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J. Biol. Chem., Vol. 277, Issue 15, 13302-13311, April 12, 2002
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,§¶, and§
From the Department of Biochemistry and
§ Howard Hughes Medical Institute, Duke University Medical
Center, Durham, North Carolina 27710
Received for publication, December 12, 2001, and in revised form, January 24, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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We have partially purified a human activity that
restores mismatch-dependent, bi-directional excision to a
human nuclear extract fraction depleted for one or more mismatch repair
excision activities. Human EXOI co-purifies with the excision activity,
and the purified activity can be replaced by near homogeneous
recombinant hEXOI. Despite the reported 5' to 3' hydrolytic polarity of
this activity, hEXOI participates in mismatch-provoked excision
directed by a strand break located either 5' or 3' to the mispair. When
the strand break that directs repair is located 3' to the mispair, hEXOI- and mismatch-dependent gap formation in
excision-depleted extracts requires both hMutS Human cells possess a strand-specific mismatch repair system that
is similar to that of Escherichia coli and depends on
structural and functional homologs of bacterial MutS and MutL (1-4).
Inactivation of genes that encode the mammalian MutS homologs MSH2 or
MSH6 or the MutL homologs MLH1 or PMS2 confers genetic instability and
a predisposition to tumor development. Eleven activities have been
implicated in E. coli methyl-directed mismatch repair, and the reaction has been reconstituted in a pure system (5-8). However, our understanding of the reaction in higher cells is limited.
Analysis of the human reaction in nuclear extracts, using model
heteroduplexes in which a strand-specific single strand break directs
repair to the incised DNA strand (9, 10), has indicated that the
reaction occurs in several steps by a mechanism similar to that of
E. coli mismatch correction (11, 12). Repair is initiated
via mismatch recognition by hMutS The excision step of E. coli methyl-directed mismatch
correction depends on DNA helicase II, as well as several 3' to 5' and 5' to 3' exonucleases that display specificity for single-stranded DNA
(6-8, 26). Several activities have been implicated in the initiation
and repair synthesis steps of eukaryotic mismatch repair, but the
nature of the excision step of the reaction is less well understood. In
yeast, deficiency of the 5' to 3' exonuclease EXOI (27) confers a weak
mutator phenotype (28, 29) that has been assigned to the
MSH2 epistasis group (29, 30). The enhancement of mutability
by exoI null defects is modest as compared with that
observed upon MSH2 inactivation, but this could be due to redundant exonuclease involvement as in the bacterial reaction (29). In
fact, Amin et al. (31) have recently demonstrated a
synergistic potentiation of mutation rates when yeast exoI
defects are combined with weakly mutagenic, missense alleles in genes that encode MLH1, PMS1, MSH2, PCNA, or DNA polymerase Analysis of dinucleotide repeat instability in yeast suggested that the
RAD27 5' to 3' exonuclease might also participate in mismatch repair
(37); however, more recent studies indicate that the contribution, if
any, of this activity to mismatch rectification is limited (38, 39).
Yeast genetic studies have also led to the suggestion that the 3' to 5'
editing exonucleases of DNA polymerases To further clarify the nature of the excision activities involved in
the mammalian reaction, we have developed an in vitro assay
for mismatch-provoked excision. Utilizing this method we have
demonstrated that hEXOI is required for mismatch repair directed by a
strand break located either 3' or 5' to the mispair, that in
vitro excision directed a 3' or 5' strand signals differ in their
requirement for hMutL Cell Culture and Nuclear Extracts--
Cell lines HeLa S3, H6 (a
subclone of HCT116), and MT1 were grown, and nuclear extracts were
prepared as described previously (9, 18, 42). Insect cells (SF9,
Invitrogen) were cultured, infected with baculovirus constructs
expressing hMutL Preparation of Excision-depleted Nuclear Extracts and Protein
Isolation--
All procedures were performed at 4 °C. Protein was
determined by Bradford assay using bovine serum albumin as standard or by absorbance at 280 nm.
For preparation of excision-depleted extracts, MT1
(MSH6
hMutS
Recombinant hEXOIa and hEXOIb were isolated from SF9 cells infected
with baculovirus-expressing constructs, which were prepared using the
pFASTBAC system (Invitrogen) according to the manufacturer's recommendations. hEXOI cDNA (EST843301, Ref. 33), kindly provided by R. Kolodner, was digested with EcoRV and KpnI
and ligated into pFASTBAC I cut with StuI and
KpnI. The resulting construct contains the complete open
reading frame of the shorter, 803-amino acid splice variant designated
hEXOIa (33). PCR mutagenesis was used to generate the corresponding
pFASTBAC I derivative of the longer, 844-residue splice variant, hEXOIb
(33).
Insect cells were infected with baculovirus expressing hEXOIa or
hEXOIb, and cleared supernatants were prepared as described previously
(43). For hEXOIa preparation, cleared supernatant (150 mg of protein)
was loaded onto a 2-ml Heparin HiTrap column (Amersham Biosciences)
equilibrated with buffer B (0.02 M KPO4, pH
7.5, 0.1 mM EDTA, 10% (v/v) glycerol) containing 0.19 M KCl. After a wash with 20 ml of starting buffer, the
column was eluted with a 20-ml gradient of 0.19-0.415 M
KCl in buffer B. hEXOIa containing fractions, which eluted about 0.3 M KCl, were supplemented with 2 mM DTT and
protease inhibitors to the levels noted above for buffer A. This
solution was diluted 2-fold with buffer A containing 10% (v/v)
glycerol and loaded onto a 1-ml MonoS column (HR 5/5, Amersham
Biosciences) equilibrated with buffer B containing 0.19 M
KCl. This column was washed and developed using the same protocol as
for the Heparin column. hEXOIa fractions, which eluted about 0.4 M KCl, were pooled, supplemented with 2 mM DTT
and protease inhibitors as described above, quick-frozen in liquid
nitrogen, and stored at
hEXOIb was purified by the same method except that the insect
cell-cleared lysate (about 300 mg of protein) was first loaded onto a
15-ml Q-Sepharose column equilibrated with buffer B containing 0.15 M KCl, 1 mM DTT, and protease inhibitors as
noted above. After washing with 75 ml of starting buffer, the column
was eluted with a gradient from 0.15-0.40 M KCl in buffer
B containing 1 mM DTT and supplemented with protease
inhibitors as above. hEXOIb-containing fractions, which eluted at 0.27 M KCl, were pooled, diluted 2-fold with buffer A containing
10% (v/v) glycerol, and subjected to fractionation on Heparin HiTrap
and MonoS columns as described above. hEXOIb eluted at ~0.3
M KCl on both of the latter columns. Final purities of
hEXOIa and hEXOIb obtained in this manner were ~95% (Fig. 4).
Purification of Mismatch-dependent Gap Formation
Activity--
1160 mg of HeLa nuclear extract was thawed on ice and
adjusted to a conductivity equivalent to 0.20 M KCl with
buffer B containing 0.40 M KCl and 1 mM DTT and
supplemented with protease inhibitors as above (Fraction I). Fraction I
was loaded onto a 90-ml phosphocellulose P-11 column equilibrated with
protease inhibitor-supplemented buffer B containing 0.20 M
KCl and 1 mM DTT. After a wash with 360 ml of starting
buffer, the column was eluted with a 10-step KCl gradient (0.22-0.40
M, 10-ml, and 0.02 M KCl increase per step).
Fractions were collected and quick-frozen in liquid nitrogen and stored
at DNA Substrates and Mismatch-directed Activity
Assays--
In vitro mismatch repair assays were performed
as described previously (9) except that reaction volume was increased
to 20 µl, salt concentration was adjusted to 0.1 M KCl,
and incubation was for 15 min at 37 °C. Assay of
mismatch-dependent gap formation was performed in the same
manner except that dNTPs were omitted and incubation was reduced to 5 min. Reactions contained 100 ng (23 fmol) of a bacteriophage
f1-derived G-T or /CA\
insertion/deletion heteroduplex (Fig. 1 and Ref. 21) or an otherwise
identical A·T homoduplex, and as indicated, 64 µg of
excision-depleted MSH6 Immunological Methods--
Rabbits were immunized with multiple
antigen peptide conjugated peptides, and sera were obtained
using methods described before (17). Antibodies were raised against
hEXOI-derived peptide 1 (TLPSKKEVERSRRERRQANL, amino acids 81-100) and
peptide 2 (RASGLSKKPASIQKRKHHNA, amino acids 762-781). The resulting
antibodies recognize both hEXOIa and hEXOIb and are referred to as AB-1
and AB-2, respectively. Antibodies raised against peptide 3, which is
only present in hEXOIb (KKPLSPVRDNIQLTPEAEED, residues 811-830), are
referred to as AB-3. Preimmune and specific IgG fractions were obtained by purification on protein A-Sepharose (Amersham Biosciences). Specific
IgGs were further purified on affinity supports prepared by
cross-linking individual peptide antigens to Reacti-Gel 6X (Pierce).
For the cross-linking, 25 mg of peptide was incubated with 1 ml of
activated resin in 0.1 M sodium carbonate, pH 10, for
24 h at 4 °C. The reaction was stopped by incubation with 1 M ethanolamine, pH 10, for 24 h. After extensive wash,
peptide columns were loaded with the protein A-purified IgG and
incubated overnight at 4 °C. The columns were washed extensively and
then eluted with 0.05 M glycine, pH 2.5, and 0.15 M NaCl. Fractions were immediately neutralized with 0.1 volume of 1 M Tris-HCl, pH 8.0, pooled, dialyzed against
0.025 M Hepes-KOH, pH 7.5, 0.15 M KCl,
aliquoted, quick-frozen in liquid nitrogen, and stored at
Gel electrophoresis and Western blotting were performed as described
previously (17). For immunodepletion, 20 µg of AB-2 was bound to 2 µl of protein A-Sepharose suspension and incubated with HeLa nuclear
extract (0.45 mg) in a total volume of 15 µl on ice for 4 h. The
resin was then removed by centrifugation, and the supernatant was used
directly in gap formation or mismatch repair assay. In mock depletion
experiments, AB-2 was replaced with preimmune IgG obtained from the
same rabbit.
Partial Purification of a Mismatch Repair Excision
Activity--
Previous assays for mismatch-provoked excision are
time-consuming (20, 21) and unsuitable as a routine assay for isolation of the required excision components. To circumvent this problem we
developed a rapid method that relies on the observation that excision
tracts produced on an incised heteroduplex extend from the strand break
to 90-170 nucleotides beyond the mispair (21). When repair DNA
synthesis is blocked, excision renders an NheI site just
beyond the mispair single-stranded and hence resistant to
endonuclease cleavage. As shown in Fig.
1, gap formation scored by this
NheI-resistant assay depends on the presence of a mismatch within the nicked, circular substrate.
Fractionation of HeLa nuclear extract indicated that one or more
activities involved in the excision step of mismatch repair elute from
phosphocellulose between 0.23 and 0.38 M KCl. This observation was exploited to prepare a crude fraction that was depleted
of excision activity for use as receptor extract for isolation of
excision activity by in vitro complementation (see "Experimental Procedures"). To avoid artifacts during
fractionation, receptor extracts were prepared from mismatch-repair
deficient MSH6
An activity that restored mismatch-dependent gap formation
to these excision-depleted extracts was purified 250-fold from HeLa nuclear extract. As summarized in Table
I, gap formation activity on 3'- and
5'-heteroduplex substrates co-purified, with activity profiles on the
two types of substrate co-eluting during the three-column steps (Fig. 3
and data not shown). The purified material (Fraction IV) was analyzed
for its activity on homoduplex and several heteroduplex DNAs using
excision-depleted extract derived from MSH6 hEXOIb Co-purifies with Mismatch Repair Excision Activity--
The
excision activity isolated by the procedure shown in Table I is not
pure and attempts to further fractionate the material failed due to
activity loss. However, we have identified a required excision activity
present in this partially purified material. Previous work in
Saccharomyces pombe and S. cerevisiae has
demonstrated that inactivation of the EXOI gene, which encodes a 5' to
3' double-stranded exonuclease (27, 29), results in a modest increase
in mutability (28, 29) that is epistatic to the larger increase in
mutability associated with msh2 mutations (29, 30).
cDNAs corresponding to two splice variants of a human EXOI
homologue have been identified, both contain the putative exonuclease
hydrolytic center (33-35), and the human EXOI polypeptide has been
shown to interact with hMSH2 and hMLH1 (36). The two hEXOI splice
variants, hEXOIa and hEXOIb, encode polypeptides of 803 and 846 amino
acids, respectively, and both contain the putative exonuclease
hydrolytic center (33). The 802 amino-terminal residues of the two
variants are identical, with the two polypeptides differing in their
C-terminal 1 and 44 amino acids (33). Potential presence of hEXOI
polypeptides in partially purified excision activity was evaluated by
use of anti-peptide hEXOI polyclonal antibodies (see "Experimental
Procedures"). Antibodies AB-1 and AB-2 recognize both splice
variants, whereas AB-3 recognizes the unique C-terminal extension
hEXOIb (see "Experimental Procedures").
As shown in Fig. 3, hEXOI polypeptide(s)
recognized by both AB-1 and AB-3 co-purified with excision activity,
co-eluting with excision activity as a 114-kDa species during each
chromatographic step (Fig. 3 and data not shown). Since antibody AB-3
is specific for hEXOIb, it is clear that this splice variant
co-purifies with excision activity. We have been unable to determine
whether hEXOIa also co-purifies with excision activity due to the fact
that only a single positive band was observed in the appropriate size
range on Western blots using antibody AB-1 that recognizes both splice variants (Fig. 3, lower panel); however, this may be due to
the small difference in the electrophoretic mobilities of the two forms
of the enzyme (see below). The AB-1-positive minor species (100 kDa)
evident in phosphocellulose and DNA cellulose fractions is not hEXOIa
and may be derived from hEXOIb by proteolysis since it was not detected
in extracts and reacts with antibody specific for this variant.
Recombinant hEXOI Supports Mismatch-directed Gap Formation--
To
assess the role of hEXOI in the excision step of mismatch repair,
cDNAs for hEXOIa and hEXOIb were expressed in a baculovirus system,
and the two proteins were isolated in near homogeneous form (Fig.
4, "Experimental Procedures"). The
electrophoretic mobilities of these two polypeptides in the presence of
sodium dodecyl sulfate correspond to molecular mass of 115 kDa
and 112 kDa, somewhat greater than those predicted from primary
sequence (94 kDa for hEXOIb and 89 kDa for hEXOIa); however, the
mobility of recombinant hEXOIb is essentially identical to that of
antibody AB-1 and AB-3-positive, 114-kDa material in purified excision activity. The specificities of these two antibodies for the several forms of the protein were confirmed using near homogeneous preparations of the recombinant activities (Fig. 4, lower panel).
Quantitative comparison of Western signals from HeLa extract with those
obtained using known quantities of purified hEXOIb indicates that
nuclear extract contains about 50-100 ng of hEXOI/mg total protein
(data not shown).
The possibility that recombinant hEXOI might substitute for purified
excision activity was tested. As shown in Table
II, 10 ng of near homogeneous hEXOIb was
nearly as efficient in restoring mismatch-dependent gap
formation to excision-depleted extracts as was 0.6 µg of the purified
excision activity (Fig. 2), which contains 7-15 ng of hEXOI as judged
by quantitative Western blot. As observed with the partially purified
excision activity, hEXOIb-supported gap formation on a 3'-heteroduplex
required both hMutS Immunological Depletion of hEXOI Reduces the Efficiency of Gap
Formation and Repair of Both 5'- and 3'-Heteroduplexes--
The use of
excision-depleted extracts in the experiments described above suggests
that hEXOI is required for excision directed by a strand break located
either 5' or 3' to the mismatch. However, this interpretation is
compromised by the fact that the crude fractionation procedure used to
produce depleted extract removes a large number of proteins. To further
clarify the requirement for hEXOI in the human strand-specific repair
reaction, affinity-purified, polyclonal anti-hEXOI peptide antibody
AB-2 (see "Experimental Procedures") was used to deplete HeLa
nuclear extract of the activity. As compared with mock depletion with
preimmune IgG, hEXOI immunodepletion reduced mismatch-provoked gap
formation by 70% on 5'- and 3'-heteroduplexes and reduced the
efficiency of mismatch repair on the two types of substrate by 50-60%
(Table III). The somewhat lower reduction in the efficiency of mismatch repair may be due to the fact that incubation time for repair assays was three times that of excision assays. Supplementation with near homogeneous hEXOIb efficiently restored mismatch repair and mismatch-dependent gap
formation to normal levels.
hEXOI Participates in Excision-directed by Strand Signals Located
3' or 5' to the Mismatch--
yEXOI and hEXOIa have been shown to
catalyze 5' to 3' hydrolysis of duplex DNA (27, 29, 35), but the
results shown in Table II suggest that the human enzyme is involved in
mismatch-provoked excision directed by a strand break that can be
located either 5' or 3' to the mismatch. To clarify the nature of the
hEXOI-dependent excision events occurring on 3'- and
5'-heteroduplexes, excision tract end points were mapped using an
indirect end-labeling procedure (21).
With a 5'-G-T heteroduplex (Fig. 5,
left panel) excision tracts produced in HeLa nuclear extract
extended from the substrate strand break to about 150 nucleotides
beyond the mismatch (lane 5), as observed previously (21).
As can be seen, the control A·T homoduplex did not support production
of this material (lane 6). Similar excision products were
observed with excision-depleted extracts upon hEXOIb supplementation
provided that extract derived from MSH6
A similar type of experiment with 3'-heteroduplex and -homoduplex
substrates is shown in the right panel of Fig. 5,
but in this case HeLa nuclear extract was immunologically depleted for hEXOI using antibody AB-2 or mock-depleted using preimmune IgG. No
significant excision was evident on homoduplex DNA, but excision products were produced on the G-T heteroduplex in mock-depleted nuclear
extract (G-T panel of upper gel, lane 16). The yield of these products (with a mobility corresponding to an average length of
about 2900 nucleotides) was reduced substantially when the extract was
depleted of hEXOI using antibody AB-2 (lane 17) but was
restored to normal levels upon supplementation with purified hEXOIb
(lane 18). Thus, hEXOI participates in mismatch-provoked excision on a 3'-heteroduplex that occurs with a directionality that is
formally 3' to 5'.
hMutS
Although not readily evident in photographic reproduction
shown in the lower panel of Fig. 6, background hEXOI
hydrolysis resulted in a low level of similar products with both A·T
homoduplex and with G-T heteroduplex, in the latter case in the absence
of hMutS
The nature of the hydrolytic products produced in a pure system with
nicked heteroduplex DNA in the presence of hEXOIb, hMutS
The basal activity of hEXOIb in the absence of hMutS
Since the oligonucleotide probes used to label the 3' termini of
incised heteroduplex strands in Fig. 7 were present in large excess,
the labeling intensities observed correspond to the recovery of these
3'-ends. Integration of hybridized label in the heteroduplex lanes
shown in upper and lower panels of
Fig. 7 demonstrated that recovery of 3' termini in all reactions that
contained hEXOI was essentially 100% as compared with control DNA
incubated in the absence of protein. Coupled with the results above,
this retention of incised strand 3' termini implies that degradation of
the incised strand was exclusively 5' to 3' irrespective of 3' or 5'
orientation of the heteroduplex. This finding is consistent with the
reported 5' to 3' polarities of yeast and human EXOI (27, 29, 35). Thus, while mismatch-, hMutS The experiments described here indicate that hEXOI participates in
the excision step of the human strand-specific mismatch repair
reaction, observations consistent with genetic studies in yeast that
have implicated yEXOI in a mutation avoidance pathway that involves
yMSH2, yMLH1, and yPMS2 (29-32) and the finding that hEXOI interacts
with both hMSH2 and hMLH1 (36). Although yeast and human EXOI have been
shown to be 5' to 3' exonucleases (27, 29, 35), the work described here
indicates that the activity has important roles in both 5'- and
3'-heteroduplex repair. If excision on these two types of heteroduplex
is assumed to be exonucleolytic initiating at the strand break as it
does in the bacterial system (6-8, 44), excision in the human system
will require both 5' to 3' and 3' to 5' hydrolytic activities. While
hEXOI clearly possesses a 5' to 3' activity, its involvement in
3'-heteroduplex repair suggests that the enzyme has a cryptic 3' to 5'
activity or that it is necessary for activation of a distinct activity that is responsible for excision on 3'-heteroduplexes.
Using near homogeneous components, we have found that hMutS In these studies we have used nuclear extract depleted of hEXOI by
fractionation or immunological removal. Depleted extract obtained by
fractionation retains 10-20% of the mismatch-provoked excision
activity observed in hEXOI-supplemented reactions (Fig. 2 and Table II)
or in undepleted extract fractions (data not shown), while
immunologically depleted extract retains 30% residual activity (Table
III). It is not clear whether this residual is due to low levels of
remaining hEXOI in the depleted fractions or whether alternate
activities can also support excision, as is the case in the bacterial
system where redundant exonuclease involvement has been demonstrated
(6-8). The fact that EXOI-deficient S. cerevisiae strains
are considerably less mutable than msh2 mutants (29, 30, 32)
may indicate that alternate activities can support the excision step in
this lower eukaryote as well. However, E. coli strains
deficient in all four of the exonucleases required for the bacterial
reaction are similarly less mutable than MutS- or MutL-deficient
strains (8), an effect that has been attributed to under-recovery of
mutants due to chromosome loss in the exonuclease-deficient background
(7). The possibility that the low mutability of yeast exoI
mutants might be due to a similar phenomenon has not been addressed.
Recent genetic studies in S. cerevisiae have suggested
several roles for yEXOI in mismatch repair. Synergistic potentiation of
mutation rates has been observed in double mutants harboring exoI mutations and weakly mutagenic alleles of genes
encoding yMLH1, yPMS1, yMSH2, yPCNA, and yeast DNA polymerase The use of excision-depleted extracts from
MSH6 There is also a precedent for hMutL The second class of hMutL While the basis of the distinct repair phenotypes of the two classes of
hMutL
and hMutL.
However, excision directed by a 5' strand break requires hMutS but
can occur in absence of hMutL. In systems comprised of pure
components, the 5' to 3' hydrolytic activity of hEXOI is activated by
hMutS in a mismatch-dependent manner. These observations
indicate a hydrolytic function for hEXOI in 5'-heteroduplex correction.
The involvement of hEXOI in 3'-heteroduplex repair suggests that it has
a regulatory/structural role in assembly of the 3'-excision complex or
that the protein possesses a cryptic 3' to 5' hydrolytic activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(the hMSH2·hMSH6 heterodimer)
(13, 14) or hMutS
(hMSH2· hMSH3 heterodimer) (15-17). hMutL
(hMLH1·hPMS2 heterodimer) and
PCNA1 are also required
during the earliest stages of the reaction since inactivation of either
of these activities blocks repair at or prior to initiation of excision
(18-20), which removes that portion of the incised strand spanning the
strand break and the mispair (21, 22). Subsequent repair DNA synthesis
depends on DNA polymerase 
and PCNA (23, 24). hRPA, the human
single-stranded DNA binding protein, has also been implicated in
mismatch repair (25), but the stages of the reaction during which this
protein functions have not been defined.
. Since all of
these activities have been implicated in mismatch repair (1-4), these
findings have been interpreted in terms of direct involvement of yeast
EXOI in the reaction. Yeast EXOI has also been shown to interact
physically with yMSH2 and yMLH1 (29, 32), and the human EXOI homolog
(33-35) interacts with hMSH2, hMSH3, and hMLH1 (36), implicating the
exonuclease in one of the several genetic stabilization pathways that
depend on these mismatch repair proteins.
and 
may participate in
the excision step of mismatch repair (40), but this conclusion has also
been questioned based on the finding that the mutator phenotype of the
pol3-01 mutant used in this study is largely due to a
checkpoint defect rather than mismatch repair deficiency (41).
, and that 5' to 3' hydrolysis by hEXOI is
activated in a mismatch- and hMutS-dependent manner in a
pure system.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits or hEXOI, and processed for protein
purification as described (43). HeLa S3 nuclear extracts (9) were
concentrated by precipitation with 420 g/liter ammonium sulfate,
dialyzed against buffer A (0.025 M Hepes-KOH, pH 7.5, 0.1 mM EDTA, 0.1% (v/v) phenylmethylsulfonyl fluoride (Sigma,
relative to a saturated stock in isopropanol), 1 µg/ml leupeptin
(Peptides International), 0.5 µg/ml E-64 (Peptides International),
0.1 µg/ml aprotinin (USB)) containing 100 mM KCl and 2 mM dithiothreitol (DTT), quick-frozen in liquid nitrogen, and stored at 
80 °C.
/) and H6
(MLH1/) nuclear extracts (about 200 mg of
protein) were adjusted to a conductivity equivalent to 225 mM KCl by the addition of 3 M KCl and loaded
onto 10 ml of P-11 phosphocellulose (Whatman) equilibrated with buffer
A containing 0.225 M KCl and 1 mM DTT. The
column was washed with 30 ml of starting buffer, and fractions
containing protein that passed through the column were pooled. After a
wash with 50 ml of buffer A containing 0.375 M KCl and 1 mM DTT, the column was step-eluted with buffer A containing
1 M KCl and 1 mM DTT. Protein-containing
fractions passing through the column in 0.225 M KCl were
combined with those eluting in the 0.375-1 M KCl step.
This material, which is depleted of mismatch-provoked excision
activity, was concentrated, dialyzed, and stored as described above for
HeLa nuclear extract. In addition to removal of excision activity, this
depletion procedure also results in partial removal of hMutS and
hMutL. To ensure that these required activities were not limiting,
depleted extract fractions were supplemented with near homogeneous
hMutL or hMutS prior to use. Thus, excision-depleted extract from
MSH6/ MT1 cells was supplemented with
hMutL (0.78 µg/mg extract protein), and the supplemented extract
is referred to below as excision-depleted MSH6/ extract. Excision-depleted extract
from MLH1/ H6 cells was supplemented
with hMutS (3.1 µg/mg) in a similar manner.
was purified to homogeneity from HeLa cells by a minor
modification of the previously described method (13). Recombinant hMutL was purified to homogeneity from a cleared supernatant prepared from insect cells infected with baculovirus constructs that
express hMLH1 and hPMS2 (43) using chromatography on phosphocellulose, hydroxyapatite, and MonoQ as described for isolation of HeLa
hMutL (19). To remove insect excision activities, the
phosphocellulose eluate was passed over single-stranded DNA-cellulose
in 0.20 M KCl prior to loading onto hydroxyapatite.
80 °C.
80 °C. phosphocellulose chromatography was repeated on a second
batch of nuclear extract (1160 mg), and fractions restoring gap
formation from the two preparations, which eluted about 0.33 KCl, were
thawed on ice and pooled (Fraction II). Fraction II was dialyzed
against 2 liters of protease inhibitor-supplemented buffer B containing
0.175 M KCl and 2 mM DTT until the conductivity was equivalent to 0.20 M KCl and loaded onto a 10-ml
single-stranded DNA-cellulose column equilibrated with buffer B
containing 0.20 M KCl, 1 mM DTT, and protease
inhibitors at 2 column volumes per hour. After a wash with 100 ml of
starting buffer, the column was eluted with a 10-step KCl gradient
(0.22-0.40 M, 1-ml, and 0.02 M KCl increase
per step). Fractions containing excision activity, which eluted about
0.33 M KCl, were quick-frozen in liquid nitrogen (Fraction
III). Fraction III was thawed, dialyzed against 1 liter of buffer B
supplemented with protease inhibitors and containing 0.075 M KCl and 2 mM DTT until the conductivity was
equivalent to 0.10 M KCl, and then loaded onto a 1-ml MonoQ
FPLC column (HR 5/5, Amersham Biosciences) equilibrated with buffer B
containing 0.10 M KCl. After a wash with 20 ml of starting
buffer, the column was eluted with a 14-ml gradient of KCl (0.10-0.50
M KCl). Active fractions, which eluted about 0.26 M KCl, were pooled and supplemented with protease
inhibitors as above, aliquoted, quick-frozen in liquid nitrogen, and
stored at 80 °C (Fraction IV). The purification is summarized in
Table I.
/ or
MLH1/ extract, 200 ng of hMutS, 50 ng of
hMutL, and purified excision activity or recombinant hEXOI. DNA
recovered from gap formation reactions was digested with 2 units each
of NheI and Bsp106I, and restriction products
were resolved by electrophoresis through 1% agarose gels, which were
stained with ethidium bromide. DNA was quantitated by ethidium
fluorescence using a cooled, photometric grade CCD imager
(Photometrics). As summarized in Fig. 1, gap formation activity is
defined as the amount of substrate linearized by Bsp106I but
resistant to cleavage by NheI. Mapping of excision tract end points was performed by indirect end-labeling as described (21) except that results were visualized and quantitated using a
PhosphorImager (Molecular Dynamics).
80 °C.
AB-1, AB-2, and AB-3 used in the work described below were all
affinity-purified in this manner.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (31K):
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Fig. 1.
Mismatch-provoked excision can be scored by
restriction endonuclease assay. The 6440-bp circular f1 DNAs used
in this work (18, 21) contained a G-T base-base mismatch, a
/CA\ dinucleotide insertion/deletion mispair,
or an A·T base pair (homoduplex control), as well as a site-specific,
strand-specific single strand break. Because mismatch-provoked excision
removes DNA spanning the shorter path between the strand break and the
mispair in these circular heteroduplexes (21), a polarity is assigned
to these substrates depending on placement of the strand break 3' or 5'
to the mismatch as viewed along the shorter path joining the two sites.
A heteroduplex containing a nick in the complementary DNA strand at the
Sau96I site 125 bp from the mismatch is referred to as a
5'-substrate, while a circle containing a nick at the gpII
cleavage site in the viral strand 181 bp from the mismatch is a
3'-substrate. As shown diagrammatically in the left panel,
mismatch-provoked gap formation directed by either type of strand break
extends beyond the NheI site that is located 10 bp from the
mismatch (21), rendering the DNA resistant to digestion by this
endonuclease. The right panel shows an example of
mismatch-dependent gap formation in HeLa cell nuclear
extract. Reactions performed in the absence of exogenous dNTPs (see
"Experimental Procedures") contained a 5'- or 3'-G-T heteroduplex
or A·T homoduplex as indicated and 75 µg of nuclear extract
protein. After digestion with NheI and Bsp106I,
DNA products were resolved by electrophoresis through 1% agarose. The
presumed nature of the DNA products is illustrated to the
left of the gel. The gapped DNA species visible near the
top of the gel migrate just slightly faster than intact,
full-length linear DNA. Quantitation of this data yielded the following
values for the extent of excision (fmol/5 min): 5'-G-T, 8.4; 5'-A·T,
1.1; 3'-G-T, 4.8; 3'-A·T, 0.5.
/ MT1 cells
(hMutS-deficient) or MLH1/ H6 cells
(hMutL-deficient), and gap formation was scored as bona
fide only if it required depleted extract, hMutS (or hMutL), and the partially purified fraction in question. Since the
fractionation method for depletion of excision activity also results in
partial removal of hMutS and hMutL, extracts prepared in this
manner were supplemented with the appropriate activity to ensure that it was not rate-limiting for repair, i.e. depleted extract
derived from MSH6/ MT1 cells was
supplemented with hMutL and is referred to here as
hMutS-deficient depleted extract, while that derived from MLH1/ H6 cells was supplemented with
hMutS and is referred to as hMutL-deficient depleted extract (see
"Experimental Procedures").
/
or MLH1/ cells. Only a background level of
gap formation occurred with homoduplex DNA when incubated with
hMutS- or hMutL-deficient depleted extract, and homoduplex
background activity was enhanced only to a limited degree by
supplementation with hMutS, hMutL, and purified excision activity
(Fig. 2, open bars). By
contrast, gap formation on 3'-G-T and 3'-/CA\
heteroduplexes was dramatically enhanced in excision-depleted
MSH6/ extracts upon supplementation with
hMutS and the purified excision activity, and a similar enhancement
of gap formation on 3'-substrates was observed with
MLH1/ extract in the presence of excision
activity and hMutL. A comparable enhancement of gap formation on
5'-heteroduplexes that required both hMutS and purified excision
activity was observed with depleted extract derived from
MSH6/ MT1 cells (Fig. 2, lower
panel). However, 5'-heteroduplex results obtained with depleted
extract derived from MLH1/ H6 cells were
surprising. In this case and in contrast to results obtained with
3'-substrates, mismatch-dependent gap formation required
only depleted extract and the purified excision activity; hMutL was
not necessary. These observations suggest that although in
vitro gap formation on a 5'-heteroduplex depends on a mismatch and
requires hMutS, this reaction can occur in the absence of hMutL.
Additional evidence supporting this view will be presented below.
Isolation of mismatch-provoked gap formation activity
/ H6 cells, 50 ng of hMutL, and samples
of fractions shown. Activities were determined from that portion of the
assay curve where product formation was linear with added protein. Note
that gap-forming activity on 3'- and 5'-heteroduplexes co-purify.
P-Cell, phosphocellulose.
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Fig. 2.
Gap formation activity supported by partially
purified excision activity requires a mismatch. Reactions (see
"Experimental Procedures") contained as indicated 64 µg of
hMutS -deficient excision-depleted extract derived from
MSH6/ MT1 cells or hMutL-deficient
depleted extract from MLH1/ H6 cells, 200 ng
of hMutS, 50 ng of hMutL, 0.6 µg of excision activity (MonoQ
fraction), and 100 ng of homoduplex DNA (open bars), G-T
heteroduplex (black bars), or /CA\
dinucleotide insertion/deletion heteroduplex (gray bars).
Gap formation was scored as NheI-resistant DNA after
digestion with NheI and Bsp106I (Fig. 1). Results
shown in the upper panel were obtained with DNAs containing
a nick in the viral DNA strand at the gpII cleavage site, while
those shown in the lower panel used DNAs containing a single
strand break in the complementary DNA strand at the Sau96I
cleavage site. Activity on the /CA\
dinucleotide insertion/deletion heteroduplex observed in
MSH6/ extracts upon supplementation with
only purified excision activity may be due to MutS (MSH2·MSH3
heterodimer), which is absent in H6 cells due to MSH3
mutation (51)
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Fig. 3.
hEXOI co-purifies with mismatch repair
excision activity. Upper panel, gap formation activity
was assayed across the MonoQ FPLC elution profile (see `Experimental
Procedures` and Table I). Reactions contained 64 µg of
excision-depleted extract from MLH1 / H6
cells, 50 ng of hMutL, 1 µl of column fraction, and 100 ng of 3'-
(closed circles) or 5'-G-T (open circles)
heteroduplex. The inset depicts a Western blot across the
gradient (5 µl, each fraction) probed with anti-hEXOI antibody AB-1
(see "Experimental Procedures") that recognizes both a and b splice
variants. The mobility of the AB-1-positive species corresponds to a
molecular mass of 114 kDa. Lower panel, samples of
extract (340 µg), phosphocellulose (24 µg), DNA cellulose (7 µg),
and MonoQ (3 µg) fractions (Table I) were resolved on a 7.5% SDS
acrylamide gel and transferred to a polyvinylidene difluoride membrane
that was probed with antibody AB-1 that recognizes a and b splice
variants (-hEXOI, see `Experimental Procedures`). In a similar
experiment DNA cellulose (7 µg), and MonoQ (3 µg) fractions were
analyzed by Western blot using antibody AB-3, which recognizes only
hEXOIb (-hEXOIb). The smaller 100-kDa immune-positive polypeptide
evident in phosphocellulose and DNA-cellulose eluates, and to a lesser
extent in the MonoQ fraction, was presumably derived from hEXOIb by
proteolysis during fractionation since it was not present in the
extract.
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Fig. 4.
Recombinant hEXOIa and hEXOIb were isolated
in near homogeneous form. Samples of hEXOIa and hEXOIb (1 µg)
purified from baculovirus-infected insect cells (see "Experimental
Procedures") were separated on 7.5% SDS acrylamide gels. The gel
shown in the upper panel was stained with Coomassie
Brilliant Blue, with molecular masses shown based on markers run in a
parallel lane. The two lower panels depict Western transfers
probed with either antibody AB-1 that recognizes both variants of hEXOI
or hEXOIb-specific antibody AB-3, confirming the specificity of the
latter antiserum for the b splice variant.
and hMutL, while efficient excision on a
5'-substrate was hMutS-dependent but largely independent
of the presence of hMutL. Gap formation activity of depleted
MLH1/ extract was increased 12-fold by
addition of hEXOIb, but the additional presence of hMutL further
enhanced activity by only 20%. Experiments like those summarized in
Table II have shown that the recombinant form of the hEXOIa also
supports mismatch-provoked excision (data not shown), implying that
hEXOIa and hEXOIb splice variants are both active in this reaction.
Recombinant hEXOI is sufficient to restore
mismatch-dependent gap formation to excision-depleted
extract
Immuno-depletion of hEXOI reduces the efficiencies of 5'- and
3'-mismatch repair to similar degrees in HeLa nuclear extract
/
cells was also supplemented with hMutS (lane 8). By
contrast, hEXOIb was sufficient to restore
mismatch-dependent 5'-excision to
MLH1/ deficient extract; hMutL was not
required (compare lanes 2 and 3), confirming
results to this effect obtained by the restriction protection assay
described above.
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Fig. 5.
Excision tracts produced in
EXOIb-complemented extracts map to the shorter path
between the strand break and the mispair. Gap formation reactions
using HeLa nuclear extract, excision-depleted extract derived from
MSH6 / or MLH1/ cells, or
HeLa nuclear extract immunologically depleted for hEXOIb were performed
as described under "Experimental Procedures" and the legends to
Fig. 2 and Table III. DNA substrates are described in Fig. 1. After
cleavage with Bsp106I and denaturation, DNA products were
separated on 1% alkaline agarose gels and transferred to nylon
membranes, which were probed with 5'-end-labeled oligonucleotides
corresponding to sequences to either side of the Bsp106I
site as described previously (21). Left panel,
5'-substrates: excision was monitored in HeLa nuclear extract, or
excision-depleted extracts derived from
MLH1/ H6 cells or
MSH6/ MT1 cells, which were supplemented as
indicated with 50 ng of hMutL, 200 ng of hMutS, and 10 ng of
hEXOIb. Probe oligonucleotides corresponded to viral strand nucleotides
2531-2551 (upper gel, black bar on DNA map) and
2510-2528 (lower gel, gray bar on DNA map). The
full-length complementary strand species (6440 nucleotides) results
from ligation of the Sau96I strand break, while the 3230 (upper gel) and the 3210 residue (lower gel)
species correspond to locations of the 5'- and 3' termini of the
Sau96I nick in the complementary DNA strand. The mobilities
of 5'-excision products evident in lanes 2, 3,
5, and 8 of the upper gel correspond
to lengths of about 2900 nucleotides as judged by size markers run in a
parallel lane. Numerical values under each lane of the upper
gel indicate the yield of these excision products (fmol)
determined by PhosphorImager quantitation. The reduced yield of the
3210 nucleotide species in lanes 1, 4,
6, 10, and to a lesser extent in lane
7, is due to ligation of the Sau96I strand break under
conditions that do not support mismatch-provoked excision. Right
panel, 3'-substrates: excision was scored in HeLa nuclear extract
that was immunologically depleted for hEXOI using antibody AB-2 or
mock-depleted using preimmune IgG (see "Experimental Procedures").
Reactions were supplemented with 10 ng of hEXOIb as indicated. Probe
oligonucleotides correspond to complementary DNA strand nucleotides
2668-2684 (upper gel, black bar on DNA map) or
2510-2528 (lower gel, gray bar on DNA map). The
3285 (upper gel) and 3155 (lower gel) nucleotide
species correspond to locations of 3'- and 5' termini in the original
substrate. Yields (in fmol) of excision products, which have mobilities
similar to that of the 2900-nucleotide marker, are indicated below each
lane of the upper gel.
Activates the 5'- to 3'-Hydrolytic Activity of hEXOI in a
Mismatch-dependent Fashion--
As described above,
mismatch-provoked gap formation on 3'-heteroduplexes in depleted
extract requires hMutS, hMutL, and either purified excision
activity or recombinant hEXOI. The gap produced under these conditions
is localized to the shorter path spanning the mismatch and the strand
break that directs repair. Upon cleavage with Bsp106I and
NheI, this gapped product migrates just slightly faster than
full-length linear DNA due to its NheI resistance and the
presence of a small gap (Fig. 6,
upper panel). Because hEXOI has been reported to interact
with hMSH2 and hMLH1 (36), we have examined the effects of hMutS and
hMutL on the hydrolytic activity of hEXOIb in a purified system.
Surprisingly, incubation of heteroduplex with just hMutS, hEXOI, and
ATP (in the presence or absence of hMutL) led to the
mismatch-dependent production of a DNA product of unusual
mobility (Fig. 6, lower panel). Production of this species
(arrow), occurred at the expense of the larger of the two
restriction fragments (asterisk) that results from cleavage
of the heteroduplex with Bsp106I and NheI. This
observation suggested that this DNA species was produced by
mismatch-dependent, 5'- to 3'-hydrolysis from the strand
break. Analysis of sensitivity to other restriction endonucleases was consistent with this view, and more direct evidence for this conclusion is presented below.
View larger version (61K):
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Fig. 6.
hMutS activates
hEXOIb in a mismatch-dependent manner. 3'-G-T
heteroduplex and A·T homoduplex DNAs contained a viral strand nick.
Upper panel, mismatch-provoked gap formation reactions (see
"Experimental Procedures") contained excision-depleted extract from
MLH1/ H6 cells, 10 ng of hEXOIb, and 50 ng
of hMutL and G-T heteroduplex/A·T homoduplex as indicated. After
reaction termination and digestion with Bsp106I and
NheI, DNA products were resolved by agarose gel
electrophoresis. Lower panel, reactions were performed as in
the upper panel but other than reaction mixture and DNA,
contained only 10 ng of hEXOIb, 50 ng of hMutL, and 200 ng of
hMutS as indicated. The asterisk indicates the
heteroduplex-derived restriction fragment that was recovered in reduced
yield when hMutS and hEXOI were both present (2nd and 4th in gel on
the right). The presumed nature of the DNA products
indicated by arrows is illustrated in the diagram on the
right. For the experiment in the lower panel,
values below each lane indicate the yield (in fmol) of the product
indicated by the arrow.
. Quantitation of this data indicates that presence of a mispair and hMutS enhances the activity of hEXOIb 5-6-fold on these
3'-substrates. This analysis also suggests that hMutL enhances hEXOIb activity on these DNAs, but this effect, which is independent of
hMutS and a mispair, is a modest 2-fold. As noted above, the hMLH1
subunit of hMutL has been shown to interact physically with hEXOI
(36).
, and
hMutL was examined using indirect end-labeling (21) to map the
location of 5' termini produced in the reaction (Fig. 7). A basal population of 3'-end-labeled
hydrolytic products was observed in all cases where hEXOIb was present.
However, the additional presence of hMutS, or both hMutS and
hMutL, led to enhanced production of a higher mobility, relatively
discrete population of fragments with 5' termini that mapped to the
vicinity of the Bsp106I cleavage site (arrows in
Fig. 7). This restriction site is located 3200-3300 bp from the strand
break in the two types of heteroduplex, indicating that the extent of
excision under these conditions is substantial. Although hMutL did
not visibly alter the yield of degradation products observed in the
presence of hMutS and hEXOIb, DNA product size was somewhat larger
when the former protein was also present (Fig. 7, compare lanes
4 and 5). This suggests that hMutL may to some
degree modulate hMutS-mediated hEXOI activation.
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Fig. 7.
hMutS -activated hEXOIb degrades with 5'
to 3' polarity without regard to heteroduplex orientation. Gap
formation reactions containing 3'- (upper panel) or 5'-
(lower panel) heteroduplex/homoduplex DNA were performed as
described under "Experimental Procedures" except that
excision-depleted extract was omitted and hMutS (200 ng), hMutL
(50 ng), and hEXOIb (20 ng for 3'-substrates and 5 ng for
5'-substrates) were present as indicated. Reactions were terminated and
DNA products were denatured (restriction digestion with NheI
and Bsp106I was omitted) and subjected to electrophoresis
through 1% agarose in the presence of 50 mM NaOH. DNA
was transferred to nylon membranes and indirectly end-labeled (21)
using 32P-end-labeled oligonucleotides corresponding to
complementary strand residues 5778-5760 (3'-substrates) or viral
strand residues 5761-5777 (5'-substrates). The former probe
(gray bar, upper map) hybridizes to the 3'
terminus of incised viral strand in 3'-substrates, while the latter
(gray bar, lower map) hybridizes to the 3'-end of
the incised complementary strand of 5'-DNAs. Recovery of 3'-terminal,
hybridization probe-positive material was estimated by quantitation of
total radiolabel in each lane. When normalized to total label present
in lane 7 (DNA only), recovery of 3' termini for lanes
2-6 was 103 ± 9% (upper gel, ± one standard
deviation) and 100 ± 6% (lower gel), implying
little if any hydrolytic removal of the 3' terminus at the strand break
in either heteroduplex. Arrows indicate the position of
probe-labeled marker products (not shown) derived from unreacted 3'-
and 5'-DNAs upon digestion with Bsp106I. These markers
correspond to 3285 and 3210 nucleotides for 3'- (upper
panel) and 5'- (lower panel) heteroduplexes,
respectively. The asterisk in the lower panel
indicates a species that was produced at elevated levels in the
presence of hEXOI and hMutL (lane 3).
on
5'-substrates is about 3-fold greater than that observed with 3'-DNAs (Fig. 7 and data not shown), and a similar phenomenon is evident with
5'- and 3'-homoduplex DNAs in extracts (Tables II and III). The basis
of this effect is not known, but it could be the consequence of
sequence context differences at the strand breaks in the two types of
substrate. The modest enhancement of hEXOI activity by hMutL alluded
to above is also evident in Fig. 7, particularly in the case of
5'-heteroduplex: hMutL in the absence of hMutS led to enhanced
production of the partial degradation product indicated by an
asterisk (compare lanes 2 and 3 of the
bottom panel).
-, hMutL-,
hEXOI-dependent excision on a 3'-heteroduplex in crude
extract occurs with a polarity that is formally 3' to 5' from the
strand break, mismatch-, hMutS-, and hEXOI-dependent
excision on this substrate in a pure system occurs from the nick with
5' to 3' polarity, effects that are depicted schematically in Fig. 6.
Furthermore, while hEXOI-dependent excision in extracts is
restricted to several hundred base pairs spanning the shorter path
between the strand break and the mismatch (Fig. 5), excision by the
mismatch- and hMutS-activated form of hEXOI rapidly removes several
thousand nucleotides (Fig. 7). These observations imply that the
polarity and the extent of hEXOI-dependent excision during
mismatch repair are regulated by as yet unidentified extract factors.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
activates the 5' to 3' hydrolytic activity of hEXOI in a
mismatch-dependent manner, an effect that is most simply
understood in terms of a physical interaction of the two proteins on a
heteroduplex. We have also observed a limited enhancement of hEXOI
activity by hMutL, but this effect does not require a mismatch.
While these simple systems are of interest in terms of potential modes
of mismatch-dependent activation of downstream repair
activities, it is also clear that hEXOI participation in the repair
reaction must be regulated by other factors that remain to be
identified. Despite its reported 5' to 3' polarity, hEXOI is involved
in the 3'- to 5'-excision reaction that occurs on 3'-heteroduplexes in nuclear extract. While hMutS is sufficient to activate hEXOI hydrolysis on a 3'-heteroduplex in a pure system, excision under these
conditions occurs with 5' to 3' polarity. Repair components other than
hMutS or hMutL must therefore regulate the activity of hEXOI when
the strand break that directs correction lies 3' to the mispair. The
hEXOI-dependent mismatch-provoked excision tracts produced
in crude fractions on the substrates used here are several hundred
nucleotides in length, spanning the shorter path between the strand
break and the mismatch and terminating as a fairly discrete species
about 150 nucleotides beyond the mispair (Fig. 5 and Ref. 21). By
contrast, mismatch-dependent hydrolysis observed in the
purified hMutS-hEXOI system (in the presence or absence of hMutL)
extend several thousand nucleotides from the strand break. Thus, in
addition to orientation-dependent regulatory effects,
unidentified repair components also control the extent of
hEXOI-dependent excision on 5'- and 3'-heteroduplexes. The
identity of these factors is under study.
(31), all of which have been implicated in eukaryotic mismatch repair (1-4).
In addition to its hydrolytic function, these effects have been
interpreted in terms of a structural role for the yEXOI polypeptide in
stabilization of a multiprotein mismatch repair assembly (31).
Overexpression of yEXOI has been shown to suppress the conditional
phenotypes associated with msh2-L560S mutation, whereas
overexpression of mutant forms of the protein containing amino acid
substitutions within the conserved nuclease domain do not. Based on
these observations, Sokolsky and Alani (30) have concluded that yEXOI
has a catalytic role in yMSH2-dependent mismatch repair.
Synergistic mutability effects have also been observed between S. cerevisiae exoI mutants and yMutL ATPase mutants with amino
acid substitution in mlh1 and pms1 ATPase motifs, findings that have also been interpreted in terms of a hydrolytic role
for yEXOI (32). Our results are consistent with hydrolytic and
structural functions for hEXOI in mismatch repair. The finding that
hEXOI is activated by hMutS in a mismatch-dependent
manner strongly suggests that the protein functions as an exonuclease in the repair reaction. Although we cannot rule out the possibility that the protein possesses a cryptic 3' to 5' hydrolytic activity, the
hEXOI requirement for 3'-heteroduplex repair could be indicative of a
structural or regulatory function of the hEXOI polypeptide in
correction of this type of substrate.
/ and MLH1/
cells has shown that while mismatch-provoked excision on
3'-heteroduplexes requires both hMutS and hMutL, excision on
5'-heteroduplexes depends on hMutS but can occur in the absence of
hMutL (Fig. 2). Since the biochemical depletion procedure used to
prepare these extracts results in removal of a large set of proteins, it is possible that this observation is an artifact of depleted extract
preparation. However, normal excision tracts are produced in such
extracts upon supplementation with hEXOI in the absence of hMutL
(Fig. 5, left panel).
-independent 5'-heteroduplex
repair in some hMLH1-deficient cells. In vitro analysis of hMutL-deficient human cell lines has demonstrated that they define two classes with respect to the biochemical nature of the repair defect. One class of hMutL-deficient cell lines includes the mismatch repair-deficient, drug-resistant derivatives of A2780 ovarian
tumor cells and the sporadic colorectal and endometrial tumor cell
lines RKO, Vaco6, and AN3CA. Each of these cell lines is
devoid of hMLH1 polypeptide and hMutL due to epigenetic silencing of
MLH1 loci by promoter methylation (45-47). Extracts
prepared from these cell lines are defective in correction of
3'-heteroduplexes. However, they display near normal levels of
5'-heteroduplex repair, and gap formation on 5'-DNAs in such extracts
is mismatch-dependent (48).2 hMutL
supplementation restores 3'-heteroduplex repair to wild type levels in
extracts, but the high level of residual 5' repair activity is enhanced
only to a modest degree.
-deficient cell line is typified by the
colorectal tumor cell lines HCT116 (and its subclone H6) and Vaco481.
H6 cells harbor nonsense mutations in the two MLH1 alleles
(49), while the PMS2 alleles in Vaco481 are inactivated by
frameshift and nonsense mutation (50). In vitro assay of extracts from these two cell lines has shown them to be defective in
repair of both 3'- and 5'-heteroduplexes, although low residual levels
of 5'-heteroduplex repair has been observed in H6 extracts with certain
mispairs (18, 19, 50). 3'- and 5'-heteroduplex correction is restored
to normal levels with both cell lines upon addition of purified
hMutL.
-deficient cell lines has not been established, the
selective repair defect in epigenetically silenced cells demonstrates that hMutL-independent 5'-heteroduplex repair can occur in
unfractionated nuclear extracts. It is pertinent to note in this
context that the failure of MLH1/ H6
extracts to support 5'-heteroduplex repair is not due to the presence
of a simple diffusible inhibitor
(18).3 Nevertheless, we have
observed apparently normal, mismatch-dependent gap
formation on 5'-heteroduplexes in excision-depleted extracts derived
from H6 extracts upon supplementation with hEXOI. This may indicate
that the failure to observe 5'-heteroduplex repair in unfractionated H6
extracts is due to a more complex form of inhibition, for example, the
stoichiometric and essentially irreversible sequestration of a required
repair activity that is disrupted upon fractionation.
| |
ACKNOWLEDGEMENTS |
|---|
We thank R. Kolodner for the generous gift of EST 843301.
| |
FOOTNOTES |
|---|
* This work was supported by Grant GM45190 from NIGMS, 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: Diosynth Research Triangle Park, Inc., 3000 Weston Parkway, Cary, NC 27513.
Investigator of the Howard Hughes Medical Institute. To whom
correspondence should be addressed: Dept. of Biochemistry and Howard
Hughes Medical Inst., Box 3711, Duke University Medical Center, Durham,
NC 27710. Tel.: 919-684-2775; Fax: 919-681-7874; E-mail:
modrich@biochem.duke.edu.
Published, JBC Papers in Press, January 24, 2002, DOI 10.1074/jbc.M111854200
2 G.-M. Li, J. Drummond, L. Bazemore, S. Littman, and P. Modrich, unpublished observations.
3 S. Littman and P. Modrich, unpublished observations.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: PCNA, proliferating cell nuclear antigen; DTT, dithiothreitol.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. |
Kolodner, R.
(1996)
Genes Dev.
10,
1433-1442 |
| 2. |
