Journal of Bacteriology, November 2001, p. 6487-6490, Vol. 183, No. 21
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.21.6487-6490.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Interaction of MutS and Vsr: Some Dominant-Negative
mutS Mutations That Disable Methyladenine-Directed Mismatch
Repair Are Active in Very-Short-Patch Repair
Margaret
Lieb,1
Shehnaz
Rehmat,1 and
Ashok S.
Bhagwat2,*
Department of Molecular Microbiology and
Immunology, Keck School of Medicine, University of Southern California,
Los Angeles, California 900331, and
Department of Chemistry, Wayne State University, Detroit,
Michigan 482022
Received 19 April 2001/Accepted 27 July 2001
 |
ABSTRACT |
In Escherichia coli and related bacteria, the
very-short-patch (VSP) repair pathway uses an endonuclease, Vsr, to
correct T · G mismatches that result from the deamination of
5-methylcytosines in DNA to C · G. The products of
mutS and mutL, which are required for adenine
methylation-directed mismatch repair (MMR), enhance VSP repair.
Multicopy plasmids carrying mutS alleles that are dominant
negative for MMR were tested for their effects on VSP repair. Some
mutS mutations (class I) did not lower VSP repair in a
mutS+ background, and most class I mutations
increased VSP repair in mutS cells more than plasmids
containing mutS+. Other plasmid-borne
mutS mutations (class II) and mutS+
decreased VSP repair in the mutS+ background.
Thus, MutS protein lacking functions required for MMR can still
participate in VSP repair, and our results are consistent with a model
in which MutS binds transiently to the mispair and then translocates
away from the mispair to create a specialized structure that enhances
the binding of Vsr.
| |
TEXT |
The product of gene vsr,
which is present in several species of enteric bacteria, is a
sequence-specific endonuclease (reviewed in reference 14).
Vsr recognizes T · G mismatches in DNA that arise as a result of
deamination of 5-methylcytosine in the sequence 5'-CmeC(A or T)GG (meC,
5-methylcytosine) and nicks 5' to the mismatched T. Following nicking,
replacement of T with C is accomplished by DNA polymerase I and DNA
ligase. T · G mispairs in related contexts are also subject to
very-short-patch (VSP) repair but at a lower frequency
(15). The removal of the mispaired T by VSP repair is
enhanced by MutS and MutL (8, 11), although these products
are not required if Vsr is supplied by a multicopy plasmid
(15). MutS, on the other hand, binds to most mismatched base pairs, with highest affinity for T · G and C · A
(20). MutL interacts with MutS, and the MutS-MutL complex
initiates repair of replication errors by stimulating nicking of one
DNA strand by the MutH protein (2, 7). The strand
discrimination signal for nicking is adenine methylation within
5'-GATC sites; hence, this is referred to as
methyladenine-directed mismatch repair (MMR) (reviewed in reference
18). We describe below experiments in which the effects of
multicopy plasmids carrying either wild-type MutS or mutant versions of
MutS on VSP repair were studied.
Overproduction of MutS+ is ineffective in restoring VSP
repair in mutS and mutL bacteria.
The
presence of a mutS+ plasmid reduces the
frequency of VSP repair in mutS+
mutL+ bacteria (Table
1, and reference 15). In
contrast, introducing a mutS+ plasmid into cells
containing chromosomal mutS+ does not disrupt
MMR (24). It was also shown that in strain GM31,
which is deficient in Vsr, excess MutS essentially abolishes VSP repair
(15) (Table 1). We have now found that a
mutS+ plasmid increased VSP repair only
marginally in a mutS strain (Table 1.) In a mutL
background, the amount of repair in the presence of plasmid
pMutS+ was substantially lower than when MutS was produced
solely by chromosomal mutS+ (Table 1). These
results suggest that MutL and MutS serve distinct functions in
promoting VSP repair and that the absence of MutL in cells cannot be
compensated for by increasing amounts of MutS. Furthermore, just as
overproduction of Vsr disrupts MMR (3, 16), overproduction
of MutS disrupts VSP repair, suggesting that both proteins must be
present in cells at optimal levels for both mismatch repair processes
to minimize mutations.
Dominant-negative mutS mutations differ with respect to
their effect on VSP repair.
Wu and Marinus (24)
isolated on multicopy plasmids a series of dominant mutS
mutations proteins that interfere with MMR. Most of these
plasmids produce a high level of spontaneous mutations when they are
introduced into mutS+ cells. In Table
2, the dominant-negative mutations of
MutS whose sites are known (24) are listed in the order of
their positions in the mutS gene. To elucidate the role of
MutS in VSP repair, we tested a number of these dominant-negative
mutant proteins for their effects on VSP repair in mutS and
mutS+ bacteria. It should be noted that none of
the mutS plasmids affected phage 
recombination in the
VSP repair assay.
The mutations can be placed into two classes based on their effects on
VSP repair in mutS+ bacteria. Class I includes
two mutations in the 5' half of mutS and several mutations
at the 3' end. In the mutS+ background, plasmids
with class I mutations either did not reduce VSP repair significantly
or produced slightly more repair than was obtained in the absence of a
mutS plasmid. This finding contrasts with the reduction in
VSP repair caused by the mutS+ plasmid in
mutS+ cells (Table 1 and reference
15). In mutS bacteria, plasmids with some of
the class I mutations increased VSP repair significantly more than
mutS+ plasmids. Notably, mutations at amino acid
(aa) 621 or 669 raised cI+ recombinant
frequencies to values comparable to those obtained in
mutS+ bacteria. We suggest that class I
mutations result in a product that retains the ability to facilitate
the binding of a T · G mismatch by Vsr but that is deficient in
another MMR function(s) not required by VSP repair.
In contrast, class II mutations strongly inhibited VSP repair. In phage
crosses made in bacteria carrying plasmids containing class II
mutations, the frequency of cI+ recombinants was
as low as 0.16% (G619D mutation). A frequency of approximately 0.15%
cI+ is expected in the absence of any VSP
repair. Thus, class II mutations have a dominant-negative phenotype for
VSP repair as well as for MMR and some class II mutations eliminate VSP
repair completely.
Overproduction of Vsr increases VSP repair in the presence of
dominant-negative mutS alleles.
It has been shown that
the frequency of VSP repair is increased when excess Vsr is supplied
(13, 15). Therefore, we expected that the addition of a
vsr+ plasmid to bacteria carrying plasmids with
dominant-negative mutS mutations might also increase VSP
repair. In the case of crosses in which one parent carries the
cI mutation am6, which is in the optimum context
for VSP repair, excess Vsr increased VSP repair in bacteria carrying
any of the plasmids with the dominant-negative mutS
mutations tested (Table 3). These results
are consistent with the idea that MutS enhances the binding of Vsr to
the mispair and that a defect in the ability of MutS to perform this
function can be compensated for by increasing the cellular
concentration of Vsr. Similar results were obtained in crosses to test
the VSP repair of cI mutation am9, which produces
a T · G mispair in a context that is less favorable for VSP
repair (5'-GTAGG). However, in bacteria with plasmids having
mutS mutations at aa 619 or 668, excess Vsr did not improve
VSP repair at am9 significantly. It is possible that these
mutations result in a MutS protein that competes very strongly with
Vsr, particularly for mispairs not in the optimum context for Vsr
binding.
What is the role of MutS in VSP repair?
The role played by
MutS in MMR is controversial. A variety of models have been proposed
for the MutS function, some of which suggest that MutS remains bound to
the mispair during its activation of nicking at 5'-GATC
sites by MutH (9, 10, 17). Others have suggested
that MutS leaves the mismatch following initial binding (1, 5,
6). Specifically, Allen et al. (1) suggested that
MutS mediates the formation of a DNA loop with the mispair at its apex.
Loop formation depends on the hydrolysis of ATP. MutL assists in this
reaction but is not necessary for it.
The crystal structure of Vsr complexed with DNA shows that Vsr
intercalates several of its residues into the DNA helix, deforming base
stacking and widening both the major and the minor groove (22). If the models of the crystal structures of both Vsr
(22) and MutS (9, 10) bound to DNA are
accurate, it is impossible to visualize simultaneous binding of Vsr and
MutS at a T · G mismatch. This means that, at least in VSP
repair, MutS must leave the mismatch to allow the binding of Vsr.
Similarly, a model in which MutS simply translocates away from the
mismatch following an ADP-to-ATP exchange (5, 6) does not
explain the beneficial effects of MutS on VSP repair. We are forced to
conclude that MutS leaves the mismatch following initial binding,
leaving behind a specialized structure that is conductive to binding by
Vsr. Creating an underwound DNA loop is one such possible way in which
MutS could promote Vsr binding to T · G, and Tsutakawa et al.
(22) previously proposed such a role for MutS in VSP repair.
We propose further that the products of dominant-negative
mutS mutants that have the ability to move the T · G
mismatch into a loop but are defective in some subsequent step in MMR
retain VSP repair-enhancing activity (class I mutants). However,
mutants whose products bind to the mismatch in an irreversible manner prevent access to the mismatch by Vsr and have a dominant-negative effect on VSP repair (class II). Although a majority of class I
mutations lie near the 3' end of mutS and fewer class II
mutations lie in this part of the gene, there is no simple correlation
between the positions of the mutS mutations and the class to
which they belong. The two classes are intermixed in terms of their
positions in the gene, and mutations in adjacent or nearby residues
often belong to different classes. This is dramatically seen at aa 117, where, depending on the substitution, the mutations can be class I or
II (Table 2).
The distribution of all tested dominant-negative mutations along the
crystal structure of MutS shows that they line the surface of the clamp
within the dimer (Fig. 1). They are seen
to cluster in two groups, with those of one group surrounding the bound
ADP molecule and with those of the other residing in a segment that connects the DNA-binding domain with the ATP-binding domain. These mutations may affect ATP binding, ATP hydrolysis, the communication between the binding sites for ATP and DNA, or the interaction between
MutS and MutL. Therefore, biochemical work is necessary to identify the
functional defects in these mutant proteins.
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FIG. 1.
The figure was drawn using coordinates kindly provided
by T. Sixma (Netherlands Cancer Institute, Amsterdam, The Netherlands)
for E. coli MutS bound to DNA containing a T · G
mismatch (10). The color scheme is as follows: gray
indicates the MutS protein, green ribbons are DNA, and yellow indicates
ADP. Residues in which class I mutations lie are shown in light blue,
while residues for class II mutations are shown in purple. Mutations in
residue 117 can either be class I or II (see the text), and this
residue is shown in red.
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|
To explain the enhancement of VSP repair in mutS bacteria by
some of the mutS plasmids, one can suppose that the
mutations affect regions of the protein where MutS normally interacts
with MutL. Consequently, these plasmid increase the amount of
MutL available to enhance Vsr binding to the mismatch. There is
evidence that MutL can interact with MutS that is not DNA bound
(23). It has been pointed out that in both wild-type and
mutL bacteria, overproduction of MutS+ reduced
VSP repair. Thus, the class I mutS mutations may fail to
reduce the amount of available MutL. The dominant-negative effect on
MMR would be attributable to the failure of MutS to interact with MutL,
an action which is required to activate MutH. It should be noted that
the mutagenic phenotype produced by the mutS mutations that
strongly enhance VSP repair in mutS bacteria is complemented
by MutL+ or by MutH+ (24).
In conclusion, our findings are compatible with the following model:
MutS binds to a T · G mispair and translocates the mispair into
a specialized DNA structure such as a loop. The affinity of Vsr for the
mismatch is increased when the mismatch is present in a loop. The
ability of Vsr to interfere with MMR (3, 16) suggests a
direct interaction of Vsr with an MMR protein. Complementation of the
mutagenic effects of Vsr overproduction by MutL and MutH, but not MutS,
indicates that Vsr can reduce the MutL available for MMR, presumably by
forming a complex that can help Vsr bind to its specific mismatch
(4). It is possible that MutH and Vsr compete for the same
site on MutL; hence, increasing the amount of MutH in cells can
overcome the mutagenic effects of Vsr. Thus, MutS and MutL may enhance
VSP repair by independent mechanisms, MutS by bending DNA and MutL by
forming a transient complex with Vsr.
| |
ACKNOWLEDGMENTS |
We thank M. Marinus, M. Radman, J. P. Claverys, and P. Modrich
for providing bacterial strains or plasmids.
The work in the laboratory of A.S.B. is supported by National
Institutes of Health grant GM 53273.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: 463 Chemistry
Building, Department of Chemistry, Wayne State University, Detroit, MI 48202. Phone: (313) 577-2547, Fax: (313) 577-8822. E-mail:
axb{at}chem.wayne.edu.
| |
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Journal of Bacteriology, November 2001, p. 6487-6490, Vol. 183, No. 21
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.21.6487-6490.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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