was detected in extracts when a null mutation in ExoVII was introduced
into the ExoI ExoX deficient background.
The results of this study differ from our earlier finding with respect
to the ability of ExoVII to support repair of a 3' heteroduplex.
Whereas previous work failed to detect 3' heteroduplex repair supported
by ExoVII in the reconstituted system (7), we now find that ExoVII can
support 3' heteroduplex repair in both cell-free extracts and the
purified system. However, in agreement with the previous study, we find
that ExoVII is much less effective in supporting 3' heteroduplex repair
as compared with its activity with 5' substrates (Fig. 4, compare
lanes 3 and 7). In agreement with earlier results
(7), we show here that RecJ or ExoVII is sufficient for removal of
mismatched bases via excision tracts initiated 5' of the mismatch. Thus
both the 3' and 5' exonuclease activities of ExoVII can be utilized in the mismatch correction system.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. Mike O'Donnell for the generous gift of DNA polymerase III holoenzyme and W. Wackernagel, R. Kolodner, and M. Marinus for strains.
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FOOTNOTES |
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* This work was supported by Grants GM43889 (to S. T. L.), GM07122 (to M. V.), and GM23719 (to P. M.) from the General Medical Institute of the National Institutes of Health.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.
§ Present address: Massachusetts Institute of Technology, 68-288D, 77 Massachusetts Ave., Cambridge, MA 02139.
** An Investigator of the Howard Hughes Medical Institute.
To whom correspondence should be addressed: Rosenstiel Basic
Medical Sciences Research Center, MS029, Waltham, MA 02454-9110. Tel.:
781-736-2497; Fax: 781-736-2405; E-mail lovett@brandeis.edu.
Published, JBC Papers in Press, June 19, 2001, DOI 10.1074/jbc.M105481200
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ABBREVIATIONS |
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The abbreviations used are: MMR, methyl-directed mismatch repair; ssDNA, single-strand DNA; Rif, rifampicin; kbp, kilobase pair(s).
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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