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1.  The Role of Nucleotide Cofactor Binding in Cooperativity and Specificity of MutS Recognition 
Journal of molecular biology  2008;384(1):31-47.
Mismatch repair (MMR) is essential for eliminating biosynthetic errors generated during replication or genetic recombination in virtually all organisms. The critical first step in Escherichia coli MMR is the specific recognition and binding of MutS to a heteroduplex, either containing a mismatch or an insertion/deletion loop of up to four nucleotides. All known MutS homologs recognize a similar broad spectrum of substrates. Binding and hydrolysis of nucleotide cofactors by the MutS-heteroduplex complex is required for downstream MMR activity, although the exact role of the nucleotide cofactors is less clear. Here we showed that MutS bound to a 30-bp heteroduplex containing an unpaired-T with a binding affinity ≈ 400-fold stronger than to a 30-bp homoduplex, a much higher specificity than previously reported. The binding of nucleotide cofactors decreased both MutS specific and non-specific binding affinity, with the later marked by a larger drop, further increasing MutS specificity by ≈ 3-fold. Kinetic studies showed that the difference in MutS KD for various heteroduplexes was attributable to the difference in intrinsic dissociation rate of a particular MutS-heteroduplex complex. Furthermore, the kinetic association event of MutS binding to heteroduplexes was marked by positive cooperativity. Our studies showed that the positive cooperativity in MutS binding was modulated by the binding of nucleotide cofactors. The binding of nucleotide cofactors transformed E. coli MutS tetramers, the functional unit in E. coli MMR, from a cooperative to a non-cooperative binding form. Finally, we found that E. coli MutS bound to single-strand DNA with significant affinity, which could have important implication for strand discrimination in eukaryotic MMR mechanism.
doi:10.1016/j.jmb.2008.08.052
PMCID: PMC2666446  PMID: 18773911
MMR; MutS specificity; binding cooperativity; binding kinetics; nucleotide cofactors
2.  Different Roles of Eukaryotic MutS and MutL Complexes in Repair of Small Insertion and Deletion Loops in Yeast 
PLoS Genetics  2013;9(10):e1003920.
DNA mismatch repair greatly increases genome fidelity by recognizing and removing replication errors. In order to understand how this fidelity is maintained, it is important to uncover the relative specificities of the different components of mismatch repair. There are two major mispair recognition complexes in eukaryotes that are homologues of bacterial MutS proteins, MutSα and MutSβ, with MutSα recognizing base-base mismatches and small loop mispairs and MutSβ recognizing larger loop mispairs. Upon recognition of a mispair, the MutS complexes then interact with homologues of the bacterial MutL protein. Loops formed on the primer strand during replication lead to insertion mutations, whereas loops on the template strand lead to deletions. We show here in yeast, using oligonucleotide transformation, that MutSα has a strong bias toward repair of insertion loops, while MutSβ has an even stronger bias toward repair of deletion loops. Our results suggest that this bias in repair is due to the different interactions of the MutS complexes with the MutL complexes. Two mutants of MutLα, pms1-G882E and pms1-H888R, repair deletion mispairs but not insertion mispairs. Moreover, we find that a different MutL complex, MutLγ, is extremely important, but not sufficient, for deletion repair in the presence of either MutLα mutation. MutSβ is present in many eukaryotic organisms, but not in prokaryotes. We suggest that the biased repair of deletion mispairs may reflect a critical eukaryotic function of MutSβ in mismatch repair.
Author Summary
DNA mismatch repair is a major pathway that prevents both base substitution and insertion or deletion errors during replication. Most eukaryotes have two recognition complexes, MutSα and MutSβ, homologues of prokaryotic MutS and differing in their affinity for mismatches, with MutSα recognizing base-base mismatches and small insertion/deletion loops and MutSβ recognizing larger loops. We show that repair mediated by these complexes has opposite biases for insertion versus deletion mispairs with MutSα-directed repair favoring insertion loops and MutSβ-directed repair favoring deletion loops. This bias is mediated by differing interactions with downstream MutL complexes. We suggest that MutSα represents a prokaryotic MutS biased for repair of insertion loops and that MutSβ represents a new eukaryotic activity biased for repair of deletion loops.
doi:10.1371/journal.pgen.1003920
PMCID: PMC3814323  PMID: 24204320
3.  Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA 
Nucleic Acids Research  2012;40(12):5448-5464.
Mismatch repair (MMR) corrects replication errors such as mismatched bases and loops in DNA. The evolutionarily conserved dimeric MMR protein MutS recognizes mismatches by stacking a phenylalanine of one subunit against one base of the mismatched pair. In all crystal structures of G:T mismatch-bound MutS, phenylalanine is stacked against thymine. To explore whether these structures reflect directional mismatch recognition by MutS, we monitored the orientation of Escherichia coli MutS binding to mismatches by FRET and anisotropy with steady state, pre-steady state and single-molecule multiparameter fluorescence measurements in a solution. The results confirm that specifically bound MutS bends DNA at the mismatch. We found additional MutS–mismatch complexes with distinct conformations that may have functional relevance in MMR. The analysis of individual binding events reveal significant bias in MutS orientation on asymmetric mismatches (G:T versus T:G, A:C versus C:A), but not on symmetric mismatches (G:G). When MutS is blocked from binding a mismatch in the preferred orientation by positioning asymmetric mismatches near the ends of linear DNA substrates, its ability to authorize subsequent steps of MMR, such as MutH endonuclease activation, is almost abolished. These findings shed light on prerequisites for MutS interactions with other MMR proteins for repairing the appropriate DNA strand.
doi:10.1093/nar/gks138
PMCID: PMC3384296  PMID: 22367846
4.  Nuclease activity of the MutS homologue MutS2 from Thermus thermophilus is confined to the Smr domain 
Nucleic Acids Research  2007;35(3):850-860.
MutS homologues are highly conserved enzymes engaged in DNA mismatch repair (MMR), meiotic recombination and other DNA modifications. Genome sequencing projects have revealed that bacteria and plants possess a MutS homologue, MutS2. MutS2 lacks the mismatch-recognition domain of MutS, but contains an extra C-terminal region called the small MutS-related (Smr) domain. Sequences homologous to the Smr domain are annotated as ‘proteins of unknown function’ in various organisms ranging from bacteria to human. Although recent in vivo studies indicate that MutS2 plays an important role in recombinational events, there had been only limited characterization of the biochemical function of MutS2 and the Smr domain. We previously established that Thermus thermophilus MutS2 (ttMutS2) possesses endonuclease activity. In this study, we report that a Smr-deleted ttMutS2 mutant retains the dimerization, ATPase and DNA-binding activities, but has no endonuclease activity. Furthermore, the Smr domain alone was stable and functional in binding and incising DNA. It is noteworthy that an endonuclease activity is associated with a MutS homologue, which is generally thought to recognize specific DNA structures.
doi:10.1093/nar/gkl735
PMCID: PMC1807967  PMID: 17215294
5.  Binding of MutS protein to oligonucleotides containing a methylated or an ethylated guanine residue, and correlation with mutation frequency 
Mutation Research  2008;640(1-2):107-112.
The MutS-based mismatch repair (MMR) system has been conserved from prokaryotes to humans, and plays important roles in maintaining the high fidelity of genomic DNA. MutS protein recognizes several different types of modified base pairs, including methylated guanine-containing base pairs. Here, we looked at the relationship between recognition and the effects of methylating versus ethylating agents on mutagenesis, using a MutS-deficient strain of E. coli. We find that while methylating agents induce mutations more effectively in a MutS-deficient strain than in wild-type, this genetic background does not affect mutagenicity by ethylating agents. Thus, the role of E. coli MMR with methylation-induced mutagenesis appears to be greater than ethylation-induced mutagenesis. To further understand this difference an early step of repair was examined with these alkylating agents. A comparison of binding affinities of MutS with O6-alkylated guanine base paired with thymine, which could lead to transition mutations, versus cytosine which could not, was tested. Moreover, we compared binding of MutS to oligoduplexes containing different base pairs; namely, O6-MeG:T, O6-MeG:C, O6-EtG:T, O6-EtG:C, G:T and G:C. Dissociation constants (Kd), which reflect the strength of binding, followed the order G:T- > O6-MeG:T- > O6-EtG:T- = O6-EtG:C- ≥ O6-MeG:C- > G:C. These results suggest that a thymine base paired with O6-methyl guanine is specifically recognized by MutS and therefore should be removed more efficiently than a thymine opposite O6-ethylated guanine. Taken together, the data suggest that in E. coli, the MMR system plays a more significant role in repair of methylation-induced lesions than those caused by ethylation.
doi:10.1016/j.mrfmmm.2007.12.009
PMCID: PMC2365708  PMID: 18243250
MutS; Mismatch repair; O6-methylguanine; O6-ethylguanine; Mutation
6.  A phylogenomic study of the MutS family of proteins. 
Nucleic Acids Research  1998;26(18):4291-4300.
The MutS protein of Escherichia coli plays a key role in the recognition and repair of errors made during the replication of DNA. Homologs of MutS have been found in many species including eukaryotes, Archaea and other bacteria, and together these proteins have been grouped into the MutS family. Although many of these proteins have similar activities to the E.coli MutS, there is significant diversity of function among the MutS family members. This diversity is even seen within species; many species encode multiple MutS homologs with distinct functions. To better characterize the MutS protein family, I have used a combination of phylogenetic reconstructions and analysis of complete genome sequences. This phylogenomic analysis is used to infer the evolutionary relationships among the MutS family members and to divide the family into subfamilies of orthologs. Analysis of the distribution of these orthologs in particular species and examination of the relationships within and between subfamilies is used to identify likely evolutionary events (e.g. gene duplications, lateral transfer and gene loss) in the history of the MutS family. In particular, evidence is presented that a gene duplication early in the evolution of life resulted in two main MutS lineages, one including proteins known to function in mismatch repair and the other including proteins known to function in chromosome segregation and crossing-over. The inferred evolutionary history of the MutS family is used to make predictions about some of the uncharacterized genes and species included in the analysis. For example, since function is generally conserved within subfamilies and lineages, it is proposed that the function of uncharacterized proteins can be predicted by their position in the MutS family tree. The uses of phylogenomic approaches to the study of genes and genomes are discussed.
PMCID: PMC147835  PMID: 9722651
7.  Structural and functional analysis of the MutS C-terminal tetramerization domain 
Nucleic Acids Research  2006;34(18):5270-5279.
The Escherichia coli DNA mismatch repair (MMR) protein MutS is essential for the correction of DNA replication errors. In vitro, MutS exists in a dimer/tetramer equilibrium that is converted into a monomer/dimer equilibrium upon deletion of the C-terminal 53 amino acids. In vivo and in vitro data have shown that this C-terminal domain (CTD, residues 801–853) is critical for tetramerization and the function of MutS in MMR and anti-recombination. We report the expression, purification and analysis of the E.coli MutS-CTD. Secondary structure prediction and circular dichroism suggest that the CTD is folded, with an α-helical content of 30%. Based on sedimentation equilibrium and velocity analyses, MutS-CTD forms a tetramer of asymmetric shape. A single point mutation (D835R) abolishes tetramerization but not dimerization of both MutS-CTD and full-length MutS. Interestingly, the in vivo and in vitro MMR activity of MutSCF/D835R is diminished to a similar extent as a truncated MutS variant (MutS800, residues 1–800), which lacks the CTD. Moreover, the dimer-forming MutSCF/D835R has comparable DNA binding affinity with the tetramer-forming MutS, but is impaired in mismatch-dependent activation of MutH. Our data support the hypothesis that tetramerization of MutS is important but not essential for MutS function in MMR.
doi:10.1093/nar/gkl489
PMCID: PMC1636413  PMID: 17012287
8.  Molecular Cloning and Functional Analysis of the MutY Homolog of Deinococcus radiodurans 
Journal of Bacteriology  2001;183(21):6151-6158.
The mutY homolog gene (mutYDr) from Deinococcus radiodurans encodes a 39.4-kDa protein consisting of 363 amino acids that displays 35% identity to the Escherichia coli MutY (MutYEc) protein. Expressed MutYDr is able to complement E. coli mutY mutants but not mutM mutants to reduce the mutation frequency. The glycosylase and binding activities of MutYDr with an A/G-containing substrate are more sensitive to high salt and EDTA concentrations than the activities with an A/7,8-dihydro-8-oxoguanine (GO)-containing substrate are. Like the MutYEc protein, purified recombinant MutYDr expressed in E. coli has adenine glycosylase activity with A/G, A/C, and A/GO mismatches and weak guanine glycosylase activity with a G/GO mismatch. However, MutYDr exhibits limited apurinic/apyrimidinic lyase activity and can form only weak covalent protein-DNA complexes in the presence of sodium borohydride. This may be due to an arginine residue that is present in MutYDr at the position corresponding to the position of MutYEc Lys142, which forms the Schiff base with DNA. The kinetic parameters of MutYDr are similar to those of MutYEc. Although MutYDr has similar substrate specificity and a binding preference for an A/GO mismatch over an A/G mismatch, as MutYEc does, the binding affinities for both mismatches are slightly lower for MutYDr than for MutYEc. Thus, MutYDr can protect the cell from GO mutational effects caused by ionizing radiation and oxidative stress.
doi:10.1128/JB.183.21.6151-6158.2001
PMCID: PMC100089  PMID: 11591657
9.  Stoichiometry of MutS and MutL at unrepaired mismatches in vivo suggests a mechanism of repair 
Nucleic Acids Research  2012;40(9):3929-3938.
Mismatch repair (MMR) is an evolutionarily conserved DNA repair system, which corrects mismatched bases arising during DNA replication. MutS recognizes and binds base pair mismatches, while the MutL protein interacts with MutS–mismatch complex and triggers MutH endonuclease activity at a distal-strand discrimination site on the DNA. The mechanism of communication between these two distal sites on the DNA is not known. We used functional fluorescent MMR proteins, MutS and MutL, in order to investigate the formation of the fluorescent MMR protein complexes on mismatches in real-time in growing Escherichia coli cells. We found that MutS and MutL proteins co-localize on unrepaired mismatches to form fluorescent foci. MutL foci were, on average, 2.7 times more intense than the MutS foci co-localized on individual mismatches. A steric block on the DNA provided by the MutHE56A mutant protein, which binds to but does not cut the DNA at the strand discrimination site, decreased MutL foci fluorescence 3-fold. This indicates that MutL accumulates from the mismatch site toward strand discrimination site along the DNA. Our results corroborate the hypothesis postulating that MutL accumulation assures the coordination of the MMR activities between the mismatch and the strand discrimination site.
doi:10.1093/nar/gkr1298
PMCID: PMC3351158  PMID: 22241777
10.  Physical and Functional Interactions between Escherichia coli MutY Glycosylase and Mismatch Repair Protein MutS▿ †  
Journal of Bacteriology  2006;189(3):902-910.
Escherichia coli MutY and MutS increase replication fidelity by removing adenines that were misincorporated opposite 7,8-dihydro-8-oxo-deoxyguanines (8-oxoG), G, or C. MutY DNA glycosylase removes adenines from these mismatches through a short-patch base excision repair pathway and thus prevents G:C-to-T:A and A:T-to-G:C mutations. MutS binds to the mismatches and initiates the long-patch mismatch repair on daughter DNA strands. We have previously reported that the human MutY homolog (hMYH) physically and functionally interacts with the human MutS homolog, hMutSα (Y. Gu et al., J. Biol. Chem. 277:11135-11142, 2002). Here, we show that a similar relationship between MutY and MutS exists in E. coli. The interaction of MutY and MutS involves the Fe-S domain of MutY and the ATPase domain of MutS. MutS, in eightfold molar excess over MutY, can enhance the binding activity of MutY with an A/8-oxoG mismatch by eightfold. The MutY expression level and activity in mutS mutant strains are sixfold and twofold greater, respectively, than those for the wild-type cells. The frequency of A:T-to-G:C mutations is reduced by two- to threefold in a mutS mutY mutant compared to a mutS mutant. Our results suggest that MutY base excision repair and mismatch repair defend against the mutagenic effect of 8-oxoG lesions in a cooperative manner.
doi:10.1128/JB.01513-06
PMCID: PMC1797285  PMID: 17114250
11.  Structural and Functional Divergence of MutS2 from Bacterial MutS1 and Eukaryotic MSH4-MSH5 Homologs†  
Journal of Bacteriology  2005;187(10):3528-3537.
MutS homologs, identified in nearly all bacteria and eukaryotes, include the bacterial proteins MutS1 and MutS2 and the eukaryotic MutS homologs 1 to 7, and they often are involved in recognition and repair of mismatched bases and small insertion/deletions, thereby limiting illegitimate recombination and spontaneous mutation. To explore the relationship of MutS2 to other MutS homologs, we examined conserved protein domains. Fundamental differences in structure between MutS2 and other MutS homologs suggest that MutS1 and MutS2 diverged early during evolution, with all eukaryotic homologs arising from a MutS1 ancestor. Data from MutS1 crystal structures, biochemical results from MutS2 analyses, and our phylogenetic studies suggest that MutS2 has functions distinct from other members of the MutS family. A mutS2 mutant was constructed in Helicobacter pylori, which lacks mutS1 and mismatch repair genes mutL and mutH. We show that MutS2 plays no role in mismatch or recombinational repair or deletion between direct DNA repeats. In contrast, MutS2 plays a significant role in limiting intergenomic recombination across a range of donor DNA tested. This phenotypic analysis is consistent with the phylogenetic and biochemical data suggesting that MutS1 and MutS2 have divergent functions.
doi:10.1128/JB.187.10.3528-3537.2005
PMCID: PMC1112012  PMID: 15866941
12.  Asymmetric ATP Binding and Hydrolysis Activity of the Thermus aquaticus MutS Dimer Is Key to Modulation of Its Interactions with Mismatched DNA† 
Biochemistry  2004;43(41):13115-13128.
Prokaryotic MutS and eukaryotic Msh proteins recognize base pair mismatches and insertions or deletions in DNA and initiate mismatch repair. These proteins function as dimers (and perhaps higher order oligomers) and possess an ATPase activity that is essential for DNA repair. Previous studies of Escherichia coli MutS and eukaryotic Msh2–Msh6 proteins have revealed asymmetry within the dimer with respect to both DNA binding and ATPase activities. We have found the Thermus aquaticus MutS protein amenable to detailed investigation of the nature and role of this asymmetry. Here, we show that (a) in a MutS dimer one subunit (S1) binds nucleotide with high affinity and the other (S2) with 10-fold weaker affinity, (b) S1 hydrolyzes ATP rapidly while S2 hydrolyzes ATP at a 30–50-fold slower rate, (c) mismatched DNA binding to MutS inhibits ATP hydrolysis at S1 but slow hydrolysis continues at S2, and (d) interaction between mismatched DNA and MutS is weakened when both subunits are occupied by ATP but remains stable when S1 is occupied by ATP and S2 by ADP. These results reveal key MutS species in the ATPase pathway; S1ADP–S2ATP is formed preferentially in the absence of DNA or in the presence of fully matched DNA, while S1ATP–S2ATP and S1ATP–S2ADP are formed preferentially in the presence of mismatched DNA. These MutS species exhibit differences in interaction with mismatched DNA that are likely important for the mechanism of MutS action in DNA repair.
doi:10.1021/bi049010t
PMCID: PMC2839884  PMID: 15476405
13.  Two new subfamilies of DNA mismatch repair proteins (MutS) specifically abundant in the marine environment 
The ISME Journal  2011;5(7):1143-1151.
MutS proteins are ubiquitous in cellular organisms and have important roles in DNA mismatch repair or recombination. In the virus world, the amoeba-infecting Mimivirus, as well as the recently sequenced Cafeteria roenbergensis virus are known to encode a MutS related to the homologs found in octocorals and ɛ-proteobacteria. To explore the presence of MutS proteins in other viral genomes, we performed a genomic survey of four giant viruses (‘giruses') (Pyramimonas orientalis virus (PoV), Phaeocystis pouchetii virus (PpV), Chrysochromulina ericina virus (CeV) and Heterocapsa circularisquama DNA virus (HcDNAV)) that infect unicellular marine algae. Our analysis revealed the presence of a close homolog of Mimivirus MutS in all the analyzed giruses. These viral homologs possess a specific domain structure, including a C-terminal HNH-endonuclease domain, defining the new MutS7 subfamily. We confirmed the presence of conserved mismatch recognition residues in all members of the MutS7 subfamily, suggesting their role in DNA mismatch repair rather than DNA recombination. PoV and PpV were found to contain an additional type of MutS, which we propose to call MutS8. The MutS8 proteins in PoV and PpV were found to be closely related to homologs from ‘Candidatus Amoebophilus asiaticus', an obligate intracellular amoeba-symbiont belonging to the Bacteroidetes. Furthermore, our analysis revealed that MutS7 and MutS8 are abundant in marine microbial metagenomes and that a vast majority of these environmental sequences are likely of girus origin. Giruses thus seem to represent a major source of the underexplored diversity of the MutS family in the microbial world.
doi:10.1038/ismej.2010.210
PMCID: PMC3146287  PMID: 21248859
mimivirus; girus; virus; DNA repair; MutS
14.  Saccharomyces cerevisiae MutLα IS A MISMATCH REPAIR ENDONUCLEASE* 
The Journal of biological chemistry  2007;282(51):37181-37190.
MutL homologs are crucial for mismatch repair and genetic stability, but their function is not well understood. Human MutLα (MLH1-PMS2 heterodimer) harbors a latent endonuclease that is dependent on integrity of a PMS2 DQHA(X)2E(X)4E motif (Kadyrov et al. (2006) Cell 126, 297-308). This sequence element is conserved in many MutL homologs, including the PMS1 subunit of Saccharomyces cerevisiae MutLα, but is absent in MutL proteins from bacteria like Escherichia coli that rely on d(GATC) methylation for strand directionality. We show that yeast MutLα is a strand-directed endonuclease that incises DNA in a reaction that depends on a mismatch, yMutSα, yRFC, yPCNA, ATP, and a pre-existing strand break, whereas E. coli MutL is not. Amino acid substitution within the PMS1 DQHA(X)2E(X)4E motif abolishes yMutLα endonuclease activity in vitro and confers strong genetic instability in vivo, but does not affect yMutLα ATPase activity or the ability of the protein to support assembly of the yMutLα•yMutSα•heteroduplex ternary complex. The loaded form of yPCNA may play an important effector role in directing yMutLα incision to the discontinuous strand of a nicked heteroduplex.
doi:10.1074/jbc.M707617200
PMCID: PMC2302834  PMID: 17951253
15.  Depletion of the cellular amounts of the MutS and MutH methyl-directed mismatch repair proteins in stationary-phase Escherichia coli K-12 cells. 
Journal of Bacteriology  1996;178(8):2388-2396.
The MutL, MutS, and MutH proteins mediate methyl-directed mismatch (MDM) repair and help to maintain chromosome stability in Escherichia coli. We determined the amounts of the MDM repair proteins in exponentially growing, stationary-phase, and nutrient-starved bacteria by quantitative Western immunoblotting. Extracts of null mutants containing various amounts of purified MDM repair proteins were used as quantitation standards. In bacteria growing exponentially in enriched minimal salts-glucose medium, about 113 MutL dimers, 186 MutS dimers, and 135 MutH monomers were present per cell. Calculations with the in vitro dissociation constants of MutS binding to different mismatches suggested that MutS is not present in excess, and may be nearly limiting in some cases, for MDM repair in exponentially growing cells. Remarkably, when bacteria entered late stationary phase or were deprived of a utilizable carbon source for several days, the cellular amount of MutS dropped at least 10-fold and became barely detectable by the methods used. In contrast, the amount of MutH dropped only about threefold and the amount of MutL remained essentially constant in late-stationary-phase and carbon-starved cells compared with those in exponentially growing bacteria. RNase T2 protection assays showed that the amounts of mutS, mutH, and mutL, but not miaA, transcripts decreased to undetectable levels in late-stationary-phase cells. These results suggested that depletion of MutS in nutritionally stressed cells was possibly caused by the relative instability of MutS compared with MutL and MutH. Our findings suggest that the MDM repair capacity is repressed in nutritionally stressed bacteria and correlate with conclusions from recent studies of adaptive mutagenesis. On the other hand, we did not detect induction of MutS or MutL in cells containing stable mismatches in multicopy single-stranded DNA encoded by bacterial retrons.
PMCID: PMC177950  PMID: 8636043
16.  Binding of MutS protein to oligonucleotides containing a methylated or an ethylated guanine residue, and correlation with mutation frequency 
Mutation research  2007;640(1-2):107-112.
The MutS-based mismatch repair (MMR) system has been conserved from prokaryotes to humans, and plays important roles in maintaining the high fidelity of genomic DNA. MutS protein recognizes several different types of damaged base pairs, including methylated guanine-containing base pairs. Here, we looked at the relationship between MutS and the effects of methylating versus ethylating agents on mutagenesis, using a MutS deficient strain of E. coli. We found that methylating agents induce mutations more effectively in a MutS-deficient strain than in wild-type, whereas the genetic background did not affect mutagenicity of ethylating agents. Thus, the role of the MMR system in repair with methylation-induced mutagenesis appears to be greater than its role with ethylation-induced mutagenesis. To further understand E. coli MutS, we compared binding of the protein to O6-alkylated guanine paired with thymine, which could lead to mutation, versus cytosine, which cannot. Moreover, we compared binding of MutS to oligoduplexes containing different base pairs; namely, O6-MeG:T, O6-MeG:C, O6-EtG:T, O6-EtG:C, G:T and G:C. The dissociation constants (Kd's), which reflect the strength of binding, followed the order G:T- > O6-MeG:T- > O6-EtG:T- = O6-EtG:C- ≥ O6-MeG:C- > G:C. These results suggest that a thymine paired with O6-methylated guanine is recognized as a mismatch by MutS and removed more efficiently than a thymine opposite O6-ethylated guanine. Taken together, the data suggest that in E. coli, the MMR system plays a more significant role in repair of methylation-induces lesions than those caused by ethylation.
doi:10.1016/j.mrfmmm.2007.12.009
PMCID: PMC2365708  PMID: 18243250
MutS; mismatch repair; O6-methylguanine; O6-ethylguanine; mutation
17.  Self-assembly of E. coli MutL and its complexes with DNA 
Biochemistry  2011;50(37):7868-7880.
The E. coli MutL protein regulates the activity of several enzymes, including MutS, MutH and UvrD, during methyl-directed mismatch repair (MMR) of DNA. We have investigated the self association properties of MutL and its binding to DNA using analytical sedimentation velocity and equilibrium. Self association of MutL is quite sensitive to solution conditions. At 25°C in Tris at pH 8.3 MutL assembles into a heterogeneous mixture of large multimers. In the presence of potassium phosphate at pH 7.4, MutL forms primarily stable dimers, with the higher order assembly states suppressed. The weight averaged sedimentation coefficient of MutL dimer in this buffer is equal to s̅20,w = 5.20 ± 0.08, suggesting a highly asymmetric dimer (f/fo = 1.58 ± 0.02). Upon binding the non-hydrolyzable ATP analogue, AMPPNP/Mg2+, the MutL dimer becomes more compact (s̅20,w = 5.71 ± 0.08 S, f/fo = 1.45 ± 0.02), probably reflecting reorganization of the N-terminal ATPase domains. A MutL dimer binds to an 18 bp duplex with a 3’-(dT20) single stranded flanking region, with apparent affinity in the micromolar range. AMPPNP binding to MutL increases its affinity for DNA by a factor of ~10. These results indicate that the presence of phosphate minimizes further MutL oligomerization beyond a dimer and that differences in solution conditions likely explain apparent discrepancies in previous studies of MutL assembly.
doi:10.1021/bi200753b
PMCID: PMC3172365  PMID: 21793594
DNA repair; self-association; protein-DNA interaction; analytical ultracentrifugation
18.  The effects of nucleotides on MutS-DNA binding kinetics clarify the role of MutS ATPase activity in mismatch repair 
Journal of molecular biology  2006;366(4):1087-1098.
SUMMARY
MutS protein initiates mismatch repair with recognition of a non-Watson-Crick base pair or base insertion/deletion site in DNA, and its interactions with DNA are modulated by ATPase activity. Here we present a kinetic analysis of these interactions, including the effects of ATP binding and hydrolysis, reported directly from the mismatch site by 2-aminopurine fluorescence. When free of nucleotides, the T. aquaticus MutS dimer binds a mismatch rapidly (kON = 3 x 106 M−1s−1) and forms a stable complex with a half-life of 10 seconds (kOFF = 0.07 s−1). When one or both nucleotide-binding sites on the MutS•mismatch complex are occupied by ATP, the complex remains fairly stable, with a half-life of 5 – 7 seconds (kOFF = 0.1 – 0.14 s−1), although MutSATP becomes incapable of (re-)binding the mismatch. When one or both nucleotide-binding sites on the MutS dimer are occupied by ADP, the MutS•mismatch complex forms rapidly (kON = 7.3 x 106 M−1s−1) and also dissociates rapidly, with a half-life of 0.4 seconds (kOFF = 1.7 s−1). Integration of these MutS DNA-binding kinetics with previously described ATPase kinetics reveals that: a) in the absence of a mismatch, MutS in the ADP-bound form engages in highly dynamic interactions with DNA—perhaps probing base pairs for errors; b) in the presence of a mismatch, MutS stabilized in the ATP-bound form releases the mismatch slowly—perhaps allowing for onsite interactions with downstream repair proteins; c) ATP-bound MutS then moves off the mismatch—perhaps as a mobile clamp facilitating repair reactions at distant sites on DNA—until ATP is hydrolyzed (or dissociates) and the protein turns over.
doi:10.1016/j.jmb.2006.11.092
PMCID: PMC1941710  PMID: 17207499
19.  Structural, molecular and cellular functions of MSH2 and MSH6 during DNA mismatch repair, damage signaling and other noncanonical activities 
Mutation research  2013;0:53-66.
The field of DNA mismatch repair (MMR) has rapidly expanded after the discovery of the MutHLS repair system in bacteria. By the mid 1990s yeast and human homologues to bacterial MutL and MutS had been identified and their contribution to hereditary non-polyposis colorectal cancer (HNPCC; Lynch Syndrome) was under intense investigation. The human MutS homologue 6 protein (hMSH6), was first reported in 1995 as a G:T binding partner (GTBP) of hMSH2, forming the hMutSα mismatch-binding complex. Signal transduction from each DNA-bound hMutSα complex is accomplished by the hMutLα heterodimer (hMLH1 and hPMS2). Molecular mechanisms and cellular regulation of individual MMR proteins are now areas of intensive research. This review will focus on molecular mechanisms associated with mismatch binding, as well as emerging evidence that MutSα and in particular, MSH6, is a key protein in MMR-dependent DNA damage response and communication with other DNA repair pathways within the cell. MSH6 is unstable in the absence of MSH2, however it is the DNA lesion-binding partner of this heterodimer. MSH6, but not MSH2, has a conserved Phe-X-Glu motif that recognizes and binds several different DNA structural distortions, initiating different cellular responses. hMSH6 also contains the nuclear localization sequences required to shuttle hMutSα into the nucleus. For example, upon binding to O6meG:T, MSH6 triggers a DNA damage response that involves altered phosphorylation within the N-terminal disordered domain of this unique protein. While many investigations have focused on MMR as a post-replication DNA repair mechanism, MMR proteins are expressed and active in all phases of the cell cycle. There is much more to be discovered about regulatory cellular roles that require the presence of MutSα and, in particular, MSH6.
doi:10.1016/j.mrfmmm.2012.12.008
PMCID: PMC3659183  PMID: 23391514
DNA mismatch repair; MSH2; MSH6; DNA damage signaling; N-terminal disordered domain
20.  Requirement for Phe36 for DNA binding and mismatch repair by Escherichia coli MutS protein 
Nucleic Acids Research  2000;28(18):3564-3569.
The MutS family of DNA repair proteins recognizes base pair mismatches and insertion/deletion mismatches and targets them for repair in a strand-specific manner. Photocrosslinking and mutational studies previously identified a highly conserved Phe residue at the N-terminus of Thermus aquaticus MutS protein that is critical for mismatch recognition in vitro. Here, a mutant Escherichia coli MutS protein harboring a substitution of Ala for the corresponding Phe36 residue is assessed for proficiency in mismatch repair in vivo and DNA binding and ATP hydrolysis in vitro. The F36A protein is unable to restore mismatch repair proficiency to a mutS strain as judged by mutation to rifampicin or reversion of a specific point mutation in lacZ. The F36A protein is also severely deficient for binding to heteroduplexes containing an unpaired thymidine or a G:T mismatch although its intrinsic ATPase activity and subunit oligomerization are very similar to that of the wild-type MutS protein. Thus, the F36A mutation appears to confer a defect specific for recognition of insertion/deletion and base pair mismatches.
PMCID: PMC110738  PMID: 10982877
21.  Impact of mutS Inactivation on Foreign DNA Acquisition by Natural Transformation in Pseudomonas stutzeri 
Journal of Bacteriology  2005;187(1):143-154.
In prokaryotic mismatch repair the MutS protein and its homologs recognize the mismatches. The mutS gene of naturally transformable Pseudomonas stutzeri ATCC 17587 (genomovar 2) was identified and characterized. The deduced amino acid sequence (859 amino acids; 95.6 kDa) displayed protein domains I to IV and a mismatch-binding motif similar to those in MutS of Escherichia coli. A mutS::aac mutant showed 20- to 163-fold-greater spontaneous mutability. Transformation experiments with DNA fragments of rpoB containing single nucleotide changes (providing rifampin resistance) indicated that mismatches resulting from both transitions and transversions were eliminated with about 90% efficiency in mutS+. The mutS+ gene of strain ATCC 17587 did not complement an E. coli mutant but partially complemented a P. stutzeri JM300 mutant (genomovar 4). The declining heterogamic transformation by DNA with 0.1 to 14.6% sequence divergence was partially alleviated by mutS::aac, indicating that there was a 14 to 16% contribution of mismatch repair to sexual isolation. Expression of mutS+ from a multicopy plasmid eliminated autogamic transformation and greatly decreased heterogamic transformation, suggesting that there is strong limitation of MutS in the wild type for marker rejection. Remarkably, mutS::aac altered foreign DNA acquisition by homology-facilitated illegitimate recombination (HFIR) during transformation, as follows: (i) the mean length of acquired DNA was increased in transformants having a net gain of DNA, (ii) the HFIR events became clustered (hot spots) and less dependent on microhomologies, which may have been due to topoisomerase action, and (iii) a novel type of transformants (14%) had integrated foreign DNA with no loss of resident DNA. We concluded that in P. stutzeri upregulation of MutS could enforce sexual isolation and downregulation could increase foreign DNA acquisition and that MutS affects mechanisms of HFIR.
doi:10.1128/JB.187.1.143-154.2005
PMCID: PMC538834  PMID: 15601698
22.  Functions of the Mismatch Repair Gene mutS from Acinetobacter sp. Strain ADP1† 
Journal of Bacteriology  2001;183(23):6822-6831.
The genus Acinetobacter encompasses a heterogeneous group of bacteria that are ubiquitous in the natural environment due in part to their ability to adapt genetically to novel challenges. Acinetobacter sp. strain ADP1 (also known as strain BD413) is naturally transformable and takes up DNA from any source. Donor DNA can be integrated into the chromosome by recombination provided it possesses sufficient levels of nucleotide sequence identity to the recipient's DNA. In other bacteria, the requirement for sequence identity during recombination is partly due to the actions of the mismatch repair system, a key component of which, MutS, recognizes mismatched bases in heteroduplex DNA and, along with MutL, blocks strand exchange. We have cloned mutS from strain ADP1 and examined its roles in preventing recombination between divergent DNA and in the repair of spontaneous replication errors. Inactivation of mutS resulted in 3- to 17-fold increases in transformation efficiencies with donor sequences that were 8 to 20% divergent relative to the strain ADP1. Strains lacking MutS exhibited increased spontaneous mutation frequencies, and reversion assays demonstrated that MutS preferentially recognized transition mismatches while having little effect on the repair of transversion mismatches. Inactivation of mutS also abolished the marker-specific variations in transforming efficiency seen in mutS+ recipients where transition and frameshift alleles transformed at eightfold lower frequencies than transversions or large deletions. Comparison of the MutS homologs from five individual Acinetobacter strains with those of other gram-negative bacteria revealed that a number of unique indels are conserved among the Acinetobacter amino acid sequences.
doi:10.1128/JB.183.23.6822-6831.2001
PMCID: PMC95523  PMID: 11698371
23.  The C-Terminal Domain of the MutL Homolog from Neisseria gonorrhoeae Forms an Inverted Homodimer 
PLoS ONE  2010;5(10):e13726.
The mismatch repair (MMR) pathway serves to maintain the integrity of the genome by removing mispaired bases from the newly synthesized strand. In E. coli, MutS, MutL and MutH coordinate to discriminate the daughter strand through a mechanism involving lack of methylation on the new strand. This facilitates the creation of a nick by MutH in the daughter strand to initiate mismatch repair. Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH. Although the exact strategy for strand discrimination in these organisms is yet to be ascertained, the required nicking endonuclease activity is resident in the C-terminal domain of MutL. This activity is dependent on the integrity of a conserved metal binding motif. Unlike their eukaryotic counterparts, MutL in bacteria like Neisseria exist in the form of a homodimer. Even though this homodimer would possess two active sites, it still acts a nicking endonuclease. Here, we present the crystal structure of the C-terminal domain (CTD) of the MutL homolog of Neisseria gonorrhoeae (NgoL) determined to a resolution of 2.4 Å. The structure shows that the metal binding motif exists in a helical configuration and that four of the six conserved motifs in the MutL family, including the metal binding site, localize together to form a composite active site. NgoL-CTD exists in the form of an elongated inverted homodimer stabilized by a hydrophobic interface rich in leucines. The inverted arrangement places the two composite active sites in each subunit on opposite lateral sides of the homodimer. Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR. The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage.
doi:10.1371/journal.pone.0013726
PMCID: PMC2965676  PMID: 21060849
24.  A unique horizontal gene transfer event has provided the octocoral mitochondrial genome with an active mismatch repair gene that has potential for an unusual self-contained function 
Background
The mitochondrial genome of the Octocorallia has several characteristics atypical for metazoans, including a novel gene suggested to function in DNA repair. This mtMutS gene is favored for octocoral molecular systematics, due to its high information content. Several hypotheses concerning the origins of mtMutS have been proposed, and remain equivocal, although current weight of support is for a horizontal gene transfer from either an epsilonproteobacterium or a large DNA virus. Here we present new and compelling evidence on the evolutionary origin of mtMutS, and provide the very first data on its activity, functional capacity and stability within the octocoral mitochondrial genome.
Results
The mtMutS gene has the expected conserved amino acids, protein domains and predicted tertiary protein structure. Phylogenetic analysis indicates that mtMutS is not a member of the MSH family and therefore not of eukaryotic origin. MtMutS clusters closely with representatives of the MutS7 lineage; further support for this relationship derives from the sharing of a C-terminal endonuclease domain that confers a self-contained mismatch repair function. Gene expression analyses confirm that mtMutS is actively transcribed in octocorals. Rates of mitochondrial gene evolution in mtMutS-containing octocorals are lower than in their hexacoral sister-group, which lacks the gene, although paradoxically the mtMutS gene itself has higher rates of mutation than other octocoral mitochondrial genes.
Conclusions
The octocoral mtMutS gene is active and codes for a protein with all the necessary components for DNA mismatch repair. A lower rate of mitochondrial evolution, and the presence of a nicking endonuclease domain, both indirectly support a theory of self-sufficient DNA mismatch repair within the octocoral mitochondrion. The ancestral affinity of mtMutS to non-eukaryotic MutS7 provides compelling support for an origin by horizontal gene transfer. The immediate vector of transmission into octocorals can be attributed to either an epsilonproteobacterium in an endosymbiotic association or to a viral infection, although DNA viruses are not currently known to infect both bacteria and eukaryotes, nor mitochondria in particular. In consolidating the first known case of HGT into an animal mitochondrial genome, these findings suggest the need for reconsideration of the means by which metazoan mitochondrial genomes evolve.
doi:10.1186/1471-2148-11-228
PMCID: PMC3166940  PMID: 21801381
25.  Substrate recognition by Escherichia coli MutY using substrate analogs. 
Nucleic Acids Research  1999;27(15):3197-3204.
The Escherichia coli adenine glycosylase MutY is involved in the repair of 7,8-dihydro-8-oxo-2"-deoxyguanosine (OG):A and G:A mispairs in DNA. Our approach toward understanding recognition and processing of DNA damage by MutY has been to use substrate analogs that retain the recognition properties of the substrate mispair but are resistant to the glycosylase activity of MutY. This approach provides stable MutY-DNA complexes that are amenable to structural and biochemical characterization. In this work, the interaction of MutY with the 2"-deoxyadenosine analogs 2"-deoxy-2"-fluoroadenosine (FA), 2"-deoxyaristeromycin (R) and 2"-deoxyformycin A (F) was investigated. MutY binds to duplexes containing the FA, R or F analogs opposite G and OG within DNA with high affinity; however, no enzymatic processing of these duplexes is observed. The specific nature of the interaction of MutY with an OG:FA duplex was demonstrated by MPE-Fe(II) hydroxyl radical footprinting experiments which showed a nine base pair region of protection by MutY surrounding the mispair. DMS footprinting experiments with an OG:A duplex revealed that a specific G residue located on the OG-containing strand was protected from DMS in the presence of MutY. In contrast, a G residue flanking the substrate analogs R, F or FA was observed to be hypersensitive to DMS in the presence of MutY. These results suggest a major conformational change in the DNA helix upon binding of MutY that exposes the substrate analog-containing strand. This finding is consistent with a nucleotide flipping mechanism for damage recognition by MutY. This work demonstrates that duplex substrates for MutY containing FA, R or F instead of A are excellent substrate mimics that may be used to provide insight into the recognition by MutY of damaged and mismatched base pairs within DNA.
PMCID: PMC148548  PMID: 10454618

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