From the Charles A. Dana Division of Human Cancer
Genetics, Dana-Farber Cancer Institute, Harvard Medical School, Boston,
Massachusetts 02115, the ¶ Lineberger Comprehensive Cancer
Research Center, University of North Carolina, Chapel Hill, North
Carolina 27599, and the ** Ludwig Institute for Cancer Research,
Department of Medicine, and Cancer Center, University of California San
Diego School of Medicine, La Jolla, California 92093
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ABSTRACT |
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Genetic and biochemical studies have indicated
that mismatch repair proteins can interact with recombination
intermediates. In this study, gel shift assays and electron microscopic
analysis were used to show that the Saccharomyces
cerevisiae MSH2/6 complex binds to Holliday junctions and has an
affinity and specificity for them that is at least as high as it has as
for mispaired bases. Under equilibrium binding conditions, the MSH2/6
complex had a Kd of binding to Holliday junctions
of 0.5 nM. The MSH2/6 complex enhanced the cleavage of
Holliday junctions by T4 endonuclease VII and T7 endonuclease I. This
is consistent with the view that the MSH2/6 complex can function in
both mismatch repair and the resolution of recombination intermediates
as predicted by genetic studies.
Virtually all recombination models propose the formation of
heteroduplex joints during recombination in which a single strand from
one parental DNA is paired with a complementary strand from another
parental DNA (1, 2). When the two single strands differ in sequence,
the resulting heteroduplex intermediate contains mispaired bases. These
mispaired bases are generally processed by the MutHLS mismatch repair
pathway in bacteria and the related pathway in eukaryotes that utilizes
the MSH2, MSH3, MSH6, PMS1, and MLH1 gene products (3-5). Some
specific mispaired bases like T:G, A:G, and A:C can also be repaired by
the base-specific mismatch repair pathways, and some other mispaired
bases like C:C and palindromic insertion mispairs sometimes escape
repair at significant frequencies (3-6).
In addition to repairing mispaired bases, the MutHLS type of mismatch
repair pathways are also known to play other roles in recombination.
One example of this is that recombination between divergent, so-called
homologous DNA sequences occurs at reduced frequency compared with
recombination between homologous sequence (7). This regulation of
recombination occurs as a consequence of the formation of mispaired
bases in recombination intermediates and is also dependent on mismatch
repair genes such as mutS, mutL, MSH2,
MSH3, MLH1, PMS1, and probably other
such genes. Such effects have been extensively documented in bacteria,
yeast, and mammalian cells (7-18). Two models have been proposed that
could explain these observations. One is that mismatch repair
recognizes recombination intermediates that are formed from divergent
DNAs and contain multiple mismatches and destroys them as a byproduct
of repair (19, 20). Alternately, some combination of mismatch repair enzymes could recognize the formation of mispaired bases in
recombination intermediates and block the formation of the mature
recombination intermediate (21-23). Consistent with this latter
model, Escherichia coli mismatch repair enzymes can block
the RecA-mediated branch migration in vitro when the
recombining DNAs contain sequence differences (24).
A second type of regulation of recombination has been observed during
the study of gene conversion polarity gradients (6, 22, 23). Gene
conversion polarity gradients are thought to be due to a gradient of
heteroduplex DNA that is highest near the site where recombination
initiates and decreases with increasing distance away from the
initiation site. However, in mismatch repair-deficient mutants or when
mutations that form mispairs that escape mismatch repair are located at
the low end of the polarity gradient, the extent of apparent
heteroduplex formation at the low end of the gradient is increased to
the level seen at the high end of the gradient. Three models have been
presented to explain this. One suggests that polarity gradients are due
to restoration repair occurring specifically at the low end of the
gradient (6). A second is that heteroduplex DNA formed at the low end
of the gradient is specifically unwound when mispaired bases are
present (22). The third is that, once mispaired bases are formed at the
low end of the gradient, this causes the resolution of the Holliday
junctions that are extending the heteroduplex regions. This would leave
symmetrically located nicks in the recombinant DNA molecules, which
could serve to initiate compensating mismatch repair that would
eliminate the genetic consequences of heteroduplex DNA at the low end
of the polarity gradient (22). Each of these models postulates the
coordination of mismatch repair/recognition with the process of
heteroduplex formation and recombination intermediate resolution.
A different type of effect of mismatch repair-related proteins on
recombination was observed through the discovery of Saccharomyces cerevisiae MSH4 and MSH5 (25, 26). These proteins are
apparently not required for mismatch repair but are required for
meiotic crossing over, as mutations in MSH4 and
MSH5 reduce the frequency of crossing over and cause defects
in chromosome segregation during meiosis. Humans and mice also contain
MSH4 and MSH5, and mice containing mutations in
MSH5 have meiotic defects suggesting that the human and mouse proteins
act in the same processes as the S. cerevisiae proteins (27,
28).1 In both humans and
S. cerevisiae, MSH4 and MSH5 form a heterodimer consistent
with the results that mutations in each of the two S. cerevisiae genes cause similar defect in meiotic crossing over (28, 29). Recent studies have suggested that MLH1 plays a role in
meiotic crossing over similar to that proposed for MSH4 and MSH5 (30).
One possible explanation for these observations is that MSH4, MSH5, and
possibly MLH1 interact with recombination intermediates and regulate
their resolution.
Although it is clear that mismatch repair and mismatch repair-related
proteins play important roles in regulating recombination, particularly
in eukaryotes, there is little information about the mechanism of these
processes. In the first two cases discussed above, there must be some
interaction between the processes that recognize and repair mispaired
bases and the processes/intermediates that form heteroduplex DNA. In
the latter case, MutS-related proteins play a role in how recombination
intermediates are resolved, suggesting they may actually recognize
recombination intermediates. In previous studies, we demonstrated that
MSH2 can recognize Holliday junctions in DNA, suggesting it may play a
role in the processing of Holliday junctions (31). It is known that
MSH2 functions in a complex with either MSH3 or MSH6 and that the
MSH2/3 and MSH2/6 complexes have different mispair recognition
properties compared with MSH2 alone (for reviews see Refs. 3, 5, 32,
and 33). To further analyze the role of MSH2 in Holliday junction
processing, we have investigated the ability of MSH2-MSH6 complex to
recognize Holliday junctions and modify their processing by Holliday
junction resolution enzymes.
Purification of S. cerevisiae MSH2/6--
S.
cerevisiae MSH2/MSH6 was purified using a method similar to that
described by Alani (34). MSH2 and MSH6 were coexpressed in the presense
of galactose from separate 2-µm plasmids under control of
GAL10 promoters in a protease deficient strain (S. cerevisiae strain RKY2418 containing pRDK354 MSH2 and
pRDK568 MSH6). Cell extracts were made from
galactose-induced cells by grinding under liquid nitrogen, and the
MSH2-MSH6 was purified by sequential chromatography on PBE94,
single-strand DNA cellulose, and Q-Sepharose using chromatography
conditions that were essentially the same as those described by Alani
(34). Details of the purification will be described
elsewhere.2 Analysis of the
protein preparation by SDS-PAGE is presented in Fig. 1A.
Densitometric analysis demonstrated that the purity of MSH2/6 in the
final fraction was determined to be greater than 95% and the molar
ratio of MSH2 to MSH6 was 1:1. Gel
filtration analysis using a Pharmacia SMART system did not detect any
free MSH2 or MSH6 in the
preparation.4
Holliday Junction and Duplex DNA Binding
Substrates--
Holliday junction substrates (J0-J12) and the G:C
homoduplex substrate used in this study were constructed from
oligonucleotides and were purified by HPLC 3 using a Waters
GEN-PAK FAX column (Milford, MA) as described (31). The G:T
heteroduplex substrate analogous to the G:C homoduplex substrate was
constructed by annealing oligos 28756 (5'-GACGCTGCCGAATTCTGGCTTGCTAGGACATCTTTGCCCACGTTGACCC) and 28755 (5'-GGGTCAACGTGGGCAAAGATGTCTTAGCAAGCCAGAATTCGGCAGCGTC), and
was purified by HPLC as described. The Holliday junction 565 and
Holliday junction 75 DNAs (32P-labeled) used for EM and
junction cleavage were prepared as described previously (35).
Gel Mobility Shift Assay of DNA Binding--
HPLC-purified DNA
substrates were 5'-end-labeled using [ Formation of MSH2/6 -Holliday Junction Complexes for Electron
Microscopy--
Complexes of MSH2/6 with Holliday junction DNA were
formed by incubating the DNA (5 µg/ml) with MSH2/6 in a 20-µl
volume at a molar ratio of 40 MSH2/6 heterodimers per Holliday junction DNA for 15 min at room temperature in a buffer containing 40 mM NaCl, 20 mM Hepes, pH 7.6, 0.1 mM DTT, 0.1 mM EDTA, and 40 µg/ml bovine
serum albumin. The DNA-protein complexes were then prepared for EM by
fixation with 0.6% glutaraldehyde for 10 min at room temperature,
followed by filtration through 2-ml columns of BioGel A5m (Bio-Rad).
The purified DNA samples were mixed with a buffer containing 2 mM spermidine, 0.15 M NaCl, and applied for
30 s to thin carbon foils supported by 400-mesh copper grids. The
grids were washed with sequential washes of water and a graded ethanol series, air-dried, and rotary shadowcast at 10 Conditions for Cleavage of Holliday Junction Complexes with T4
and T7 Endonucleases--
The 32P-labeled Hol75 DNA was
used as a substrate for the junction cleavage assays. The DNA was
incubated in 50 mM Tris, pH 8, 5 mM
MgCl2, 1 mM DTT, 100 µg/ml bovine serum
albumin, and 10 µg/ml calf thymus DNA at room temperature with or
without MSH2/6 for 15 min, and then treated with the indicated amount
of enzyme (the kind gift of Dr. Borries Kemper, University of Koln,
Koln, Germany) for different times as described previously (35). The reactions were stopped by adding EDTA and SDS to 50 mM and
1%, respectively, followed by incubation for 5 min at 37 °C. The
cleavage products were electrophoresed on a 4% polyacrylamide gel and
autoradiographed. The percent of cleavage was measured by using a PhosphorImager.
MSH2/6 Binds to Holliday Junctions--
In previous experiments,
MSH2 was observed to bind to oligonucleotide duplexes containing
Holliday junctions (31) in addition to its ability to recognize
mispaired based present in oligonucleotide duplexes (36, 37). In
contrast, the available evidence suggests that isolated MSH6 only
interacts with DNA nonspecifically (34, 38, 39). Because MSH2 is known
to function in a complex with MSH6 (3, 5, 34, 40, 41), we tested
whether MSH2/6 heterodimers would also bind to Holliday junctions. In
the present experiments, gel mobility shift experiments were used to
assess the binding of MSH2/6 to the same series of eight Holliday
junctions (J0-J12) previously used to study binding of MSH2 (31). In
addition, binding of MSH2/6 to a control oligonucleotide homoduplex
(G:C) and an oligonucleotide heteroduplex (G:T) was assessed. The
MSH2/6 preparation (Fig. 1A)
used in these experiments was greater than 95% pure, had a molar ratio
of MSH2 to MSH6 of 1:1, and did not contain any free MSH2 or free MSH6.
In experiments where increasing amounts of MSH2/6 were incubated with a
fixed amount of different DNA substrates, increasing amounts of MSH2/6
complexed with either the J12 Holliday junction or the G:T- or
G:C-containing duplexes were observed (Fig. 1B). In this
experiment, the relative affinity of MSH2/6 for the different DNA
substrates appeared to be J12 > G:T > G:C. Similarly, in
experiments with a fixed amount of MSH2/6 and increasing amounts of
either J12 or G:T-containing duplex substrates, MSH2/6 bound to greater
amounts of J12 at lower substrate concentration than to a
G:T-containing duplex. The binding of MSH2/6 to J12 and G:T-containing
duplex was found to be saturating at 1.1 and 0.9 pmol of MSH2/6 per
pmol of substrate, assuming that the active species is a MSH2/6
heterodimer.
In previous experiments with MSH2, equilibrium binding conditions were
not achieved (31). To determine if equilibrium binding was observed
with MSH2/6, two different experiments were performed with J12 Holliday
junction, and the G:T- and G:C-containing duplexes. In one experiment,
each labeled substrate was mixed with different amounts of unlabeled
competitor substrate (molar ratios of 2.5:1 to 50:1 were tested) and
incubation with MSH2/6 at a 5:1 ratio of MSH2/6 per labeled substrate
was performed for 60 min prior to measuring the relative amount of
labeled substrate present in complex. In a second experiment, MSH2/6
was incubated with labeled J12, G:T, or G:C substrate for 60 min to
preform complexes. Then the same ratios of unlabeled competitors were
added and incubation continued. The results showed equal amounts of
competition independent of whether competitors were added before or
after the MSH2/6 complexes were formed with labeled substrate (not
shown). This demonstrated that, under the binding conditions used in
the present experiments, equilibrium binding is achieved. This allows
the data of Fig. 1C to be analyzed by Scatchard analysis
yielding Kd values of 0.5 and 0.7 nM
for MSH2/6 binding to the J12 and G:T substrates, respectively.
To further analyze the specificity of MSH2/6 for Holliday junctions, a
series of competition experiments were performed in which labeled
G:T-containing substrate was mixed with different amounts of unlabeled
G:T-, J12, or G:C-containing competitors and the amount of complex
formed between MSH2/6 and labeled G:T-containing substrates was
measured. Analysis of the results showed that J12 was a slightly better
competitor than G:T, which was a significantly better competitor than
G:C (Fig. 2A). When these data
were analyzed by the method of Chi and Kolodner (42), MSH2/6 was found
to have a 9.4- and 5.6-fold greater affinity for J12 and G:T,
respectively, compared with G:C. In a second series, a 10-fold excess
of eight different Holliday junctions, as well as G:T- and
G:C-containing duplexes, were tested for their ability to compete for
binding of MSH2/6 to labeled G:T-containing substrate (Fig.
2B). The results showed that under these conditions,
theoretical competition was observed for the G:T competitor. The G:T
competitor was about a 7-fold better competitor than G:C, and all of
the Holliday junctions tested were significantly better competitors
that the G:T competitor, with some being up to 3-fold better
competitors than the G:T competitor. These data demonstrate that MSH2/6
binds specifically to both G:T- and Holliday junction-containing
substrates compared with G:C-containing substrate and that MSH2/6
likely recognizes Holliday junctions with greater affinity than the
G:T-containing substrate tested here. In a study to be published
elsewhere, we have analyzed the ability of MSH2/6 to recognize 65 different mispaired base-containing DNAs including all possible
base-base mispairs and numerous insertion/deletion mispairs ranging
from +1 base to +16 bases. In no case did the affinity of MSH2/6 for
another mispaired base exceed the affinity seen here for the
G:T-containing substrate. Thus MSH2/6 appears to generally have a
higher affinity for the Holliday junctions studied here than for
mispaired bases.
Visualization of MSH2/6 Bound to Holliday Junctions--
The
preceding experiments demonstrate that MSH2/6 binds strongly to DNA
molecules containing a Holliday junction, but do not address the
question whether it binds directly to the junction as contrasted to
binding along an arm or as an extreme case, binding to the ends of the
four arms and then coalescing to form a more stable complex. To address
this, EM was used to examine complexes of MSH2/6 with a synthetic
Holliday junction DNA (31, 35) containing four arms of ~565 base
pairs extending from the J12 junction of Picksley et al.
(43) as described previously (31, 35). This DNA, termed Hol565, was
incubated with MSH2/6 at a ratio of 40 heterodimers per junction DNA
for 15 min and the samples then fixed with glutaraldehyde followed by
preparation for EM (44). Examination of fields of molecules revealed
Holliday junction DNAs with four extended arms and, in many cases, with
proteins bound (Fig. 3). In one
experiment, 79% of the DNA molecules scored (n = 89)
showed MSH2/6 bound while 21% of the molecules were protein-free. Of
the Holliday junction molecules containing MSH2/6, 82% showed a large
protein complex centered over the junction while 18% had protein balls
located along one or more arms or at a DNA terminus. In general, the
four DNA arms exiting the MSH2/6 complex were well separated from each
other, often taking nearly 90° trajectories. When the incubation
buffer contained 5 mM MgCl2, conditions used below for the endonuclease cleavage experiments, all four DNA arms were
most often folded back on themselves, generating a thick DNA filament
having the length of one DNA arm and with a protein complex at one end
(not shown). Reduction of the molar ratio of MSH2/6 to Holliday
junction DNA to 5:1 resulted in a lower amount (25%) of the Holliday
junction DNA-containing bound protein. From these results we conclude
that the protein is mostly localizing to the junction as contrasted to
elsewhere on the DNA and binds with very high specificity as shown
above.
Previous EM studies demonstrated that MSH2 can also bind to Holliday
junctions (31). Using the same substrates and binding conditions, 25%
of the Holliday junctions had MSH2 bound at a single site and 75% were
protein-free. Of the DNAs with MSH2 bound, 61% had MSH2 bound at the
junction, 21% had MSH2 bound at an end and 18% had MSH2 bound at a
single internal site on an arm. As described above, MSH2/6 complex
bound to over three times more DNA molecules and with a greater
proportion of binding at the junction. These results suggest that
MSH2/6 has a greater affinity and specificity for Holliday junctions
compared with MSH2 alone.
Binding of MSH2/6 to Holliday Junctions Enhances Their Cleavage by
Two Junction-specific Endonucleases--
In order to further
characterize the binding of MSH2/6 to Holliday junctions, we tested
whether the binding of MSH2/6 would occlude the Holliday junction from
access by the Holliday junction resolution enzymes T4 endo VII and T7
endo I. Complexes of MSH2/6 were formed with a 32P-labeled
Holliday junction DNA containing 75-bp arms (Hol 75 DNA) (35). The DNA
was incubated with either no MSH2/6 or 40 heterodimers per junction
DNA. T4 endo VII (1, 2, 5, or 10 ng) or T7 endo I (1, 2, 3, 5, or 12 ng) were added to each sample, and incubation continued under
conditions where the enzymes were competent for cleavage. The DNA was
deproteinized, electrophoresed on an acrylamide gel, and imaged by
autoradiography. As shown in Fig. 4
(A and B) and quantified by phosphoimager
analysis (Fig. 4C), the binding of MSH2/6 to the Hol75 DNA
significantly facilitated its cleavage by both enzymes as contrasted to
parallel incubations lacking MSH2/6. Under the salt conditions required
for enzyme cleavage, approximately half of the Holliday junctions would
have been complexed by MSH2/6. The effect of MSH2/6 was particularly evident when only 1 ng of T4 endo VII was present in the reaction; under these conditions, at least a 2-fold enhancement of cleavage was
observed in the presence of MSH2/6. A prior study of the binding of p53
to Holliday junctions (35) similarly revealed that, although the
junctions were hidden by a large mass of protein (as seen by EM), they
were nonetheless more sensitive to cleavage by two well characterized
junction-resolving enzymes, T4 endo VII and T7 endo I.
To follow the kinetics of junction cleavage, the Hol75 DNA was
preincubated with either no MSH2/6 or 40 heterodimers per DNA, followed
by the addition of 100 ng of T4 endo VII or T7 endo I for 15 min.
Aliquots were removed over this period and the cleavage patterns
analyzed by acrylamide gel electrophoresis. The results (Fig.
5) revealed that, for both enzymes, the
rate of cleavage of the Holliday junctions was markedly greater when
the Holliday junctions were first complexed by MSH2/6. The greatest
difference was noted with T7 endo I (Fig. 5, B and
C), where a roughly 3-fold difference in the rate of
cleavage was observed. These data suggest that MSH2/6 binds to Holliday
junctions and alters their structure in some way so as to either make
them more assessable to the Holliday junction resolution enzymes tested
or increase the activity of these enzymes on the substrate DNA.
Genetic studies demonstrating that mismatch repair proteins might
play a role in the resolution of recombination intermediates previously
led us to perform experiments demonstrating that MSH2 protein could
specifically recognize Holliday junction recombination intermediates
(22, 23, 31, 45). Because MSH2 is known to function as part of a
complex with other MutS-related proteins, we have now extended our
previous studies to the analysis of the interaction between the MSH2/6
complex and Holliday junctions. The results of these studies indicate
that the MSH2/6 complex can interact with DNA molecules containing
Holliday junctions due to a specific interaction with the Holliday
junction. Several observations support this view. First, a high
proportion of the Holliday junction-containing DNAs can be bound by
MSH2/6. Binding saturation occurs at a molar ratio of approximately 1 MSH2/6 complex per Holliday junction. This value is similar to that
obtained with a G:T mispair-containing substrate, a mispair known to be efficiently recognized by MSH2/6 in vitro and in
vivo. Second, competition experiments and equilibrium binding
experiments have shown that MSH2/6 complex has a higher affinity for
Holliday junctions than for either control duplex DNAs or DNAs
containing well recognized mispairs like G:T. Third, because the
binding experiments presented here were performed in the presence of
Mg2+, it is likely that MSH2/6 recognizes the junction
structure itself rather than interacting with unpaired bases at the
junction since all of the bases at the junction are base-paired under
these conditions (reviewed in Ref. 46). Finally, EM experiments
directly demonstrate that MSH2/6 primarily binds to the junction rather
than to other locations on junction-containing DNAs. Exactly what
structural feature of a Holliday junction MSH2/6 recognizes is unclear.
In previous experiments, MSH2 was observed to bind to oligonucleotide
duplexes containing Holliday junctions (31) in addition to its ability
to recognize mispaired based present in oligonucleotide duplexes (36,
37). In contrast, the available evidence suggests that isolated MSH6
only interacts with DNA nonspecifically (34, 38, 39). In previous
studies, MSH2 interactions with DNAs containing Holliday junctions were
analyzed using filter binding assays and EM (31), whereas in the
present studies, we used gel mobility shift assays and EM. While the
filter binding and gel mobility shift assays used were somewhat
different, the DNA binding conditions used in the two studies were
almost exactly the same and the EM experiments were conducted under the
same DNA binding conditions, allowing us to compare the Holliday
junction recognition properties of MSH2 and MSH2/6 complexes. Some
distinct differences between the proteins were observed. First, MSH2
and MSH2/6 preferentially recognized Holliday junctions compared with either duplex control DNAs or mispair-containing DNAs in competition experiments. However, saturation binding to Holliday junctions occurred
at 10-fold lower protein to DNA ratios for MSH2/6 complex compared with
MSH2. Second, MSH2 alone appears to form two types of complexes with
Holliday junctions, an unstable complex and a stable complex. In
contrast, MSH2/6 appears to be in equilibrium with Holliday junctions
in solution and only one type of complex is formed. Finally, EM
experiments demonstrate that, compared with MSH2, MSH2/6 shows a
greater specificity for binding at the junction compared with
interaction at other locations on the Holliday junction-containing
substrate. These observations indicate that not only does the presence
of the MSH6 subunit change the character of the interaction between
MSH2 and Holliday junctions, it also increases the specificity and
affinity for interaction with Holliday junctions. These data support
the idea that MSH6 modifies the intrinsic ability of MSH2 to recognize
Holliday junctions and further support the idea that the MutS family of
proteins interact with structures like Holliday junctions in
vivo.
A number of genetic studies have demonstrated that mismatch repair
proteins interact with recombination intermediates and alter their
resolution and/or processing in response to the extent of mispairing
between the recombining DNAs. One idea that has been suggested is that
proteins like MSH2 or, as shown here, MSH2/6 can coordinate the
resolution of recombination intermediates in response to mispairing by
virtue of their ability to recognize both mispaired bases and
structures like Holliday junctions (22, 31). The observation that the
binding of MSH2/6 to Holliday junctions enhanced the cleavage of these
structures suggests that the interaction between MSH2/6 and Holliday
junctions alters the structure of the Holliday junction in some way
that enhances their accessibility to the T4 and T7 endonucleases. It
was not possible to test the effect of MSH2/6 on the appropriate
eukaryotic Holliday junction resolution enzymes because the identity of
these proteins has not yet been clarified and they are not available in
pure form. However, the observation that MSH2/6 enhances the cleavage of Holliday junctions suggests that MSH2/6 and possibly other MutS-related proteins may be components of the Holliday junction resolution machinery. This suggests that MSH2/6 could be a useful reagent for use in the identification of other proteins that function in Holliday junction resolution.
Genetic and biochemical studies have documented three different
heterodimeric complexes of MutS-related proteins that function in the
nucleus in genetic recombination and in some cases mismatch repair.
These are MSH2/6, MSH2/3, and MSH4/5, the latter of which appears to
only be required for efficient meiotic crossing over (3, 5, 28, 29, 40,
41). MSH2 appears to have an intrinsic ability to recognize mispaired
bases and other structures in DNA and the MSH3 and MSH6 subunits appear
to alter the ability of MSH2 to interact with these structures as well
as possibly interact with other proteins (3). It is not known what
structures MSH4, MSH5, or the MSH4/5 complex recognize in DNA. However,
by analogy to the properties of MSH2, MSH3, and MSH6 and taking into account the genetics of MSH4 and MSH5, it seems likely that MSH4/5 also
recognizes some DNA structure involved in recombination. Such
recognition could occur in conjunction with other MSH proteins and
other mismatch repair proteins like MLH1 (30). It seems likely that it
is different DNA structure recognition properties of these complexes
and different abilities to interact with other proteins and
recombination/repair pathways that accounts for the different roles
these proteins appear to play in recombination.
INTRODUCTION
Top
Abstract
Introduction
References
MATERIALS AND METHODS
-32P]ATP and T4
polynucleotide kinase and purified by centrifugation through a
Centri-Sep column (Princeton Separations, Adelphia, NJ). DNA binding
assays were performed by incubating MSH2/6 (50 nM MSH2/6
heterodimer, unless otherwise described) with the
32P-labeled DNA substrate (10 nM, unless
otherwise described) in a final volume of 30 µl of Binding Buffer (25 mM NaCl, 20 mM Tris-Cl, pH 7.5, 1 mM DTT, 5 mM MgCl2, 100 µg/ml
bovine serum albumin) for 30 min at 20 °C. Glycerol was added to a
final concentration of 5% just prior to gel electrophoresis on 4.5%
polyacrylamide (60:1 acrylamide:bisacrylamide), 0.5× TBE (45 mM Tris borate, 1 mM EDTA, pH 8.0), 5%
glycerol for 5 h at 150 V at 4 °C. Gels were dried and
subsequently analyzed using a PhosphorImager and ImageQuant software
(Molecular Dynamics, Sunnyvale, CA).
7 torr with
tungsten. Samples were examined in a Philips CM12 instrument. Images
were taken on sheet film, scanned with a Nikon LS4500 AF film scanner,
and the contrast adjusted with Adobe Photoshop.
RESULTS
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Fig. 1.
Binding of MSH2/6 to Holliday junctions.
A, 2.5 µg of purified MSH2/6 analyzed by SDS-PAGE on a
7.5% acrylamide gel. Molecular weight markers are present in the
lane labeled MW. B, a fixed amount of
32P-labeled DNA substrate (10 nM each: J12
Holliday junction (closed circles), G:T
heteroduplex (open circles), and G:C homoduplex
(closed triangles)) was incubated with increasing
amounts of MSH2/6 (0-200 nM). The amount of bound
substrate was determined by gel mobility shift as described under
"Materials and Methods." C, a fixed amount of MSH2/6 (50 nM) was incubated with increasing amounts of
32P-labeled DNA substrate (0.5-100 nM each:
J12 Holliday junction (closed circles), and G:T
heteroduplex (open circles)).
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Fig. 2.
MSH2/6 binds specifically to Holliday
junctions. A, competition of binding of MSH2/6 to
32P-labeled G:T heteroduplex substrate (10 nM)
by increasing amounts of unlabeled competitor DNA (25-500
nM each: J12 Holliday junction (closed
circles), G:T heteroduplex (open
circles), and G:C homoduplex (closed
triangles). B, competition with additional
Holliday junction substrates at a fixed concentration of competitor
(100 nM each).
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Fig. 3.
Visualization of MSH2/6 bound to Holliday
junctions. MSH2/6 was incubated with a Holliday junction DNA
containing 565-570-bp arms in a buffer containing 40 mM
NaCl, 20 mM Hepes, pH 7.6, 0.1 mM DTT, 0.1 mM EDTA, and 40 µg/ml bovine serum albumin for 10 min at
room temperature and the complexes then treated with glutaraldehyde and
prepared for EM as described under "Materials and Methods,"
including dehydrating through an ethanol series, air-drying, and rotary
shadowcasting with tungsten at high vacuum. Shown in reverse contrast.
Bar is equal to a 500-bp segment of DNA.
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Fig. 4.
MSH 2/6 heterodimer binding to Holliday
junctions can enhance their cleavage. A,
32P-labeled Hol75 DNA (1 ng, 0.25 nM) was
incubated with 1 µl of protein storage buffer (lanes
1-5) or 1 µl of protein (40 heterodimer proteins/DNA,
lanes 6-10) in 10 µl of cleavage buffer as
described under "Materials and Methods" at room temperature for 15 min, and then incubated for an additional 15 min at room temperature
with 1 (lanes 2 and 7), 2 (lanes 3 and 8), 5 (lanes
4 and 9), or 10 (lanes 5 and 10) ng of T4 endo VII. The samples were deproteinized,
and the DNA electrophoresed on a 5% polyacrylamide gel and imaged by
autoradiography. The positions of Hol75 DNA (Hol75) and the
cleavage products (products) are indicated. B, a
parallel experiment to that described in A was carried out
using 1 (lanes 2 and 8), 2 (lanes 3 and 9), 3 (lanes
4 and 10), 4 (lanes 5 and
11), and 5 (lanes 6 and 12)
ng of T7 endo I without MSH2/6 (lanes 1-6), or
preincubated with MSH2/6 as in A (lanes
7-12). The positions of Hol75 DNA and the cleavage products
(products) are indicated. C and D, the
percentage of uncleaved DNA in the gels shown in A and
B was measured using a Molecular Dynamics PhosphorImager and
was calculated by dividing the intensity of the Hol75 DNA band by the
total intensity (Hol75 plus product).
C, data derived from the data presented in A.
D, data derived from the data presented in B. No
MSH2/6 added (open circles); with added MSH2/6
(closed circles).
View larger version (35K):
[in a new window]
Fig. 5.
Kinetics of Holliday junction cleavage.
A, Hol75 DNA (32P-labeled; 10 ng) was incubated
with protein storage buffer (lanes 1-10) or
MSH2/6 (40 heterodimers/DNA; lanes 11-20) in
cleavage buffer for 15 min followed by the addition of 100 ng of T4
endo VII in a 100-µl final volume. Aliquots (10 µl) were removed at
each time point, the samples deproteinized and electrophoresed on 5%
polyacrylamide gel, and the DNA imaged by autoradiography as described
under "Materials and Methods." The position of Hol75 DNA and the
cleavage products are indicated. B, a parallel experiment to
that presented in A was carried out using 100 ng of T7 endo
I instead of T4 endo VII. The positions of Hol75 DNA and the cleavage
products are indicated. C and D, the percentage
of uncleaved DNA in the gels shown in A and B
were measured and calculated. C, data derived from
A. D, data derived from B. With added
MSH2/6 (closed circles); no added MSH2/6
(open circles).
DISCUSSION
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ACKNOWLEDGEMENT |
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We thank Abhijit Datta for helpful comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants GM50006 (to R. D. K.) and GM31819 (to J. G.).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.
§ Supported by a Charles A. King Trust postdoctoral fellowship from the Medical Foundation, Inc.
Present address: Los Alamos National Laboratories, Los Alamos,
NM 87545.
To whom correspondence should be addressed: Ludwig Institute
for Cancer Research, University of California San Diego School of
Medicine, CMME-3080, 9500 Gilman Dr., La Jolla, CA 92093. Tel.: 619-534-7804; Fax: 619-534-7750; E-mail: rkolodner{at}ucsd.edu.
1 Edelmann, W., Cohen, P., Knetz, B., Winand, N., Heyer, J., Kolodner, R., Pollard, J. W., and Kucherlapati, R. (1999) Nat. Genet. 21, 123-127
2 G. T. Marsischky and R. D. Kolodner, submitted for publication.
4 H. Flores-Ruzas and R. D. Kolodner, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: HPLC, high performance liquid chromatography; EM, electron microscopy; DTT, dithiothreitol; bp, base pair(s); endo, endonuclease.
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REFERENCES |
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