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INTRODUCTION |
The strand specificity necessary for correction of DNA
biosynthetic errors by the Escherichia coli mismatch repair
system is provided by the transient absence of adenine modification of d(GATC) sequences within newly synthesized DNA (1). Repair is initiated
by binding of a MutS homodimer to a mismatch followed by addition of
MutL to this complex (2-4). Assembly of this ternary complex activates
a MutH-associated endonuclease that cleaves the unmethylated strand at
a hemimethylated d(GATC) sequence within newly replicated DNA (5). The
single-strand break introduced by MutH, which may occur either 3' or 5'
to the mismatch on the unmethylated strand, directs the excision of
that portion of the unmodified strand spanning the d(GATC) sequence and
the mispair (6, 7). Excision requires MutS, MutL, DNA helicase II (also called MutU), and depending on the strand break to mismatch
orientation, a 3'
5' or 5'
3' single-strand exonuclease (6,
8).
The accompanying manuscript (9) demonstrates that MutS and MutL greatly
enhance the activity of DNA helicase II on incised heteroduplex DNA. In
this paper we have used KMnO4 to determine the site of
initiation of mismatch-dependent helix unwinding in DNA
substrates containing a site- and strand-specific, single-strand break.
Permanganate preferentially attacks single-stranded DNA where it
oxidizes the 5,6 double bond of thymine and methylcytosine and reacts
to a lesser degree with other bases (10, 11). This single-strand
selective reagent has been used previously to detect helix opening
associated with promoter melting by bacterial and eukaryotic RNA
polymerases (12, 13). Using this approach we show that MutS-, MutL-,
and helicase II-dependent unwinding of an incised
heteroduplex initiates at the strand break, with the direction of
unwinding being biased toward the shorter path between the strand break
and the mismatch in a circular heteroduplex.
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EXPERIMENTAL PROCEDURES |
Proteins and DNA--
E. coli MutS (14), MutL (3),
and MutH (15) were purified as described previously. DNA helicase II
was isolated from an overproducing strain according to Runyon et
al. (16). Single-strand DNA-binding protein
(SSB)1 and T4 polynucleotide
kinase were purchased from Amersham Pharmacia Biotech, and restriction
endonucleases were purchased from New England Biolabs.
Circular 6440-base pair (bp) G-T heteroduplex and G·C homoduplex DNAs
containing a strand- and site-specific, single-strand break were
prepared using f1MR phage DNAs (2, 6, 17). The structure of these
molecules is illustrated in Fig. 1. DNAs with a single-strand break in
the complementary strand at the HincII site are referred to
as 5'-heteroduplexes since the nick is 5' to the mismatch as viewed
along the shorter path (808 base pairs) in the circular DNA. A second
configuration, referred to as a 3'-substrate, was prepared by MutH
incision of the viral strand at the single GATC site (1023 bp from the
mismatch, shorter path) in hemimethylated DNA (6, 15). Corresponding
control homoduplexes containing a G·C base pair instead of a mismatch at position 5632 were constructed in a similar manner. DNA size markers
were prepared by cleavage of f1MR3 (2) replicative form DNA with
appropriate restriction endonucleases.
Oligonucleotides (Table I), which were
purchased from Oligos Etc., were 5'-32P-end-labeled using
[
-32P]ATP (3000 Ci/mmol, New England Nuclear) and T4
polynucleotide kinase according to the recommendations of the
manufacturer. Labeling was terminated by addition of EDTA to 10 mM and heating at 65 °C for 10 min. Unincorporated label
was removed by passing the solution through Sephadex G-25 (Amersham
Pharmacia Biotech) equilibrated with 10 mM Tris/HCl (pH
7.6), 1 mM EDTA, and 100 mM NaCl. Labeled oligonucleotide was ethanol precipitated and resuspended in 10 mM Tris/HCl (pH 7.6), 1 mM EDTA.
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Table I
Oligonucleotide probes
Oligonucleotides are designated by strand (viral or complementary) and
location of the sequence on the f1MR replicative form duplex molecule.
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Chemical Quench Analysis--
Chemical quench experiments
utilized a KinTek apparatus (KinTek Instruments). Unless specified
otherwise, a solution (40 µl) containing 0.8 µg (0.19 pmol)
heteroduplex DNA, 1.4 µg MutL (10 pmol as dimer), 2.8 µg MutS (15 pmol as dimer), 0.8 µg of DNA helicase II (10 pmol as monomer) in 50 mM Hepes/KOH (pH 8.0), 20 mM KCl, 6 mM MgCl2, 50 µg/ml bovine serum albumin, 1 mM dithiothreitol was mixed with 40 µl of 2 mM ATP containing 4 µg of SSB in the same buffer. Mixing
syringes were maintained at 37 °C and reaction times varied between
50 ms and 5 s. Reactions were quenched by injection of 40 µl of
freshly prepared 30 mM KMnO4 in
H2O, and samples were collected in tubes on ice.
Approximately 2 s after collection, permanganate oxidation was
terminated by addition of 10 µl of 1 M dithiothreitol.
Samples were supplemented with 2 µl of 0.5 M EDTA and 10 µl of 10 mM Tris/HCl (pH 7.6), 1 mM EDTA, and
passed through a spin-column containing S-300 (Amersham Pharmacia
Biotech) equilibrated with 10 mM Tris/HCl (pH 7.6), 1 mM EDTA, 0.3 M NaCl. The column flow-through
was extracted with phenol, precipitated with ethanol, and DNA was
resuspended in 10 mM Tris/HCl (pH 7.6), 1 mM
EDTA. After hydrolysis with restriction endonucleases as indicated, DNA
digests (50 µl) were supplemented with 5.6 µl of piperidine and
incubated at 90 °C for 30 min. Piperidine-treated samples were
evaporated to dryness, the residue was dissolved in 30 µl of
H2O, and the solution taken to dryness. After repetition of
the H2O wash, the DNA pellet was dissolved in 6 µl of
85% formamide, 20 mM EDTA, 0.05% bromphenol blue, and
0.05% xylene cyanol FF, and subjected to electrophoresis through 6%
polyacrylamide gels containing 8 M urea. DNA fragments were
transferred to nylon membranes (ICN BiotranTM), and sites
of permanganate oxidation mapped by indirect end-labeling with
5'-32P oligonucleotides as indicated (18). DNA species were
visualized by autoradiography and quantitated using a Molecular
Dynamics PhosphorImager.
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RESULTS |
The excision step of methyl-directed mismatch repair requires
MutS, MutL, DNA helicase II, an appropriate exonuclease, and a
heteroduplex containing a single-strand break (6-8). The accompanying article demonstrates that MutL greatly stimulates helicase II on
conventional helicase substrates and that MutS and MutL activate the
unwinding activity of helicase II on an incised DNA containing a
mismatch (9). We show here that MutS-, MutL-, and helicase II-dependent unwinding of heteroduplex DNA initiates at the
strand break.
MutS-, MutL-, Helicase II-, and Mismatch-dependent
Unwinding Initiates at the Strand Break--
To determine the site of
initiation of heteroduplex unwinding, we have used permanganate, which
preferentially oxidizes thymidylate residues in single-stranded DNA
(10, 11). Since thymidylate oxidation renders the phosphodiester bond
subject to hydrolysis by strong base, the location of oxidized residues
can be determined. The circular 6.4-kilobase DNAs used in this work
contained a G-T mismatch (or an G·C base pair) and a site- and
strand-specific nick located 808 bp 5' or 1023 bp 3' to the mismatch as
viewed along the short path linking the two DNA sites in the circular molecules (Fig. 1). For convenience, the
two substrate orientations are referred to as 5'- or 3'-heteroduplexes,
respectively.

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Fig. 1.
Structure of f1MR heteroduplexes.
Circular heteroduplex DNAs used in this study contained a G-T mismatch
at position 5632 and a single-strand break either at position 0/6440 on
the complementary strand (C) or at position 215 on the viral strand
(V). DNAs with a nick in the C strand are designated as
5'-heteroduplexes, since the incision lies 5' to the mismatch along the
shorter path that joins the two sites in the circular molecule. DNAs
with a nick on the V strand are dubbed 3'-heteroduplexes for a similar
reason. Sites of cleavage by restriction endonucleases used in the
experiments are also indicated with coordinates shown corresponding to
the nucleotide 5' to the phosphodiester attacked. Shaded regions
correspond to oligonucleotides (Table I) used for indirect
end-labeling.
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A solution of G-T heteroduplex (or G·C homoduplex), MutL, MutS, and
helicase II was mixed in a chemical quench device with a solution of
ATP and SSB, and after brief incubation reactions were subjected to a
2 s oxidation with KMnO4 (see "Experimental Procedures"). Sites of KMnO4 hydrolysis were mapped
relative to an appropriate restriction site by indirect end-labeling
(18) after piperidine hydrolysis and electrophoresis through denaturing polyacrylamide gels. As shown in Fig. 2
(lanes 1-3), initiation of unwinding of the 5'-G-T
heteroduplex at the single-strand break was evident as judged by
conversion of either the incised (upper panel) or the
continuous (lower panel) strand of the molecule to a
permanganate-sensitive form. Conversion of that region of the molecule
to permanganate-sensitivity in the vicinity of the nick was rapid with
a maximal unwinding rate achieved in 5 s or less. An otherwise
identical G·C homoduplex did not support the reaction (lane
8), and increased permanganate reactivity was not observed in the
absence of helicase II or MutS (lanes 4 and 5) or
in the absence of MutL (not shown). Consequently, unwinding observed at
the strand break is dependent on MutS, MutL, and helicase II and on the
recognition of a mismatch 808-bp distant. Analysis of permanganate
sensitivity of the continuous strand of the heteroduplex (Fig. 2,
lower panel) revealed that mismatch-provoked unwinding of
5'-heteroduplex occurred to either side of the nick. This observation will be considered further below.

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Fig. 2.
Helix unwinding initiates at the nick with
5'-heteroduplex DNA. 5'-G-T heteroduplex or 5'-G·C homoduplex
DNA containing a nick in the C strand at the HincII site
(Fig. 1) was incubated as indicated with MutS, MutL, DNA helicase II,
SSB, and ATP followed by a 2 s quench with 10 mM
KMnO4 (see "Experimental Procedures"). After cleavage
with DraI and piperidine treatment to cleave strands at the
sites of permanganate oxidation, DNA samples were subjected to
electrophoresis through denaturing polyacrylamide gels and
electrotransferred to a nylon membrane. Fragments of interest were
visualized by indirect end-labeling (7) using
32P-oligonucleotides V6287 and C6289 as probes. As
summarized in Table I, these probes hybridize to individual strands of
the heteroduplex near the DraI site. Upper panel,
permanganate reactive sites on the incised C strand; lower
panel, permanganate reactive sites on the continuous V strand.
Lanes: 1, complete system, 50 msec reaction;
2, complete, 1 s reaction; 3, complete,
5 s reaction; 4, helicase II omitted, 1 s
reaction; 5, MutS omitted, 1 s reaction; 6,
complete system, 1 s reaction but no KMnO4 quench;
7, all proteins omitted, 1 s reaction; 8,
complete system, 1 s reaction but G·C homoduplex substituted for
heteroduplex; 9, marker for location of the strand break.
The strong band that runs with the marker for the strand break in the
lower panel is the result of permanganate oxidation of the closed
circular V strand near the nick. It was not produced in the absence of
the oxidizing agent (lane 6).
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As shown in Fig. 3, virtually identical
results were obtained with a 3'-G-T heteroduplex in which the mismatch
and strand break were separated by 1023 bp. However, in contrast to
results obtained with the 5'-heteroduplex described above, the degree of unwinding of the 3'-substrate increased significantly between 1 and
5 s, perhaps due to the increased distance between the two DNA
sites in the latter molecule.

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Fig. 3.
Helix unwinding initiates at the nick with
3'-heteroduplex DNA. Substrates were 3'-G-T heteroduplex or
3'-G·C homoduplex DNA containing a nick in the V strand at the
MboI site (Fig. 1). Reactions were performed as described
under "Experimental Procedures" and in the legend to Fig. 2. DNAs
were cleaved with HincII prior to electrophoresis, and
oligonucleotides C005 and V005 were used for indirect
end-labeling. Upper panel, permanganate reactive sites on
the V strand; lower panel, permanganate reactive sites on
the C strand. Lanes: 1, complete system, 50 msec
reaction; 2, complete system, 1 s reaction;
3, complete system, 5 s reaction; 4,
helicase II omitted, 1 s reaction; 5, MutS omitted,
1 s reaction; 6, complete system, 1 s reaction but
no KMnO4 quench; 7, all proteins omitted, 1 s reaction; 8, complete system, 1 s reaction but G·C
homoduplex substituted for heteroduplex; 9, marker for
location of nick. As noted in the legend to Fig. 2, the strong band in
the lower panel results from permanganate oxidation of the
covalently continuous strand near the site of the strand break in the
open strand.
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Although presence of a G-T mismatch is known to enhance helix dynamics
in the vicinity of the mispair (19, 20), the mismatched thymidylate in
the G-T heteroduplex did not detectably react with permanganate under
the mild oxidation conditions
used.2 Furthermore, under
conditions where single-strand character was rapidly generated at the
strand break in the G-T heteroduplex in the presence of MutS, MutL,
helicase II, and SSB, permanganate oxidation products were not detected
in the vicinity of the mismatch after 5 s incubation. This is
illustrated in Fig. 4 for the 5'-G-T heteroduplex, and identical results were obtained with the 3'-substrate (not shown). These results imply that the single-strand character that
develops at the strand break in an incised heteroduplex is not the
consequence of an unwinding event that initiates at the mispair and
propagates to the mismatch. Consequently, we have concluded that
unwinding by activated helicase II initiates at the strand break. The
extent of unwinding observed with 5'- and 3'-heteroduplexes was about
50-100 nucleotides.

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Fig. 4.
Helix opening does not occur near the
mismatch early in the repair reaction. Reactions with the 5'-G-T
heteroduplex containing a nick in the C strand at the HincII
site were performed as described under "Experimental Procedures"
and the legend to Fig. 2. DNAs were cleaved with HgiAI prior to
electrophoresis, and indirect end-labeling with oligonucleotides V5470
or C5470 was used to visualize permanganate reactive sites. Upper
panel, permanganate reactive sites on the C strand; lower
panel, permanganate reactive sites on the V strand.
Lanes: 1, complete system, 50 msec reaction;
2, complete system, 1 s reaction; 3,
complete system, 5 s reaction; 4, marker for location
of nick; 5, marker for location of mismatch.
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Bias in the Direction of the DNA Helicase II Unwinding--
As
mentioned above and shown in the lower panels of Figs. 2 and
3, analysis of permanganate sensitivity of the continuous heteroduplex
strand demonstrated that unwinding occurs in both directions from the
strand break. However, analysis of mismatch-provoked methyl-directed
excision tracts produced in extracts and in a purified system has
demonstrated that excision is largely restricted to the shorter path
between the strand signal and the mismatch in circular heteroduplexes
similar to those used here (7). The unwinding reactions described above
utilized only a subset of the proteins required for methyl-directed
mismatch repair, and helicase II is known to load preferentially onto
single-strand regions within otherwise duplex DNA (21). Consequently,
it was possible that a directional unwinding preference was masked to some degree by secondary events in which the helicase loaded onto single-stranded DNA produced by orientation-dependent
unwinding from the strand break. This possibility was assessed in
several ways with potential unwinding preference estimated by summing integrated band intensities of oxidation products produced on the
continuous heteroduplex strand to either side of the nick.
As shown in Figs. 5 and
6 (upper panels), directional
unwinding from the strand break toward the mismatch via the shorter path in the circular heteroduplex could be demonstrated with both 3'-
and 5'-heteroduplexes, but the magnitude of preference for the shorter
path decreased monotonically with increasing helicase II concentration.
A decrease in preferential unwinding along the shorter path with both
substrates also occurred as reaction time increased (Figs. 5 and 6,
lower panels), and whereas heteroduplex unwinding from the
strand break did not require SSB, presence of the protein conferred a
modest increase in directional unwinding (not shown). MutS and MutL are
therefore not only sufficient to activate helicase II unwinding from
the strand break of an incised heteroduplex, but they also confer
directionality on this process.

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Fig. 5.
Helicase II unwinding is biased toward the
shorter path between the nick and mismatch with a 3'-heteroduplex.
Complete reactions utilized a 3'-G-T heteroduplex containing a nick in
the V strand at the MboI site (Fig. 1) and were performed by
a modification of the procedure described under "Experimental
Procedures" and in the legend to Fig. 2. Helicase II, which was
omitted from the DNA syringe, was present in the syringe containing SSB
and ATP, and incubation was for 1 s at 37 °C prior to
permanganate quench. Unwinding bias to either side of the strand break
was estimated by summing radiolabel present in oxidized species
produced to either side of the strand break on the continuous C strand
(e.g. the species evident in the lower panels of
Figs. 2 and 3 that migrate below or above the position of the strand
break). Error bars are ± 1 S.D. Upper panel, helicase
II was varied as indicated. Reaction time was 1 s. Lower
panel, SSB and helicase II were present at 8 and 0.6 µg,
respectively, and reaction time was varied as indicated.
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Fig. 6.
Helicase II unwinding is biased toward the
shorter path between the nick and mismatch with a 5'-heteroduplex.
Complete reactions, which contained a 5'-G-T heteroduplex with a nick
in the C strand at the HincII site (Fig. 1), were performed
as described under "Experimental Procedures" and in the legend to
Fig. 5. SSB was present at 8 µg. Upper panel, helicase II
was varied as indicated, and reactions were stopped after 350 ms.
Lower panel, helicase II was present at 0.6 µg, and
reaction time was varied as shown.
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DISCUSSION |
A single hemimodified d(GATC) sequence, which may reside to either
side of the mismatch, is sufficient to provide strand specificity to
heteroduplex repair by the E. coli methyl-directed pathway (8, 22), with mismatch-provoked incision of the unmethylated strand of
the d(GATC) site providing a strand break that directs removal of that
portion of the new strand spanning the nick and the mispair (5-7).
Analysis of reaction intermediates has suggested that excision
initiates at the strand break by a mechanism in which helicase II
displacement renders the incised strand sensitive to an appropriate 3'
5' or 5'
3' single-strand exonuclease, depending on location of
the nick 3' or 5' to the mispair (7). The experiments described here
are compatible with this mechanism and demonstrate that MutS and MutL
are sufficient to coordinate mismatch recognition to activation of
helicase II unwinding at a single-strand break that can be located
800-1,000 bp from the mismatch.
As noted previously (7, 8), the bidirectional nature of the
methyl-directed system requires loading of the appropriate hydrolytic
activity at the incised d(GATC) sequence to ensure that excision
proceeds toward the mispair. The finding that helicase II activation by
MutS and MutL results in a significant degree of
orientation-dependent unwinding on a nicked heteroduplex
suggests that the latter proteins are sufficient to evaluate placement of the strand break 3' or 5' to the mismatch. Despite their separation distance, interaction of the mismatch and the strand break during the
course of this reaction is fast with maximal initiation of unwinding
achieved after 1-5 s under conditions of MutS, MutL, and helicase II
excess. Although the molecular events responsible for interaction of
the two DNA sites are not fully understood, recent electron microscopy
experiments (4) have suggested that MutS translocation along the
heteroduplex contour may play a role in this process. Whereas the MutS
dimer initially binds to heteroduplex DNA at the mismatch, this complex
is converted in the presence of ATP to an
-shaped DNA structure that
is stabilized by MutS at the base. The mismatch in such complexes is
usually found in the DNA loop. This rearrangement has been attributed
to a mechanism in which the two subunits of the MutS dimer act as
ATP-driven divergent motors that translocate from the mispair in a
bidirectional fashion along the helix contour. MutL stimulates this
reaction and when present migrates along the helix with MutS. Under the buffer conditions used for the experiments described here, the rate of
MutS-catalyzed formation of
-shaped DNA loops approaches 10,000 bp
per min in the absence of MutL (4), sufficiently fast to account for
the interaction of the two sites observed in the experiments described
here.
Fig. 7 illustrates a mechanism for MutS-
and MutL-dependent activation of helicase II unwinding that
incorporates these electron microscopy results, as well as the findings
presented here and in the accompanying paper (9). Helicase II
activation initiates by binding of a MutS dimer to the mismatch (4).
MutL adds to the MutS·DNA complex in a reaction that requires ATP but
apparently not ATP hydrolysis (3). Although MutL exists as a dimer in solution (3), the stoichiometry of MutL addition has not been established. In a reaction that depends on ATP hydrolysis, the subunits
of the MutS dimer leave the mismatch, usually in a bidirectional manner, with MutL moving along the helix with MutS. At a stage in the
reaction that remains to be determined, helicase II adds to
MutS·MutL·DNA complex, and when a strand break is encountered the
activity enters the helix in such a way that unwinding tends to proceed
toward the mismatch, irrespective of placement of the nick 3' or 5' to
the mispair on the incised strand. Since MutL greatly stimulates the
activity of helicase II and since the two proteins interact physically
(9, 23), it is likely that MutL directly promotes initiation of
unwinding by helicase, perhaps by physically facilitating helix entry
of the activity at the strand break.

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Fig. 7.
Model for mismatch and
orientation-dependent excision during mismatch repair.
A description of the mechanism is provided in the text.
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DNA helicases show a preferred polarity during initiation of unwinding
(24). The orientation-dependent unwinding from the nick
toward the mismatch described here can be understood in terms of this
polarity preference, which for helicase II is 3' to 5' (25). Thus, one
need only invoke loading of the unwinding activity onto the incised
strand when the nick is 3' to the mismatch or onto the continuous
strand when nick is 5' to the mispair.
The suggestion that MutL may have an important role in activating the
excision step of bacterial mismatch repair may have implications for
the eukaryotic reaction. The mammalian pathway has a mispair
specificity similar to that of the bacterial reaction and occurs by a
similar bidirectional mechanism. Defects in this system have been
implicated in both inherited and sporadic cancers, as well as in
cellular resistance to certain DNA damaging agents (26-29). However,
in contrast to the MutS and MutL homodimers that are active in the
bacterial pathway, human mismatch repair is dependent on MutS
, a
heterodimer of the MutS homologs MSH2 and MSH6, and MutL
, a
heterodimer of the MutL homologs MLH1 and PMS2 (30-32). Certain
MLH1 and PMS2 mutations confer selective
directional defects in mismatch repair. Thus, some MLH1
mutations are selectively defective in repair directed by a strand
break located 3' to the mismatch but are proficient in mismatch
correction directed by a 5'-strand signal
(33).3 Conversely, a
PMS2 mutation has been identified that blocks repair from
the 5'-side of the mismatch but not from the 3'-side (34). One
interpretation of these findings is that like bacterial MutL, human
MutL
functions to activate the mismatch repair excision system, but
in the case of the human pathway the two subunits of MutL
differentially function to load a 3' to 5' or 5' to 3' excision system,
depending on the location of the strand break that directs the
reaction.
We thank Dwayne Allen and Keith Bjornson for
comments on the manuscript, and Sam Wilson (National Institute of
Environmental Health Sciences) for suggesting the use of permanganate
as a probe for single-stranded DNA.