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INTRODUCTION |
The methyl-directed mismatch repair pathway in Escherichia
coli functions to correct DNA biosynthetic errors that arise
during chromosomal replication and to discourage recombination between substantially diverged DNA sequences (1). Inactivation of the mismatch
repair system results in elevated spontaneous mutation rates (2). The
pathway has been reconstituted in vitro and involves the
action of eight proteins (3).
Initiation of mismatch repair requires MutS, MutL, and MutH in addition
to a DNA mismatch, ATP, and Mg2+, and results in the
generation of a nick in the unmethylated (nascent) strand of a nearby
hemimethylated d(GATC) sequence (4). The transient hemimethylated state
of d(GATC) sequences after replication serves as a signal to direct
repair to the nascent DNA strand (5, 6). MutS recognizes and binds the
mismatched base (7, 8). MutL binds the MutS-mismatch complex (9), and
MutH is stimulated to catalyze the endonucleolytic cleavage at the
d(GATC) site in the presence of MutL and MutS (4). After the initiation
stage of mismatch repair, DNA unwinding is initiated at the nick by DNA
helicase II (UvrD) and proceeds to a point beyond the error (10, 11).
Excision of the error-containing DNA strand is facilitated by the
action of one of several exonucleases (depending on the polarity of the
reaction) which serve to degrade the single-stranded DNA
(ssDNA)1 as it is unwound by
UvrD (11, 12). In the presence of ssDNA-binding protein, DNA polymerase
III holoenzyme catalyzes repair synthesis on the resulting gapped DNA
molecule to restore the correct sequence, and DNA ligase seals the
final nick (3).
The E. coli MutH protein possesses a weak endonuclease
activity that is specific for unmethylated d(GATC) sequences (13). In
the presence of ATP, MutS, MutL, and a hemimethylated DNA substrate containing a mismatched base pair, the MutH-associated endonuclease activity is greatly stimulated (4). However, the mechanism by which the
MutH endonuclease activity is activated by the MutS-MutL complex is not known.
Recently, we identified a physical interaction between the MutL and
UvrD proteins using a yeast two-hybrid screen with UvrD as bait (14).
Simultaneously, a biochemical interaction was reported between MutL and
UvrD (15). To identify other potential interactions involving E. coli mismatch repair proteins, all possible pairwise combinations
of MutS, MutL, MutH, and UvrD were tested for interactions using the
yeast two-hybrid system. An interaction was identified between MutL and
MutH which was subsequently confirmed by affinity chromatography. The
weak endonuclease activity of MutH on unmethylated d(GATC) sequences
was greatly stimulated by MutL. Surprisingly, this stimulation of the
activity of MutH occurred in the absence of MutS and a mismatched base
pair, suggesting that MutL is the component of the MutS-MutL complex
responsible for activating MutH during mismatch repair in
vivo and that the activation occurs via a direct physical
interaction. In addition, the stimulation of MutH by MutL was dependent
on ATP but not ATP hydrolysis. These results suggest an additional role
for the MutL protein in coordinating activities during mismatch repair
in E. coli.
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EXPERIMENTAL PROCEDURES |
Materials
pGAD424 and pGBT9, and yeast HF7c and SFY526 were from the
Matchmaker two-hybrid system (CLONTECH). pCYB1,
pCYB2, and all components of the Impact I protein purification system
were from New England Biolabs. E. coli
GE1752
uvrD (16) and GE1752mutS::Tn5 (17) were constructed previously in this laboratory. HMS174 (recA1 hsdR (rK12-mK12+)
RifR) was from Novagen.
BL21(DE3)mutS::Tn5 was constructed by P1
transduction (18) using GE1752mutS::Tn5 as the
donor strain and BL21(DE3) as the recipient. Several Kanr
transductants were selected, colony purified, and screened for a
mutator phenotype. To ensure that the mutator phenotype was caused by
the mutant mutS allele, complementation experiments were
performed using a high copy number plasmid that expressed MutS.
To prepare M13mp18 ssDNA, phage infection of E. coli XL-1
Blue (Stratagene) and collection of phage particles were performed as
described (19). Phage particles were purified on a CsCl gradient (0.438 g of CsCl/ml; 83,000 × g for 24 h at 25 °C).
After isolation from the gradient and dialysis against 10 mM Tris-HCl (pH 8.0) to remove CsCl, the phage particles
were treated with 200 µg/ml proteinase K and 0.1% SDS for 1 h
at 50 °C. M13mp18 ssDNA was purified from phage particles by
sequential extractions with buffered phenol, 25:24:1
phenol:chloroform:isoamyl alcohol, and 24:1 chloroform:isoamyl alcohol
followed by ethanol precipitation. M13mp18 RF DNA was prepared from
phage-infected XL-1 Blue cells as described (20).
T7 DNA polymerase was purified previously according to a published
procedure (21). All enzymes used for cloning and PCR were from New
England Biolabs with the exception of T4 DNA ligase, which was from
Boehringer Mannheim. Nucleotides were from Amersham Pharmacia Biotech.
Methods
Cloning Mismatch Repair Genes--
Construction of pGAD424-UvrD,
pGAD424-MutL, pGBT9-UvrD, and pGBT9-MutL was described previously (14).
The coding regions of mutS and mutH were
amplified by PCR from E. coli K-12 genomic DNA using Vent
DNA polymerase. Oligonucleotide primers for amplifying the
mutS gene contained restriction enzyme sites that allowed cloning of the mutS coding sequence into the
EcoRI and BamHI sites of pGAD424 and pGBT9,
creating a translational fusion with the Gal4 transcriptional
activation domain and DNA binding domain, respectively, for use in the
yeast two-hybrid system. In addition, these primers contained
restriction enzyme sites that allowed cloning of mutS into
the NdeI and SmaI sites of pCYB2 for
overexpression and purification of MutS using the Impact I protein
purification system. Likewise, primers for amplifying the
mutH coding sequence contained restriction enzyme sites that
allowed cloning of mutH into the EcoRI and
BamHI sites of pGAD424 and pGBT9 and into the NdeI and SapI sites of pCYB1. pET3c-MutL was
constructed by subcloning the NdeI-BamHI fragment
containing the mutL coding sequence from pGAD424 into the
NdeI and BamHI sites of pET3c (Novagen).
Deletion Constructions--
Deletions from each end of the
mutL gene in pGAD424 were constructed previously (14). To
construct mutH
38N and mutH
10C, the
appropriate portion of the mutH gene was amplified by PCR using Vent DNA polymerase. The oligonucleotide primers used in these
reactions contained restriction enzyme sites that allowed cloning of
each PCR product into the EcoRI and BamHI sites
of pGBT9, creating a translational fusion with the Gal4 DNA binding domain.
Yeast Two-hybrid Assays--
Potential interactions between
mismatch repair genes were tested in yeast HF7c by cotransformation of
all possible combinations of pGAD424 and pGBT9 harboring the
uvrD, mutL, mutS, and mutH genes. After selection of cotransformants on complete synthetic media
lacking tryptophan and leucine, cells were transferred to complete
synthetic media lacking tryptophan, leucine, and histidine and
supplemented with 1 mM 3-amino-1,2,4-triazole. HF7c
contains a HIS3 reporter gene that requires a two-hybrid
interaction for expression. Yeast SFY526 containing a lacZ
reporter gene was used to confirm any interactions by monitoring
-galactosidase activity in the presence of the color-producing
substrate
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside.
-Galactosidase activity was quantified (where indicated) as
described by the supplier using the substrate o-nitrophenyl
-D-galactopyranoside, and results were expressed as
Miller units (18).
Protein Purifications--
Purification of MutL from
GE1752
uvrD was described previously (14). To overexpress
MutL in an E. coli strain lacking a functional
mutS gene product, a 10.5-liter culture of
BL21(DE3)mutS::Tn5 containing pET3c-MutL was grown
at 30 °C in 2 × YT medium. Protein expression was induced with
0.5 mM isopropyl
-D-thiogalactopyranoside at
an A600 nm of 2.0, and incubation at 30 °C
was continued for 5 h. Cells (118 g) were harvested by
centrifugation and washed with M9 minimal medium salts. Cell lysis and
protein purification were performed essentially as described previously
(9) with a few exceptions. First, 10% glycerol was included in all
column buffers. Second, a Bio-Rex 70 column was used as the initial
chromatographic step. Subsequently, the first hydroxylapatite
chromatographic step was used, and the second hydroxylapatite column
was eliminated. Third, a Superose 12 HR 10/30 high performance liquid
chromatography sizing column (Amersham Pharmacia Biotech) was used for
the final purification step instead of a Sephadex G-150 column. Storage buffer for MutL was 25 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM 2-mercaptoethanol, 0.1 mM EDTA, and 50% glycerol.
To overexpress MutH, three 1-liter cultures of HMS174 containing
pCYB1-MutH were grown at 37 °C to an A600 nm
of 1.2 in 2 ×YT medium. Protein expression was induced by the addition
of isopropyl
-D-thiogalactopyranoside to 0.5 mM, and growth was continued at 30 °C for 5 h.
Cells (12 g) were collected by centrifugation and resuspended in column
buffer (20 mM Tris-HCl (pH 8.0), 750 mM NaCl,
0.1 mM EDTA, 0.1% Triton X-100, 10% glycerol, and 0.1 mM phenylmethylsulfonyl fluoride). Cells were lysed by
sonication, and protein purification was performed using a 20-ml chitin
column (4.1 cm × 4.9 cm2) equilibrated in column
buffer essentially as recommended for the Impact I system.
To overexpress MutH in an E. coli strain lacking a
functional mutS gene product, a 10.5-liter culture of
GE1752mutS::Tn5 containing pCYB1-MutH was grown at
30 °C to an optical density of 3.0 in 2 × YT medium. Protein
expression was induced by the addition of isopropyl
-D-thiogalactopyranoside to 0.5 mM, and
growth was continued for 5 h at 30 °C. Cells were harvested by
centrifugation, washed once with M9 minimal medium salts, and
resuspended in column buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 0.2% Triton X-100, 0.5 mM EDTA, 10%
glycerol, and 0.1 mM phenylmethylsulfonyl fluoride). Cells
(70 g) were lysed by sonication and purified on a 20-ml chitin column
(4.1 cm × 4.9 cm2) according to the Impact I
purification protocol. The chitin column was equilibrated and washed
with column buffer, and intein-induced self-cleavage was initiated with
cleavage buffer (20 mM Tris-HCl (pH 8.0), 200 mM NaCl, 0.1 mM EDTA, 10% glycerol, and 100 mM 2-mercaptoethanol). The cleavage reaction was allowed to
proceed for 72 h before elution of MutH from the chitin column.
Pooled MutH (18 mg in 27.5 ml) was dialyzed extensively against 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, 0.1 mM EDTA, and 10% glycerol to remove the 2-mercaptoethanol. MutH was precipitated with 60% ammonium sulfate and resuspended in 4 ml of 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, 0.1 mM EDTA, and 10% glycerol and dialyzed against two 500-ml
volumes of this buffer to remove ammonium sulfate. MutH was loaded in
1-ml aliquots onto a Superose 12 HR 10/30 sizing column at a flow rate
of 0.2 ml/min to separate MutH from a prominent contaminating protein of approximately 75 kDa. Both preparations of MutH were stored in 25 mM Tris-HCl (pH 8.0), 200 mM NaCl, 0.1 mM EDTA, and 50% glycerol.
To overexpress MutS, four 750-ml cultures of HMS174 containing
pCYB2-MutS were grown at 37 °C to an A600 nm
of 0.7 in 2 × YT medium. Protein expression was induced by the
addition of isopropyl
-D-thiogalactopyranoside to 0.5 mM, and growth was continued for an additional 5 h at
30 °C. Cells were harvested by centrifugation and resuspended in
column buffer (20 mM Tris-HCl (pH 8.0), 750 mM
NaCl, 0.1 mM EDTA, 0.1% Triton X-100, 10% glycerol, and
0.1 mM phenylmethylsulfonyl fluoride). Cells (9 g) were
lysed by sonication and purified using a 20-ml chitin column (4.1 cm × 4.9 cm2) essentially as recommended for the
Impact I system. Purified MutS was stored in 25 mM Tris-HCl
(pH 8.0), 200 mM NaCl, 0.1 mM EDTA, and 50%
glycerol. All protein concentrations were determined using the Bio-Rad
protein assay. Because of the nature of the intein cleavage reaction in
the Impact I system, purified MutS contained two extra amino acids on
the COOH terminus (proline and glycine). The amino acid sequence of
MutH purified using the Impact I system was identical to native MutH.
Preparation of DNA Substrates--
The oligonucleotides
5'-GGTACCGAGTTCGAATTCG-3' and
5'-GGTACCGAGCTCGAATTCG-3' were used to generate
covalently closed duplex M13mp18 DNA containing a single G-T mismatch
(heteroduplex) and no mismatch (homoduplex), respectively. The G-T
mismatch in the heteroduplex substrate disrupted a SacI site
in the polylinker of M13mp18. The presence of the mismatch was
confirmed by digestion of the heteroduplex substrate with
SacI.
Both oligonucleotides anneal to identical positions in the M13mp18
polylinker, and all manipulations used to generate the heteroduplex and
homoduplex substrates were identical. Before annealing,
oligonucleotides were phosphorylated using T4 polynucleotide kinase.
Annealing mixtures (65 µl) contained 100 mM Tris-HCl (pH 8.0), 20 mM MgCl2, 140 pmol of oligonucleotide,
and 4.6 pmol of M13mp18 ssDNA molecules. These mixtures were heated to
94 °C for 3 min and cooled 1 °C/min to 30 °C in a Perkin-Elmer
2400 thermal cycler. Components of the extension reaction were added to
the annealing mixtures such that a final volume of 130 µl was
achieved containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 8 µg/ml bovine serum albumin (BSA),
500 µM each dNTP, and 8.3 mM dithiothreitol. Extension reactions were incubated at 30 °C for 30 min with enough T7 DNA polymerase to achieve complete conversion of M13mp18 ssDNA to
duplex molecules. To achieve covalently closed molecules, 750 µM ATP and 1 unit of T4 DNA ligase (Boehringer Mannheim)
were added to each extension reaction. Extension reactions were pooled and covalently closed, and nicked circular DNA was separated on a
CsCl/EtBr gradient as described (19).
Endonuclease Assays--
MutH-catalyzed endonuclease reactions
(16 µl) contained 20 mM Tris-HCl (pH 7.6), 4 mM MgCl2, 20 mM NaCl, 50 µg/ml
BSA, and 50 ng of the appropriate DNA substrate. When present, ATP,
ATP
S, and AMP-PNP were 1.25 mM. When present, MutL and
MutS were added immediately before initiation of the reactions with the
indicated concentration of MutH. All protein dilutions were made in 10 mM Tris-HCl (pH 8.0). All reactions were incubated at
37 °C for 15 min and quenched with 4 µl of 5 × dye solution
(25% glycerol, 100 mM EDTA, and 0.025% bromphenol blue).
Reaction products were subjected to electrophoresis on 0.8% agarose
gels in the presence of 0.5 µg/ml EtBr to separate covalently closed
and nicked circular DNA species. Agarose gels were subsequently
irradiated with a hand-held UV (254 nm) lamp for 30 min, restained for
30 min with 0.5 µg/ml EtBr, and destained with deionized and
distilled water. Gels were illuminated with UV light and photographed
using an Eagle Eye II still video imaging system (Stratagene).
Affinity Chromatography--
4.5 mg of purified MutH was
covalently coupled to approximately 750 µl of Affi-Gel 10 resin as
described by the supplier (Bio-Rad) in 25 mM MES (pH 6.4),
200 mM NaCl, and 20% glycerol for 12 h at 4 °C.
The coupling reaction was quenched with 25 mM ethanolamine (pH 8.0) for 1 h, and the resin was transferred to a
chromatography column (inner diameter = 0.75 cm). The coupling
efficiency was greater than 50% based on quantitation of protein in
the initial column flow-through using the Bio-Rad protein assay. The
column was equilibrated with affinity buffer (25 mM
Tris-HCl (pH 7.5), 10% glycerol, 2.5 mM 2-mercaptoethanol,
and 3 mM MgCl2) containing 50 mM
NaCl. Approximately 100 µg of the indicated protein, diluted to a
1-ml volume in affinity buffer plus 50 mM NaCl, was applied to the MutH affinity column at a flow rate of 10 ml/h. The column was
washed four times with 500 µl of affinity buffer plus 50 mM NaCl, collecting each wash as an individual fraction.
The column was eluted with four 500-µl volumes of affinity buffer
plus 1 M NaCl, collecting each as an individual fraction.
Fractions were analyzed for protein content by electrophoresis on a
10% polyacrylamide gel in the presence of SDS followed by staining
with Coomassie Brilliant Blue. A control column containing chicken egg
white lysozyme covalently coupled to Affi-Gel 10 resin was constructed previously (14). Experiments using this column were performed exactly
as described for the MutH affinity column.
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RESULTS |
MutL and MutH Interact in the Yeast Two-hybrid
System--
Previously, we identified a physical interaction between
the methyl-directed mismatch repair proteins MutL and UvrD using a
yeast two-hybrid screen of an E. coli genomic library with
UvrD as bait (14). To identify other potential interactions between components of the mismatch repair system, the mutS and
mutH genes were amplified by PCR from E. coli
K-12 genomic DNA and cloned into the two-hybrid system vectors pGAD424
and pGBT9 as described under "Experimental Procedures." All
possible pairings of uvrD, mutL, mutS,
and mutH were tested for interactions in yeast HF7c cells
containing a HIS3 two-hybrid reporter gene. Although the previously described dimerization of MutL (9), oligomerization of MutS
(7), and interaction between MutL and MutS (9) were not detected using
the yeast two-hybrid system (data not shown), a potential interaction
between MutL and MutH was observed (Fig. 1).

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Fig. 1.
MutL and MutH interact in the yeast
two-hybrid system. Yeast HF7c cells containing pGBT9 and pGAD424
with or without the mutH and mutL genes were
grown at 30 °C on complete synthetic media lacking tryptophan,
leucine, and histidine and supplemented with 1 mM
3-amino-1,2,4-triazole. Cells in each quadrant were streaked from a
single transformant that was colony-purified. Labels represent the
fusion proteins present in the HF7c cells in the order: DNA binding
domain fusion/transcriptional activation domain fusion. A minus
sign indicates the absence of MutH or MutL from the fusion
protein.
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Interestingly, the interaction between MutL and MutH was only observed
when MutL was fused to the Gal4 transcriptional activation domain and
MutH was fused to the Gal4 DNA binding domain. To confirm that the
pGAD424-MutH construct expressing MutH as a fusion with the Gal4
transcriptional activation domain did not contain a mutation resulting
in the loss of an interaction with the MutL-Gal4 DNA binding domain
fusion, mutH was subcloned from pGBT9-MutH into pGAD424.
Again, an interaction was not observed with the MutL-Gal4 DNA binding
domain fusion. These results were confirmed using yeast SFY526,
containing a lacZ reporter gene under the control of a
promoter other than the HIS3 reporter gene in HF7c. The
reason for the observed "polarity" in the two-hybrid interaction
between MutL and MutH is not known.
Purified MutL Is Specifically Retained on a MutH Affinity
Column--
Purified MutH protein (Fig.
2) was covalently coupled to an activated
agarose resin (Affi-Gel 10) as described under "Experimental Procedures." To confirm a physical interaction between MutL and MutH
in vitro, 100 µg of purified MutL was applied to the MutH affinity column. The column was washed with buffer containing 50 mM NaCl, and bound protein was eluted with buffer
containing 1 M NaCl. A large fraction of the applied MutL
was retained on the MutH column after the 50 mM NaCl wash
steps and was eluted with 1 M NaCl (Fig.
3A). In contrast, when an
identical experiment was performed using an Affi-Gel 10 column
covalently coupled to chicken egg white lysozyme, the applied MutL was
found exclusively in the flow-through and 50 mM NaCl wash
fractions (Fig. 3B). Therefore, MutL was specifically
retained on the MutH affinity column because of a physical interaction
with MutH.

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Fig. 2.
Purified MutL and MutH proteins.
Proteins were subjected to electrophoresis through a 12%
polyacrylamide gel in the presence of SDS and visualized with Coomassie
Brilliant Blue. Lane 1, 1 µg of MutL purified using the
Impact I system. Lane 2, 1 µg of MutL purified from
GE1752mutS::Tn5 as described under "Experimental
Procedures." Lane 3, 1 µg of MutH purified using the
Impact I system. Molecular mass markers were: rabbit muscle
phosphorylase b, 97.4 kDa; BSA, 66.2 kDa; hen egg white
ovalbumin, 45.0 kDa; bovine carbonic anhydrase, 31.0 kDa; soybean
trypsin inhibitor, 21.5 kDa; and lysozyme, 14.4 kDa.
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Fig. 3.
MutL is specifically retained on a MutH
affinity column. Approximately 100 µg of MutL (panel
A), BSA (panel C), or MutS (panel D) was
applied to a 750-µl Affi-Gel 10 column to which purified MutH had
been covalently coupled as described under "Experimental
Procedures." Likewise, 100 µg of MutL was applied to an Affi-Gel 10 column containing covalently coupled chicken egg white lysozyme
(panel B). In all panels: lane 1,
flow-through (FT); lanes 2-5, 50 mM
NaCl wash fractions; lanes 6-9, 1 M NaCl
elution fractions. Each lane contains 36 µl of the
corresponding fraction. All fractions were 500 µl with the exception
of the flow-through, which was 1 ml. Molecular mass markers were:
rabbit muscle phosphorylase b, 97.4 kDa; BSA, 66.2 kDa.
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To ensure further that the interaction between MutL and MutH observed
using affinity chromatography was specific, 100 µg each of BSA and
MutS were applied to the MutH affinity column. Using the same
experimental protocol used for MutL, neither BSA nor MutS was retained
to a significant extent on the column (Fig. 3, C and
D). These results support the yeast two-hybrid results and
suggest that a physical interaction exists between MutL and MutH.
The COOH Terminus of MutL Contains the MutH Interaction
Domain--
To identify the regions of MutL and MutH responsible for
the two-hybrid interaction, a series of truncations was made from the
NH2 and COOH termini of both proteins. Truncated
mutL alleles were generated in pGAD424 and tested for an
interaction in the presence of pGBT9-MutH in yeast SFY526 (Fig.
4). Likewise, truncated mutH
alleles were generated in pGBT9 and tested for an interaction in the
presence of pGAD424-MutL. SFY526 contains a lacZ reporter gene encoding
-galactosidase. The relative strengths of interactions were measured using a spectrophotometric assay that monitors the cleavage of o-nitrophenyl
-D-galactopyranoside by
-galactosidase. Results are
reported as Miller units (18).

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Fig. 4.
The COOH terminus of MutL is sufficient for
the two-hybrid interaction with MutH. Yeast SFY526 cells
containing pGBT9 and pGAD424 with various alleles of the
mutH and mutL genes were grown at 30 °C on
complete synthetic media lacking tryptophan and leucine.
-Galactosidase activity was measured as described under
"Experimental Procedures." Panel A, truncations of the
mutL gene were constructed in pGAD424 and were tested for an
interaction in the presence of pGBT9-MutH. Panel B,
truncations of the mutH gene were constructed in pGBT9 and
tested for an interaction in the presence of pGAD424-MutL. A plus
sign indicates the presence of an interaction, and a minus
sign indicates the absence of one. Results represent the average
of at least three trials using independent transformants when an
interaction was detected and at least two trials using independent
transformants when an interaction was not detected. ND, not
detectable.
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Removal of 293, 344, or 397 amino acids from the NH2
terminus of MutL (MutL
293N, MutL
344N, and MutL
397N) did
not eliminate the two-hybrid interaction with MutH. In contrast,
removal of 438 amino acids from the NH2 terminus
(MutL
438N) or 59 amino acids from the COOH terminus (MutL
59C) of
MutL completely eliminated the two-hybrid interaction with MutH. These
results indicate that the COOH-terminal 218 amino acids of MutL are
necessary and sufficient to maintain this interaction and therefore
contain the MutH interaction interface.
Removal of 38 amino acids from the NH2 terminus
(MutH
38N) or 10 amino acids from the COOH terminus (MutH
10C) of
MutH eliminated the two-hybrid interaction with MutL. Thus, we were
unable to define the interaction interface of MutH in more detail. It
is possible that both ends of MutH contribute to the interaction domain. Alternatively, one or both of these truncation mutants may not
be expressed or maintained as stable proteins in the yeast cells. These
results are strikingly similar to those observed for the MutL-UvrD
interaction (14). The COOH-terminal 218 amino acids of MutL were also
sufficient to maintain the two-hybrid interaction with UvrD, whereas
both the NH2 and COOH termini of UvrD were required for the
interaction with MutL.
Purified MutL Stimulates the Endonuclease Activity of Purified MutH
in the Absence of MutS or a Mispaired Base--
The d(GATC)-specific
endonuclease activity of MutH is relatively weak in the absence of
other components of the mismatch repair system (13). However, this
activity is markedly stimulated in the presence of MutS, MutL, ATP,
Mg2+, and a DNA substrate containing a mispaired base (4).
MutL is known to stimulate the helicase activity of UvrD (15), with which it physically interacts (14). In an effort to identify the
functional role of the interaction between MutL and MutH, we examined
the effect of MutL on the endonuclease activity of MutH in the absence
of MutS, ATP, and/or a mismatch-containing DNA substrate.
The MutH endonuclease activity is specific for unmethylated d(GATC)
sequences (13) and is not dependent on superhelicity in the DNA (4).
One strand of the homoduplex substrate (see "Experimental
Procedures"), which was synthesized in vitro using T7 DNA
polymerase, was completely unmethylated. The template strand was at
least partially methylated since it was prepared directly from a
dam+ E. coli strain (XL-1 Blue). MutH
endonuclease activity was evaluated by monitoring the conversion of
covalently closed M13mp18 molecules to nicked circular molecules based
on their different migration rates during agarose gel electrophoresis
in the presence of EtBr. At high enzyme concentrations purified MutH
catalyzed the complete conversion of the homoduplex substrate to nicked
circular and a small fraction of linear molecules, as expected (data
not shown). The appearance of a linear species in the reaction products
was likely caused by incomplete methylation of the M13mp18 ssDNA
molecules, leaving a fraction of available d(GATC) sites subject to
cleavage on both strands of the substrate. MutH exhibited no activity
on a fully methylated M13mp18 circular duplex (data not shown). A concentration of MutH (1.7 nM) which catalyzed barely
detectable conversion of covalently closed homoduplex molecules to
nicked circular molecules in a 15-min reaction at 37 °C was chosen
to examine the potential stimulation of this reaction by MutL (Fig. 5, lane 2).

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Fig. 5.
MutL, but not MutS, stimulates the
endonuclease activity of MutH. Endonuclease assays containing the
components indicated above each lane were performed as
described under "Experimental Procedures." When present, MutH,
MutL, and MutS were at concentrations of 1.7, 19.0, and 33.8 nM, respectively. When present, ATP was 1.25 mM. Each reaction contained 50 ng of the M13mp18 homoduplex
DNA substrate. NC, nicked circular DNA. CCC,
covalently closed circular DNA.
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The addition of MutL greatly stimulated the level of MutH endonuclease
activity but only in the presence of ATP (Fig. 5, lanes 5 and 6). Purified MutL alone exhibited no detectable
endonuclease activity (Fig. 5, lane 3), and MutS had no
effect on the MutH endonuclease activity in the presence of ATP (Fig.
5, lane 7). The level of endonuclease activity in reactions
containing MutH alone was not altered by the presence of ATP (data not shown).
A titration with MutL demonstrated that stimulation of the
MutH-associated endonuclease activity was dependent on MutL
concentration (Fig. 6). The maximal level
of conversion of covalently closed molecules to nicked circular
molecules occurred at a MutL concentration of 38 nM. The
specificity of the MutH-catalyzed endonuclease reaction for
unmethylated d(GATC) sequences has been well defined (4). Consistent
with this, stimulation of MutH endonuclease activity by MutL was
dependent on the presence of unmethylated d(GATC) sites in the DNA
because use of M13mp18 RF DNA prepared from a dam+ E. coli strain did not result in
significant endonuclease activity (Fig.
7B). Results were identical
using a heteroduplex DNA substrate that differed only in the existence
of a single G-T mismatch (data not shown).

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Fig. 6.
Stimulation of MutH is dependent on the MutL
concentration. The endonuclease activity of MutH in the presence
of increasing concentrations of MutL was measured as described under
"Experimental Procedures." Lane 1, unreacted DNA. All
reactions contained 1.7 nM MutH, 50 ng of the M13mp18
homoduplex DNA substrate, and 1.25 mM ATP. The
concentration of MutL in each reaction is indicated. NC,
nicked circular DNA. CCC, covalently closed circular
DNA.
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Fig. 7.
Stimulation of the MutH endonuclease activity
by MutL does not require ATP hydrolysis and is dependent on the
methylation state of d(GATC) sites. Endonuclease reactions were
performed as described under "Experimental Procedures." Panel
A, all reactions contained 50 ng of homoduplex DNA substrate and
19.0 nM MutL. Lanes 2-5, 1.7 nM
MutH was also present in the reactions. Lane 2, nucleotide
was omitted from the reaction. Lanes 3, 4, and
5, ATP, AMP-PNP, and ATP S were 1.25 mM each,
respectively, in the reactions. Panel B: lane 1,
unreacted homoduplex DNA substrate; lane 3, unreacted
M13mp18 RF DNA. Lanes 2 and 4 contained 50 ng of
the homoduplex substrate or M13mp18 RF DNA, respectively, 1.7 nM MutH, 19.0 nM MutL, and 1.25 mM
ATP. NC, nicked circular DNA. CCC, covalently
closed circular DNA.
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ATP Hydrolysis Is Not Required for Stimulation of the MutH
Endonuclease Activity by MutL--
The clear requirement for ATP to
achieve MutL stimulation of the MutH endonuclease activity was
surprising because neither protein has been previously demonstrated to
bind or hydrolyze ATP. We were unable to detect ATP hydrolysis in
either the purified MutH or MutL preparations (data not shown).
Furthermore, no detectable ATP hydrolysis was observed during the
MutL-stimulated endonuclease assays described above (data not shown).
To determine if ATP hydrolysis was required for the
MutL-dependent stimulation of the MutH endonuclease activity, AMP-PNP and ATP
S were substituted for ATP. AMP-PNP and
ATP
S are either not hydrolyzed or are poorly hydrolyzed by most
ATPases. Fig. 7A demonstrates that both ATP analogs
supported the MutL-stimulated endonuclease activity of MutH, suggesting that ATP hydrolysis was not required. The apparent
Km for ATP in the MutL-stimulated nicking reaction
was approximately 265 µM (data not shown). A titration of
the endonuclease reaction with AMP-PNP was qualitatively identical to
that with ATP (data not shown), ruling out the possibility that
contaminating ATP present in the AMP-PNP affected the results.
Purified MutS Has No Effect on the MutL-stimulated MutH
Endonuclease Activity in the Absence of a Mispaired Base or
ATP--
Because it was believed that MutS, MutL, and a DNA mismatch
were required for stimulation of the MutH-associated endonuclease activity, it was necessary to rule out the possibility that a MutS
contaminant existed in the MutL and/or MutH preparations. To address
this concern MutL and MutH were purified from an E. coli
strain containing an insertion in the mutS gene (see
"Experimental Procedures"). Results using these protein
preparations were indistinguishable from those using the original
preparations, eliminating the possibility that MutS was a contributing factor.
To evaluate the effect of purified MutS on the MutL-stimulated
endonuclease activity directly, reactions containing either homoduplex
or heteroduplex DNA were titrated with MutS in the presence of either
ATP or AMP-PNP. The concentration of MutH was 0.7 nM, and
the concentration of MutL was 9.5 nM in these reactions and
resulted in a low level of endonuclease activity. MutS had no effect on
the stimulation of MutH endonuclease activity by MutL when the
homoduplex substrate was used in the presence of ATP (Fig.
8A). In stark contrast, MutS
further stimulated the endonuclease activity of MutH when the
heteroduplex DNA substrate containing a single G-T mismatch was used
(Fig. 8B). However, stimulation of the endonuclease activity
by MutS using the heteroduplex required ATP hydrolysis because
substitution of AMP-PNP for ATP eliminated the stimulatory effect (Fig.
8C). The fact that MutS required a hydrolyzable form of ATP,
coupled with the observation that MutS had no effect on reactions
containing the homoduplex substrate, provided further support that MutL
alone is capable of stimulating the MutH-associated endonuclease
activity in the absence of MutS and a DNA mismatch.

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Fig. 8.
MutS has no effect on the stimulation of MutH
endonuclease activity by MutL in the absence of ATP or a DNA
mismatch. Endonuclease assays were performed as described under
"Experimental Procedures." The concentration of MutH in all
reactions was 0.7 nM. The concentration of MutL in all
reactions was 9.5 nM. When present, MutS was included at
the concentration indicated. Lane 1 in all three panels
represents 50 ng of unreacted DNA. Panel A, all reactions
contained 50 ng of the homoduplex DNA substrate and 1.25 mM
ATP. Panel B, all reactions contained 50 ng of the
heteroduplex DNA substrate and 1.25 mM ATP. Panel
C, all reactions contained 50 ng of the heteroduplex DNA substrate
and 1.25 mM AMP-PNP. NC, nicked circular DNA.
CCC, covalently closed circular DNA.
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DISCUSSION |
In this report, a physical interaction between the E. coli methyl-directed mismatch repair proteins MutL and MutH was
demonstrated using the yeast two-hybrid system and protein affinity
chromatography. MutL also interacts with MutS (9) and with UvrD (14,
15). Taken together, these results support the previously proposed hypothesis that MutL acts to bring together other protein components of
the mismatch repair pathway (15, 22, 23). Interestingly, MutL has been
demonstrated to stimulate the helicase activity of UvrD (14, 15), the
rate of MutS-mediated DNA loop formation at the site of a mismatched
base (24), and in this report, the endonuclease activity of MutH. Thus,
one can envision MutL as a master coordinator of the mismatch repair
pathway. Its ability to interact with the other mismatch repair
proteins and stimulate their respective activities, presumably in a
coordinated fashion, might serve to restrict these activities to the
mismatch repair pathway itself. For example, inappropriate
endonucleolytic cleavage by the MutH protein when action of the
mismatch repair pathway is not required would be prevented. The
observation that the MutL-stimulated MutH endonuclease activity is
further enhanced in the presence of MutS, a mismatch, and ATP supports
this notion.
A previous study suggested that MutS, MutL, ATP, Mg2+, and
a DNA mismatch were all required to stimulate the endonuclease activity of the MutH protein in the context of the mismatch repair system (4).
The results presented here are largely in agreement with these results
but demonstrate that the MutL subunit of the MutL-MutS complex is
responsible for the stimulation of MutH and that this stimulation
likely is effected through a protein-protein interaction. Two major
lines of evidence indicate that stimulation of the MutH-associated endonuclease activity by MutL is independent of the MutS protein. First, neither ATP hydrolysis nor the presence of a mispaired base was
required to observe stimulation of the MutH-associated endonuclease
activity by MutL. Thus, the two major activities ascribed to MutS, ATP
hydrolysis and mismatch binding, were not required to observe this
stimulation. Further stimulation of reactions containing MutL and MutH
by MutS did require ATP hydrolysis and a mispaired base. Second,
experiments performed using MutL and MutH purified from an E. coli strain containing an insertion in the mutS gene
were indistinguishable from those performed with MutL and MutH purified
from E. coli containing a wild-type mutS gene.
Thus, MutS did not contribute to the reaction as a minor contaminant of
the purified MutH or MutL preparations. Consistent with these results,
we were unable to detect a physical interaction between MutH and MutS
using the yeast two-hybrid system and affinity chromatography, and MutS
had no effect on the MutH-catalyzed endonuclease reaction in the
absence of MutL. We conclude that MutL stimulates the endonuclease
activity of MutH and that this stimulation is further enhanced in the
complete mismatch repair system. The mechanism by which MutS further
stimulates the endonuclease activity of MutH in the presence of MutL is
unknown at this time. However, it is clearly related to the mismatch
binding activity of MutS because a mismatch must be present to observe
this stimulation. It is possible that the MutL-MutH interaction is
facilitated when MutL is targeted to a DNA substrate via its
interaction with a MutS-DNA mismatch complex. Thus, a series of
protein-protein interactions provides a mechanism for specifically
targeting MutH-catalyzed DNA incisions to hemimethylated d(GATC)
sequences when a mismatch is present.
Surprisingly, the stimulation of MutH by MutL requires the presence of
ATP or a nonhydrolyzable ATP analog. The nucleotide dependence of the
MutL-stimulated MutH endonuclease activity suggests that either MutH or
MutL possesses a nucleotide binding activity. The ability to bind ATP
has not been demonstrated for either of these proteins. However,
computer-assisted sequence comparisons have predicted the presence of a
nucleotide binding motif in MutL related to those contained by type II
topoisomerases and a class of chaperone proteins (25). We were able to
demonstrate UV light-induced cross-linking of
[
-32P]ATP to MutL. However, because the efficiency of
cross-linking was extremely low, we were unable to demonstrate
convincingly specificity for the cross-linking event. Furthermore, we
observed cross-linking of [
-32P]ATP to MutH under
identical reaction conditions.
In addition to the computer-predicted nucleotide binding motif, two
pieces of circumstantial evidence suggest that MutL is the more likely
candidate for possession of a nucleotide binding activity. First, the
basal endonuclease activity of MutH was not increased in the presence
of 1 mM ATP in our reactions. In fact, at high ATP
concentrations, an inhibition of activity was observed which may be
caused by depletion of free Mg2+, an essential cofactor for
the MutH-catalyzed endonuclease reaction (13), or competition between
ATP and DNA for the DNA binding site on MutH. Second, ATP binding, but
not hydrolysis, was shown to be required for the association of MutL
with a MutS-DNA mismatch complex (9). This ATP binding requirement
likely is not caused solely by MutS for the following reason. The
Km for ATP hydrolysis catalyzed by our purified MutS
preparation was 23 µM (data not shown), consistent with a
recent report (26). The Km for ATP during the
overall mismatch repair reaction in E. coli is approximately
300 µM (4). The large difference between these two values
suggests that either the affinity of MutS for ATP is altered in the
context of the complete mismatch repair pathway or another protein is
contributing to the observed Km value. The results
presented in this study argue in favor of the latter possibility,
especially when one considers that the Km observed
here for the MutL-stimulated MutH endonuclease reaction (265 µM) is very consistent with that for the complete
mismatch repair system (300 µM). Taken together, the
available evidence suggests that the MutL protein is more likely to
possess a nucleotide binding activity than MutH, although this remains
to be demonstrated directly.
The localization of the region of MutL responsible for interacting with
MutH to the COOH-terminal 218 amino acids was interesting in light of
the fact that this same portion of MutL contains the interface for
interacting with UvrD (14). Eukaryotic mismatch repair systems contain
homologs of the E. coli mutL gene. Extensive amino acid
sequence conservation between MutL and its eukaryotic homologs is
restricted to a region near the NH2 terminus of MutL (27,
28). It is likely that the highly conserved regions are involved in
activities common to both prokaryotic and eukaryotic MutL proteins such
as dimerization and interaction with the mismatch recognition proteins
(MutS and MutS homologs). Thus, the presence of a domain at the
nonconserved COOH terminus of MutL which mediates interactions with
MutH and UvrD suggests that these interactions may be unique to the
E. coli system. In support of this notion, mutH
and uvrD homologs have not been identified as components of
any eukaryotic mismatch repair pathway. Although the region of MutL
containing the MutS interaction interface has not been defined, one
might expect it to reside near the NH2 terminus. This line
of reasoning also suggests that the prokaryotic and eukaryotic mismatch
repair mechanisms diverge extensively after the mismatch recognition
steps. Indeed, recent evidence supports a novel model for strand
discrimination and mismatch excision in eukaryotes. An interaction
between eukaryotic mismatch repair proteins and proliferating cell
nuclear antigen was identified recently in a yeast two-hybrid screen
(29). Because this protein acts as a polymerase processivity clamp
during DNA replication, this interaction suggests that a physical link
may exist between the mismatch repair and replication machinery. Such
an interaction provides one possible mechanism by which the newly
replicated strand can be identified and may eliminate the need for a
helicase dedicated solely to mismatch excision as is the case in
E. coli. However, other mechanisms are possible given the
current data, and additional studies will be required to understand
fully the mechanism of mismatch excision in eukaryotic cells.
The mechanism by which MutL stimulates the MutH-associated endonuclease
activity is still unknown, although the results presented here suggest
that the physical interaction between these two proteins is likely to
be involved. In future experiments it will be necessary to examine the
correlation between the physical interaction and the biochemical
stimulation. In addition, understanding the role of ATP binding in this
interaction will be an important goal.