Multicopy single-stranded DNAs with mismatched base pairs are mutagenic in Escherichia coli

Retrons are genetic elements that encode multicopy single-stranded DNAs called msONAs. They are clonally distributed in Escherichia coli and retrons in different clones produce DNAs with different nucleotide sequences. msDNAs consist of an RNA molecule covalently linked to a single-stranded DNA molecule. The latter contains an inverted repeat, resulting in a stem-loop structure. In two retrons, Ec83 and Ec78, the DNA is cleaved off from the RNA. All known retrons except Ec78, have one or more mismatched base pairs in the stem-loop structure. We found that two retrons, Ec86 and Ec83, when present in high copy numbers are mutagenic. The ratios of mutation frequencies observed in Lac indicator strains were similar to the ratios observed for a mutant defective in mismatch repair. It is known that some proteins required for mismatch repair bind to mismatched base pairs prior to carrying out repair. The similarity in the mutation frequency ratios suggested that the mutagenesis caused by msDNAs of retrons Ec86 and Ec83 might be due to seqestration of a mismatch repair protein by msDNA. Strong support for this interpretation was obtained from the finding that the msDNA produced by retron Ec78 is not mutagenic.     

A mutational study of the site-specific cleavage of EC83, a multicopy single-stranded DNA (msDNA): nucleotides at the msDNA stem are important for its cleavage.

Multicopy single-stranded DNA (msDNA) molecules consist of single-stranded DNA covalently linked to RNA. Such molecules are encoded by genetic elements called retrons. Unlike other retrons, retron EC83 isolated from Escherichia coli 161 produces RNA-free msDNA by site-specific cleavage of msDNA at 5'-TTGA/A-3', where the slash indicates the cleavage site. In order to investigate factors responsible for the msDNA cleavage, retron EC83 was treated with hydroxylamine and colonies were screened for cleavage-negative mutants. We isolated three mutants which were defective in msDNA cleavage and produced RNA-linked msDNA. They were all affected in msd, a gene for msDNA, with a base substitution at the bottom part of the msDNA stem. In contrast, base substitution at and around the cleavage site has no marked effect on msDNA synthesis or its cleavage. From these results, we concluded that the nucleotides at the bottom of the msDNA stem, but not the nucleotides at the cleavage site, play a major role in the recognition and cleavage of msDNA EC83.     

Dual role of MutS glutamate 38 in DNA mismatch discrimination and in the authorization of repair

MutS plays a critical role in DNA mismatch repair in Escherichia coli by binding to mismatches and initiating repair in an ATP-dependent manner. Mutational analysis of a highly conserved glutamate, Glu38, has revealed its role in mismatch recognition by enabling MutS to discriminate between homoduplex and mismatched DNA. Crystal structures of MutS have shown that Glu38 forms a hydrogen bond to one of the mismatched bases. In this study, we have analyzed the crystal structures, DNA binding and the response to ATP binding of three Glu38 mutants. While confirming the role of the negative charge in initial discrimination, we show that in vivo mismatch repair can proceed even when discrimination is low. We demonstrate that the formation of a hydrogen bond by residue 38 to the mismatched base authorizes repair by inducing intramolecular signaling, which results in the inhibition of rapid hydrolysis of distally bound ATP. This allows formation of the stable MutS-ATP-DNA clamp, a key intermediate in triggering downstream repair events.     

DNA binding and protein-protein interaction sites in MutS, a mismatched DNA recognition protein from Thermus thermophilus HB8

The mismatch repair system repairs mismatched base pairs, which are caused by either DNA replication errors, DNA damage, or genetic recombination. Mismatch repair begins with the recognition of mismatched base pairs in DNA by MutS. Protein denaturation and limited proteolysis experiments suggest that Thermus thermophilus MutS can be divided into three structural domains as follows: A (N-terminal domain), B (central domain), and C (C-terminal domain) (Tachiki, H., Kato, R., Masui, R., Hasegawa, K., Itakura, H., Fukuyama, K., and Kuramitsu, S. (1998) Nucleic Acids Res. 26, 4153-4159). To investigate the functions of each domain in detail, truncated genes corresponding to the domains were designed. The gene products were overproduced in Escherichia coli, purified, and assayed for various activities. The MutS-MutS protein interaction site was determined by size-exclusion chromatography to be located in the B domain. The B domain was also found to possess nonspecific double-stranded DNA-binding ability. The C domain, which contains a Walker's A-type nucleotide-binding motif, demonstrated ATPase activity and specific DNA recognition of mismatched base pairs. These ATPase and specific DNA binding activities were found to be dependent upon C domain dimerization.     

Functional characterization of the DNA mismatch binding protein MutS from Haemophilus influenzae

This investigation demonstrates DNA mismatch repair activity in Haemophilus influenzae cell free extracts. The mutS gene as well as purified protein of H. influenzae restored repair activity in complementation assays performed with mutS deficient Escherichia coli strain. The difference in affinity for GT and AC mismatched bases by H. influenzae MutS was reflected in the efficiency with which these DNA heteroduplexes were repaired in vitro, with GT being repaired well and AC the least. Unlike E. coli MutS, the H. influenzae homolog failed to give protein-DNA complex with homoduplex DNA. Interestingly, MutS was found to bind single-stranded DNA but with lesser affinity as compared to heteroduplex DNA. Apart from the nucleotide- and DNA-mediated conformational transitions, as monitored by circular dichroism and limited proteolysis, our data suggest a functional role when H. influenzae MutS encounters single-stranded DNA during exonucleolytic step of DNA repair process. We propose that, conformational changes in H. influenzae MutS not only modulate mismatch recognition but also trigger some of the down stream processes involved in the DNA mismatch repair process.     

Structural and functional divergence of MutS2 from bacterial MutS1 and eukaryotic MSH4-MSH5 homologs

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.     

Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA

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.     

Atomic force microscopy captures the initiation of methyl-directed DNA mismatch repair

In Escherichia coli, errors in newly-replicated DNA, such as the incorporation of a nucleotide with a mis-paired base or an accidental insertion or deletion of nucleotides, are corrected by a methyl-directed mismatch repair (MMR) pathway. While the enzymology of MMR has long been established, many fundamental aspects of its mechanisms remain elusive, such as the structures, compositions, and orientations of complexes of MutS, MutL, and MutH as they initiate repair. Using atomic force microscopy, we—for the first time—record the structures and locations of individual complexes of MutS, MutL and MutH bound to DNA molecules during the initial stages of mismatch repair. This technique reveals a number of striking and unexpected structures, such as the growth and disassembly of large multimeric complexes at mismatched sites, complexes of MutS and MutL anchoring latent MutH onto hemi-methylated d(GATC) sites or bound themselves at nicks in the DNA, and complexes directly bridging mismatched and hemi-methylated d(GATC) sites by looping the DNA. The observations from these single-molecule studies provide new opportunities to resolve some of the long-standing controversies in the field and underscore the dynamic heterogeneity and versatility of MutSLH complexes in the repair process.     

Mechanism for verification of mismatched and homoduplex DNAs by nucleotides‐bound MutS analyzed by molecular dynamics simulations

In order to understand how MutS recognizes mismatched DNA and induces the reaction of DNA repair using ATP, the dynamics of the complexes of MutS (bound to the ADP and ATP nucleotides, or not) and DNA (with mismatched and matched base‐pairs) were investigated using molecular dynamics simulations. As for DNA, the structure of the base‐pairs of the homoduplex DNA which interacted with the DNA recognition site of MutS was intermittently disturbed, indicating that the homoduplex DNA was unstable. As for MutS, the disordered loops in the ATPase domains, which are considered to be necessary for the induction of DNA repair, were close to (away from) the nucleotide‐binding sites in the ATPase domains when the nucleotides were (not) bound to MutS. This indicates that the ATPase domains changed their structural stability upon ATP binding using the disordered loop. Conformational analysis by principal component analysis showed that the nucleotide binding changed modes which have structurally solid ATPase domains and the large bending motion of the DNA from higher to lower frequencies. In the MutS–mismatched DNA complex bound to two nucleotides, the bending motion of the DNA at low frequency modes may play a role in triggering the formation of the sliding clamp for the following DNA‐repair reaction step. Moreover, MM‐PBSA/GBSA showed that the MutS–homoduplex DNA complex bound to two nucleotides was unstable because of the unfavorable interactions between MutS and DNA. This would trigger the ATP hydrolysis or separation of MutS and DNA to continue searching for mismatch base‐pairs. Proteins 2016; 84:1287–1303. © 2016 Wiley Periodicals, Inc.     

Insights into protein - DNA interactions, stability and allosteric communications: a computational study of mutSα-DNA recognition complexes

DNA mismatch repair proteins (MMR) maintain genetic stability by recognizing and repairing mismatched bases and insertion/deletion loops mistakenly incorporated during DNA replication, and initiate cellular response to certain types of DNA damage. Loss of MMR in mammalian cells has been linked to resistance to certain DNA damaging chemotherapeutic agents, as well as to increase risk of cancer. Mismatch repair pathway is considered to involve the concerted action of at least 20 proteins. The most abundant MMR mismatch-binding factor in eukaryotes, MutSα, recognizes and initiates the repair of base-base mismatches and small insertion/deletion. We performed molecular dynamics simulations on mismatched and damaged MutSα-DNA complexes. A comprehensive DNA binding site analysis of relevant conformations shows that MutSα proteins recognize the mismatched and platinum cross-linked DNA substrates in significantly different modes. Distinctive conformational changes associated with MutSα binding to mismatched and damaged DNA have been identified and they provide insight into the involvement of MMR proteins in DNA-repair and DNA-damage pathways. Stability and allosteric interactions at the heterodimer interface associated with the mismatch and damage recognition step allow for prediction of key residues in MMR cancer-causing mutations. A rigorous hydrogen bonding analysis for ADP molecules at the ATPase binding sites is also presented. Due to extended number of known MMR cancer causing mutations among the residues proved to make specific contacts with ADP molecules, recommendations for further studies on similar mutagenic effects were made.     

Physical and functional interactions between Escherichia coli MutY glycosylase and mismatch repair protein MutS

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, hMutSalpha (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.     

Asymmetric ATP binding and hydrolysis activity of the Thermus aquaticus MutS dimer is key to modulation of its interactions with mismatched DNA

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; S1(ADP)-S2(ATP) is formed preferentially in the absence of DNA or in the presence of fully matched DNA, while S1(ATP)-S2(ATP) and S1(ATP)-S2(ADP) 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.     

Formation of a DNA mismatch repair complex mediated by ATP

The mismatch repair proteins, MutS and MutL, interact in a DNA mismatch and ATP-dependent manner to activate downstream events in repair. Here, we assess the role of ATP binding and hydrolysis in mismatch recognition by MutS and the formation of a ternary complex involving MutS and MutL bound to a mismatched DNA. We show that ATP reduces the affinity of MutS for mismatched DNA and that the modulation of DNA binding affinity by nucleotide is even more pronounced for MutS E694A, a protein that binds ATP but is defective for ATP hydrolysis. Despite the ATP hydrolysis defect, E694A, like WT MutS, undergoes rapid, ATP-dependent dissociation from a DNA mismatch. Furthermore, MutS E694A retains the ability to interact with MutL on mismatched DNA. The recruitment of MutL to a mismatched DNA by MutS is also observed for two mutant MutL proteins, E29A, defective for ATP hydrolysis, and R266A, defective for DNA binding. These results suggest that ATP binding in the absence of hydrolysis is sufficient to trigger formation of a MutS sliding clamp. However, recruitment of MutL results in the formation of a dynamic ternary complex that we propose is the intermediate that signals subsequent repair steps requiring ATP hydrolysis.     

Structures of mismatched base pairs in DNA and their recognition by the Escherichia coli mismatch repair system.

The Escherichia coli mismatch repair system does not recognize and/or repair all mismatched base pairs with equal efficiency: whereas transition mismatches (G X T and A X C) are well repaired, the repair of some transversion mismatches (e.g. A X G or C X T) appears to depend on their position in heteroduplex DNA of phage lambda. Undecamers were synthesized and annealed to form heteroduplexes with a single base-pair mismatch in the centre and with the five base pairs flanking each side corresponding to either repaired or unrepaired heteroduplexes of lambda DNA. Nuclear magnetic resonance (n.m.r.) studies show that a G X A mismatch gives rise to an equilibrium between fully helical and a looped-out structure. In the unrepaired G X A mismatch duplex the latter predominates, while the helical structure is predominant in the case of repaired G X A and G X T mismatches. It appears that the E. coli mismatch repair enzymes recognize and repair intrahelical mismatched bases, but not the extrahelical bases in the looped-out structures.