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Bacterial EndoMS/NucS acts as a clamp-mediated mismatch endonuclease to prevent asymmetric accumulation of replication errors

Abstract Mismatch repair (MMR) systems based on MutS eliminate mismatches originating from replication errors. Despite extensive conservation of mutS homologues throughout the three domains of life, Actinobacteria and some archaea do not have genes homologous to mutS. Here, we report that EndoMS/NucS of Corynebacterium glutamicum is the mismatch-specific endonuclease that functions cooperatively with a sliding clamp. EndoMS/NucS function in MMR was fully dependent on physical interaction between EndoMS/NucS and sliding clamp. A combination of endoMS/nucS gene disruption and a mutation in dnaE, which reduced the fidelity of DNA polymerase, increased the mutation rate synergistically and confirmed the participation of EndoMS in replication error correction. EndoMS specifically cleaved G/T, G/G and T/T mismatches in vitro, and such substrate specificity was consistent with the mutation spectrum observed in genome-wide analyses. The observed substrate specificity of EndoMS, together with the effects of endoMS gene disruption, led us to speculate that the MMR system, regardless of the types of proteins in the system, evolved to address asymmetrically occurring replication errors in which G/T mismatches occur much more frequently than C/A mismatches.


INTRODUCTION
All living organisms are equipped with a DNA replication and a DNA repair system for the maintenance of genomic information. Although the DNA replication system has high fidelity, it sometimes misincorporates incorrect nucleobases. The DNA mismatch repair (MMR) system is known to correct such replication errors (1). Dysfunction in the MMR system increases the spontaneous mutation rate and can lead to cancer in humans or drug resistance in pathogenic microorganisms. In the MMR system, cells utilize MutS to detect mismatches, and the binding of MutS to a mismatched nucleotide initiates the correction pathway by activating MutL protein, which works as an activator of MutH methyl-directed endonuclease (in Escherichia coli and closely related species) or as a nickase by itself (in other organisms) (2). One of the hallmarks of the MutS-MutL-dependent MMR system compared to other DNA repair systems is the breadth of target mutations. MutS has been reported to bind to a broad range of mismatched nucleotides (3,4) and to correct G/T, C/A, C/T, A/A, T/T and G/G mismatches with similar efficiencies (5). Such situations enable organisms to repair all types of mutations (6,7); however, the breadth of targets makes it difficult to determine the origin of mutations. For example, because G/T and C/A mismatches have possibility to lead to the same mutation depending on the strand in which the replication error occurs, the ability of MutS-based system to repair both types of mismatches prevents us from determining which of the two errors occurred in the absence of the system.
Although mutS gene is known to be widely conserved throughout the three domains of life, two bacterial groups, belonging to the phylum Actinobacteria and to the phylum mollicutes, and some Archaea has no gene homologous to mutS (8). Interestingly, despite the absence of mutS homolog, mutation rate of organisms belonging to phylum Actinobacteria is comparable to that of E. coli with intact MMR system (9). Recently, it has been revealed that NucS of Pyrococcus abyssi (an organism belonging to the archaeal phylum Euryarchaeota), which had been reported to be a flapped DNA endonuclease (10), has mismatch-dependent nuclease activity, and the name EndoMS (Endonuclease Mismatch-Specific) has been proposed for this enzyme (11). Because endoMS/nucS is preferentially conserved in organisms lacking mutS, the authors suggested that EndoMS/NucS works in non-canonical MMR systems. In addition, the disruption of endoMS/nucS gene in Mycobacterium smegmatis and Streptomyces coelicolor, which are both Actinobacteria, was recently reported to result in an increased spontaneous mutation rate, although biochemical analysis of the bacterial EndoMS/NucS protein failed to show enzymatic activity (12). Thus, although EndoMS/NucS has been suggested to function in a noncanonical MMR pathway, the link between the phenotype of the endoMS/nucS gene disruptant and the biochemical properties of the EndoMS/NucS protein is not clear. Finally, whether the EndoMS/NucS-dependent pathway corrects replication errors similar to the MutS-dependent canonical system has not been analyzed.
In the present study, we found that disruption of endoMS/nucS increased the spontaneous mutation rate in Corynebacterium glutamicum. Our biochemical and genetic analyses demonstrated that C. glutamicum EndoMS/NucS is a mismatch-specific endonuclease that works cooperatively with the sliding clamp of the replisome to correct replication errors. Although our data clearly indicated that the EndoMS-pathway is a counterpart of the MutSdependent MMR system in Actinobacteria, unlike MutSdependent systems, the EndoMS-pathway is highly specific to G/T, G/G and T/T mismatches. These findings led us to hypothesize about the nature of replication in vivo and about the biological significance of MMR systems.

In silico screening
A protein database that included all proteins expected from 785 whole genome sequences of bacterial and archaeal type strains was constructed on the CLC Genomics workbench (CLC Bio). Among the 785 type strains, 105 strains belong to the phylum Actinobacteria. The type strains analyzed in this study are listed in Supplementary Table S1. M. tuberculosis H37Rv proteins were categorized according to Clusters of Orthologous Groups of proteins (COGs) using in silico molecular cloning (In Silico Biology, Inc.) as described by the manufacture's protocol. Using the M. tuberculosis nucleotide sequence of genes categorized to COG 'L' group as a query, a BLASTX search was performed on the CLC Genomics workbench against the protein database with BLO-SUM45 matrix (E-value < 0.001, gap existence and extension penalties of 16 and 1, respectively). The number of strains with BLAST hits was counted for each query and used to find genes conserved among Actinobacteria. The threshold for genes predominant in Actinobacteria were those for which BLAST hits were found in >70% of actinobacterial species (>74 species) and in <35% of other species (<238 species).

Strains, plasmids, and growth conditions
Strains and plasmids used in this study are listed in Supplementary Table S2. Oligonucleotide primers used in this study are listed in Supplementary Table S3. Escherichia coli were grown at 37 • C in Luria-Bertani medium while C. glutamicum was grown aerobically at 30 • C in brain heart in-fusion (BHI; BD) medium with shaking at 200 rpm. When appropriate, kanamycin was added to the media at a concentration of 12.5 g/ml (C. glutamicum) or 50 g/ml (E. coli) and ampicillin at 100 g/ml (E. coli). In-frame deletion mutants of C. glutamicum and codon exchanges in chromosomal genes were constructed by using a two-step homologous recombination protocol as described previously (13) using suicide vector.

Overexpression and purification of His-tagged proteins
The endoMS Cg gene and C-terminal region of the dnaN Cg gene (S98-G394) were amplified from the chromosomal DNA of C. glutamicum ATCC13032 by PCR with primer pairs NCgl1168(nucS) F2(NdeI)/NCgl1168(nucS) R2(EcoRI-XhoI) and NCgl0002 F1(SphI)/NCgl0002 Cterm EcoRI-NotI R, respectively. The PCR product of endoMS Cg was cloned into the NdeI-EcoRI site of expression vector pColdI (Takara), yielding pColdI-Cg-NucS. The PCR product of the C-terminal region of the dnaN Cg gene was cloned into the BamHI-NotI site of the expression vector pColdI (Takara), yielding pColdI-Cg-dnaN-Cterm. His-tagged proteins were overexpressed in E. coli BL21-CodonPlus (DE3)-RIPL (Agilent Technologies) by cold shock and purified by affinity chromatography as described by the manufacture's protocol. The concentration of the purified protein was determined using a Bio-Rad protein assay (Bio-Rad Laboratories) with bovine serum albumin (BSA) as a standard.
His-DnaN Cg was overproduced in E. coli BL21 Codon-Plus (DE3)-RIPL harboring pQE2-Cg-dnaN. The cells were grown to OD600 = ∼0.8 at 37 • C in LB medium containing 50 g/ml carbenicillin, and further incubated with 1 mM IPTG at 37 • C for 20 h. Cell lysis, polyethyleneimine treatment and ammonium-sulfate precipitation were performed by the same procedures as for EndoMS Cg . The ammonium-sulfate precipitation was resuspended in Nicolumn buffer (20 mM Tris-HCl, pH7.5, 500 mM NaCl, 2 mM 2-mercapt ethanol, 10% glycerol, 0.2 mM EDTA, 10 mM Imidazole). The resuspended fraction was applied to a HisTrap HP column (GE Healthcare), and His-DnaN Cg was eluted with linear gradient of 10-500 mM Imidazole in Ni-column buffer. The protein concentrations were measured by the Bradford assay.

In vitro cleavage assay
Mismatch DNAs were prepared by annealing of oligonucleotides (Supplementary Table S3). The mismatch DNA (50 nM) was incubated at 37 • C for 60 min in 10 l of cleavage buffer (20 mM Tris-HCl, pH 8.0, 10 mM Mg[oAc] 2 , 4 mM dithiothreitol, 0.071 mg/ml BSA, and 100 mM [or indicated concentration] K[oAc]) in the presence of 1 M EndoMS Cg and/or 1 M His-DnaN Cg , unless otherwise indicated. The reaction was then stopped by the addition of an equal volume of 2 × Stop buffer (50 mM Tris-HCl, pH 8.0, 50 mM EDTA, 0.2% sodium dodecyl sulfate, 0.1 mg/ml proteinase K, 10% glycerol, 0.02% bromophenol blue) and further incubated at 37 • C for 30 min. An aliquot (5 l) of this mixture was analyzed by 12% native PAGE followed by SYBR Green I staining (Molecular Probes). For the denaturing PAGE analysis of 5 -FAM-labeled DNA, the reaction sample (1 l) was added to 4 l of denaturing buffer (100 mM Tris-borate, pH 8.3, 2 mM EDTA, 10 M urea), incubated 95 • C for 5 min, and then analyzed by 8 M Urea 20% PAGE. Oligonucleotides used as 5 -FAM-labeled size markers are listed (Supplementary Table S5). The images were acquired with a Typhoon FLA 9500 (GE Healthcare).

Preparation of polyclonal antibodies
One hundred g of purified His-EndoMS Cg and His-DnaN Cg -Cterm proteins was used as antigen to immunize rabbits or rats with Freund's adjuvant (Difco). After four times of booster, animals were sacrificed to prepare antiserum. The animal experiments were approved by the Ethical Committee for Animal Experiments at the Research Institute of the National Center for Global Health and Medicine.

Immunoprecipitation followed by immunoblot analysis
C. glutamicum ATCC13032 WT and nucS-Cdel strains were grown aerobically in 250 ml of BHI medium for 4h. Formaldehyde was added at final concentration of 1% (W/V) and incubated for 30 min at room temperature. Crosslinking reaction was quenched by adding of 4 ml of 1M Tris-HCl (pH7.5). Cells were collected by centrifugation (6000 × g, 10 min) and washed twice with 20 ml of cell wash buffer (50 mM Tris-HCl, H7.5, 50 mM NaCl). Cells were resuspended in HNTG buffer (20 mM HEPES buffer pH 7.5, containing 150 mM NaCl, 0.1% (w/v) Triton X-100 and 10% (w/v) glycerol) supplemented with cOm-plete™ Protease Inhibitor Cocktail (Roche) and sonicated using handy-type sonicator (UR-20P, Tomy). Cell lysates were centrifuged (20 000 × g, 30 min) and supernatants were collected as cell free extracts (CFEs). CFEs were incubated with anti-EndoMS Cg antibody raised in rats for at 4 • C. After 1 h incubation, 50 l of protein G-Sepharose 4FF (GE healthcare) was added for a further 1 h. The precipitates were washed five times with HNTG buffer and SDS-PAGE 4× sample buffer was added.
Cell-free extracts (Input) and immunoprecipitated fraction (IP) were analyzed on 12% polyacrylamide gels. After transfer to Immobilon-P polyvinylidene fluoride membranes (Millipore), EndoMS Cg and DnaN Cg were detected with anti-EndoMS Cg and anti-DnaN Cg antibodies raised in mice.

Bio-layer interferometry (BLI) analysis
The BLItz system with Ni-NTA (NTA) biosensor (Pall For-teBio) was used to monitor BLI response. Analysis steps were consists of 30 s initial baseline, 120 s DnaN-loading, 120 s baseline, 120 s EndoMS association and 120 s En-doMS dissociation followed by Ni-NTA biosensor regeneration steps (

Electrophoretic mobility shift assay (EMSA)
Double stranded DNA with or without G/T mismatch was incubated with indicated components at 37 • C for 5 min in cleavage buffer. Then the samples were chilled on ice for 2 min and UV crosslink was conducted as described previously (3 × 10 5 J) (16). Resultant samples were subjected to native-PAGE in Tris-borate buffer (without EDTA).

Estimation of mutation rates by fluctuation tests
Mutation rates to rifampicin resistance was estimated using fluctuation tests as described (17). Mutant frequencies for each indicated strain were determined by starting 20 cultures (2 ml of BHI) from single colonies and growing them to saturation at 30 • C. Appropriate dilutions of the cultures were plated on BHI plates containing 2 g/ml rifampicin to determine the number of rifampicin resistant mutant and on BHI plates to determine the total cell count. The mutation rate was calculated using the Ma-Sandri-Sarkar maximum likelihood method (18) and confidence limits (CL) (17) were calculated as described using FALCOR web tool found at www.mitochondria.org/protocols/FALCOR.html (19).

Mutation accumulation
MA lines originated from single colonies isolated on BHI agar plates. Every day, a single colony isolated from each line was streaked onto a new BHI agar plate, with four lines per plate, and incubated at 30 • C for 24 h. Culture was initiated with 40 lines of the wild-type strain, 20 lines of the endoMS strain, 16 lines of the dnaE (D647G) strain, and 16 lines of the endoMS -dnaE (D647G) strain. MA lines were passaged through 82 single-cell bottlenecks for the wild-type strain, 7 bottlenecks for the endoMS strain, 7 bottlenecks for the dnaE (D647G) strain and 5 bottlenecks for the endoMS -dnaE (D647G) strain. For all strains, the number of cells in a colony ranged from 2.2-3.8 × 10 7 , with a log average of 3.0 × 10 7 cells, which indicated 24.8 generations.

Genomic DNA preparation and whole genome resequencing analysis
Single colonies from the endpoints of MA lines and from a parental stock of each strain were isolated and grown in BHI medium, and genomic DNA was extracted using a Qiagen DNeasy Blood & Tissue Kit. The genomes were subjected to MiSeq sequencing using Nextera XT library kits (Illumina, Inc., San Diego, CA, USA) according to the manufacturer's instructions. Approximately one million pair-end reads (301 base pairs [bp] × 2) were obtained from each genome. Sequence data obtained for the MA lines were archived in DDBJ under BioProject PRJDB6606.
Data analyses were performed using CLC Genomics Workbench software (Qiagen, Hilden, Germany). The reads from each line were trimmed by screening for base quality (quality score limit = 0.05; reads that contained greater than two ambiguous nucleotides or that were less than 15 bp in length were removed). Trimmed reads were mapped to the C. glutamicum ATCC13032 genome (NC 003450) with the following parameters: no masking mode, match score = 3, mismatch cost = 3, linear gap cost, insertion cost = 3, deletion cost = 3, length fraction = 0.5, similarity fraction = 0.99 and no global alignment. A basic variants detection analysis was performed with the following parameters: ploidy = 1, ignore positions with coverage above 100,000, restrict calling to target regions = not set, ignore broken pairs = no, ignore non-specific matches = reads, minimum coverage = 7, minimum count = 2, minimum frequency (%) = 90, base quality filter = yes, neighboring radius = 5, minimum central quality = 30, minimum neighboring quality = 30, read direction filter = no, relative read direction filter = yes, significance (%) = 1, read position filter = no and remove pyro-error variants = no. Single nucleotide polymorphisms (SNPs) detected in MA lines were compared with those in the parental stock, and SNPs detected only in MA lines were counted as mutations in the MA experiment. Because of the probable loss due to homologous recombination in some MA lines, mutations that occurred in the region from 1 958 664 to 1 994 456 were not counted. In the case of wild-type and endoMS strains, mapping of all mutation sites was manually confirmed, and SNPs that detected in ambiguous mapping locations (probably because of the existence of highly homologous sequences) were ignored. In the case of strains dnaE (D647G) and endoMS -dnaE (D647G), consensus bases across all MA lines were compared, and SNPs was counted as mutations if they were observed only in that line but not in the rest (6). To represent mutations as nucleotide changes in the leading strand, SNPs were categorized by the relative position to the ori (replication initiation site; position 1 or 3 309 401) and dif sites (replication termination site; position 1 551 515 (20,21)). When a mutation was located between positions 1 551 515 and 3 309 401 (the so-called left replicore), the mutation was represented as the mutational change of the bottom strand, which corresponded to the leading strand in the left replicore.

In silico screening of genes predominantly conserved in Actinobacteria
M. tuberculosis is the most important pathogen in Actinobacteria. Therefore, to identify alternative genes that compensate for the lack of a canonical mismatch repair system in Actinobacteria, BLAST searches against a protein database with whole genome sequences of 785 type strains of bacteria and archaea (Supplementary Table S1) were performed using the nucleotide sequence of M. tuberculosis H37Rv genes categorized as Clusters of Orthologous Groups (COGs) 'L' (replication, DNA repair and recombination) as queries, except for genes encoding mobile elements. From 142 queries, 20 candidate genes were identified that matched the defined criteria (see Methods and Supplementary Table S4).

Identification of genes working for the maintenance of low levels of spontaneous mutation
Because of the fast growth and simplicity of the method to construct a markerless gene deletion mutant, C. glutamicum was used as a model organism. Among 20 candidate genes, homologues of three genes (Rv1267c, Rv3114 and Rv3908) were not found in C. glutamicum. The C. glutamicum homologues of Rv2412 (NCgl2261) and Rv2738c (NCgl1806) were annotated as ribosomal protein and phage integrase, respectively, and were not analyzed further. We attempted to obtain a C. glutamicum in-frame deletion mutant strains with the remaining 15 genes, and deletion mutants of 12 genes, except for those of NCgl0004, NCgl2264 and NCgl2982, were obtained (Supplementary Table S4). An examination of the frequency of spontaneous rifampicin resistance mutations indicated that deletion of NCgl1168 (endoMS/nucS), corresponding to Rv1321 (endoMS/nucS) in M. tuberculosis (endoMS/nucS Mt ), increased the frequency of rifampicin resistance mutations (Supplementary Figure S1).
The rate of rifampicin resistance mutations estimated from the fluctuation assay was 8.2 × 10 −9 per cell per division in wild-type C. glutamicum. Thus, deletion of NCgl1168 (endoMS/nucS Cg ) showed a mutator phenotype, in which the rate of spontaneous mutation increased ∼280-fold. Complementation with a plasmid containing endoMS/nucS Cg completely restored the mutator phenotype, indicating the essentiality of endoMS/nucS Cg in maintaining the low spontaneous mutation rate in C. glutamicum ( Figure. 1A).

C. glutamicum EndoMS is mismatch-specific endonuclease and its nuclease activity is essential to maintaining a lower spontaneous mutation rate
EndoMS/NucS homologues have a RecB-like nuclease domain (10)(11)(12). Thermococcus kodakarensis En-doMS (EndoMS Tk ) has been reported to have mismatchdependent double-stranded DNA (dsDNA) cleavage activity, and a mutational analysis showed that point mutations in the RecB-nuclease motif resulted in the loss of activity (11). In contrast, purified Mycobacterium smegmatis EndoMS/NucS (EndoMS Ms ) has been reported not to have the ability to cleave mismatch-containing dsDNA (12). To examine whether EndoMS/NucS Cg has nuclease activity, we performed an in vitro cleavage assay using purified EndoMS/NucS Cg protein (Supplementary Figure S2). An analysis of EndoMS/NucS Cg cleavage products by native PAGE revealed that EndoMS/NucS Cg digested dsDNA in a mismatch-dependent manner ( Figure 1B, lanes 2-6). Although we found that the EndoMS/NucS Cg -dependent cleavage of G/T mismatch was more efficient under low salt condition containing more than 0.3 mM Mn 2+ ion, we used 100 mM potassium acetate with 10 mM Mg 2+ condition to examine the enzymatic activity under physiologically possible condition (Supplementary Figure S3). As observed for EndoMS Tk , our in vitro cleavage assay revealed that mutations in the RecB-nuclease motif (D144A) completely diminished the ability to cleave G/T mismatchcontaining dsDNA ( Figure 1B, lanes 8-11). These results clearly demonstrated that cleavage of mismatch containing dsDNA was dependent on intact EndoMS/NucS Cg enzyme but not on any other contaminated nuclease in purified EndoMS/NucS Cg fraction. Therefore, we use the name EndoMS Cg to refer to this enzyme hereafter. An examination of overexpression of a D144A mutant of EndoMS Cg in endoMS cells showed that the D144A mutant protein did not restore the mutator phenotype ( Figure 1A), although the expression level of the D144A mutant was comparable to that of the wild-type protein (Supplementary Figure S4), indicating the essentiality of nuclease activity to lower mutation rate. The number of EndoMS Cg molecules in a wildtype cell was estimated to be 14 molecules per one cell, given that 1 bacterial cell contains 0.15 pg protein. Although this value was about one order lower than that of T. kodakarensis (11), our observation that the mutation rate of cells overexpressing EndoMS Cg (Supplementary Figure S4) was not different from that of WT cells ( Figure 1A, 15 versus 8.3 × 10 −9 per cell per division, respectively, P = 0.17 (n = 20), Mann-Whitney test) suggests that the expression level of EndoMS Cg in wild-type C. glutamicum cells is sufficient. To determine the cleavage sites, 5 -FAM-labeled dsDNA was subjected to an in vitro cleavage assay followed by denaturing PAGE ( Figure 1C). The results showed that EndoMS Cg cleaved the 5 side of the mismatched base in both strands to produce a cohesive end with a three-nucleotide long 5protrusion.
When M. tuberculosis EndoMS/NucS (EndoMS Mt ) or EndoMS Tk was overexpressed in endoMS of C. glutamicum, the mutator phenotype was partially restored, whereas the RecB-nuclease motif mutant (D137A of EndoMS Mt or D165A of EndoMS Tk ) did not decrease mutation rate, confirming the importance of the RecB-nuclease motif maintaining the low mutation rate ( Figure 1A).

Interaction of EndoMS with the sliding clamp (beta-clamp) enhances EndoMS activity and is essential to maintaining a lower mutation rate
The existence of a beta-clamp (DnaN) binding motif in the C-terminus of EndoMS ( Figure 2A) prompted us to examine whether EndoMS interacts with DnaN. Although an immunoblot analysis following immunoprecipitation of either EndoMS Cg or DnaN Cg under normal conditions did not result in the detection of their counterparts in the immunoprecipitated (IP) fraction (data not shown), crosslinking treatment of cells using formaldehyde enabled us to detect DnaN Cg in the EndoMS Cg IP fraction ( Figure 2B, lower panel). As expected, when the endoMS-Cdel strain was subjected to immunoprecipitation assay, co-recovery of the DnaN Cg protein with EndoMS-Cdel was severely reduced. Direct interaction between EndoMS Cg and DnaN Cg was confirmed by Bio-layer interferometry (BLI) analysis using purified proteins (Supplementary Figure S5). Loading of EndoMS Cg wild-type and D144A mutant proteins after DnaN Cg preloading showed BLI signal at association step (Supplementary Figure S5A and B). In contrast, as expected, such binding signal was not observed when DnaN pre-loading was not conducted or EndoMS Cg Cdel mutant was used (Supplementary Figure S5C), although baseline shift caused by unknown reason was observed between association and dissociation steps. Estimated KD values of EndoMS Cg wild-type and D144A mutant proteins were 185 nM and 182 nM, respectively, under our assay condition.
We next examined whether disruption of DnaN-EndoMS interactions affected the mutation rate in vivo. Surprisingly, the mutation rate of the endoMS-Cdel strain was almost the same as that of the endoMS gene disruptant ( Figure  2C). We also confirmed that overexpression of EndoMS Cg -Cdel mutant in endoMS Cg cells using strong gapA promoter could not restore the mutation rate at all (Supplementary Figures S4 and S6A). This result indicates that DnaN-EndoMS interaction itself is essential for EndoMS to maintain a lower spontaneous mutation rate. An in vitro cleavage assay using the EndoMS Cg -Cdel mutant protein showed that EndoMS Cg -Cdel cleaved mismatched ds-DNA as efficiently as intact EndoMS Cg did ( Figure 2D, −DnaN Cg ). We next examined the effect of the DnaN Cg protein on activity of the nuclease. As expected, the addition of DnaN Cg considerably enhanced the cleavage efficiency of intact EndoMS Cg but not that of EndoMS Cg -Cdel ( Figure  2D Figure S6E). In consistent with these idea, cleavage efficiency of EndoMS Cg -Cdel mutant was slightly but reproducibly inhibited by addition of DnaN Cg , probably due to the masking effect of DnaN Cg loaded on to the substrate DNA ( Figure 2D, lanes  7-11 and lanes 18-22). A quantitative analysis of the specific activity of EndoMS Cg in the presence or absence of DnaN Cg revealed that DnaN Cg increased EndoMS Cg activity about 30-fold ( Figure 2E and Supplementary Figure S7A). We then tried to reveal the effects of DnaN Cg -EndoMS Cg interaction on the affinity of EndoMS Cg to the DNA by performing electrophoretic mobility shift assay (EMSA). Although both wild-type (Supplementary Figure  S7B) and D144A mutant (data not shown) of EndoMS Cg were used, any upshifted bands were not detected. Instead, in the presence of DnaN Cg , cleavage products were detected. Consequently, we could not clarify whether DnaN Cg binding to EndoMS Cg increase the affinity of EndoMS Cg to DNA or not. However, it should be noted that these results were quite different from that of EndoMS Tk in which band shift caused by EndoMS Tk -DNA complex formation could be detected (11). These observations suggest that regardless of the presence or absence of DnaN Cg , the dissoci- ation of EndoMS Cg from DNA is much faster than that of EndoMS Tk . When the preference for mismatch base-pair type was examined, EndoMS Cg digested G-G, G-T and T-T mismatches flanking to A residues, but not others ( Figure  2F). Even when nucleotides flanking to the mismatch were changed to G residues, the mismatch base-pair specificity was not changed (Supplementary Figure S7C), confirming that EndoMS Cg is able to digest G-G, G-T and T-T mismatches independent of the flanking nucleotides.

The EndoMS-dependent pathway corrects replication errors
Since the canonical mismatch repair system works to repair replication errors, we attempted to determine whether the EndoMS-dependent mutation repair pathway was involved in the correction of replication errors. For this purpose, we examined the relationship between DNA polymerase fidelity and the effects of endoMS gene disruption. If EndoMS participates in the correction of replication errors, the combination of endoMS gene disruption and de-Nucleic Acids Research, 2018, Vol. 46, No. 12 6159 creased DNA polymerase fidelity would have synergistic effects on the mutation rate (23). Conversely, if the EndoMSdependent mutation repair pathway was independent of replication, the effects would be additive.
Overexpression of the dnaE E612K mutant in E. coli has been reported to increase mutation frequency (24) and confer hyper-processivity to the polymerase (25,26). In C. glutamicum dnaE, although the amino acid residue of the corresponding position was aspartic acid (D647) rather than glutamate, overexpression of the D647G mutant protein increased mutation frequency (Supplementary Figure S8). In the course of our examinations, we noticed that the plasmid containing the D647G mutant was instable. Therefore, we introduced the D647G mutation into the chromosomal dnaE locus. The mutation rate of the resulting strain, determined by fluctuation assay, was 0.85 × 10 −6 per cell per division, which was ∼100-fold higher than the rate of the wild-type strain ( Figure 3A). The mutation rate in the dnaE (D647G) and endoMS double mutant strain was 8.9 × 10 −6 per cell per division, which was ∼2.7-fold higher than the sum of the mutation rate in the dnaE (D647G) strain and the endoMS strain (0.85 × 10 −6 and 2.3 × 10 −6 per cell per division, respectively), indicating that the effects of reducing replication fidelity and disrupting the endoMS gene on the mutation rate are synergistic (23).

The EndoMS-dependent pathway is specific to transitions and some transversions
A mutation accumulation (MA) assay enabled us to detect neutral mutations, including base-pair substitutions (BPSs) and short insertions and deletions (indels) that occur throughout the genome (6,9,(27)(28)(29). Therefore, the MA assay was performed in wild-type, dnaE (D647G), endoMS, and endoMS-dnaE (D647G) strains. The results of the MA assay are summarized in Supplementary Table S5 and  Table 1, and the rates of each mutation are presented (Tables  2 and 3). A whole genome re-sequencing analysis of 37 lines of wild-type strains passaged for about 2000 generations resulted in the detection of 240 BPSs and 25 indels. The overall mutation rate was 1.1 × 10 −9 per nucleotide per generation (Table 2). This value was almost comparable to that observed in M. smegmatis (9). The ratio of the number of BPSs that occurred in the coding versus non-coding DNA was 4.3 (195/45), which is similar to the expected value of 6.6 (chi-squared test 2 = 2.6, P = 0.11). In addition, the ratio of nonsynonymous to synonymous nucleotide changes determined by the MA assay was 2.0 (131/64), which was not significantly different from that expected based on the codon usage in C. glutamicum ATCC13032 strain (3.1 [nonsynonymous/synonymous], chi-squared test 2 = 3.4, P = 0.07) (30). These observations suggest that the effects of selective pressure were minimal in the MA assay. Two-thirds of the BPSs (168/240) observed in the analysis of the wild-type strain were transitions, indicating a bias towards transitions as observed in other species (6,9,31,32). The remaining one-third of BPSs (72/240) were transversion mutations, and, among them, 75% (54/72) were A:T ↔ G:C mutations (Table 1).
When replication fidelity was reduced by the D647G mutation in dnaE gene, rates of all types of mutations increased more than 50-folds, except for that of G:C ↔ C:G transversions (Table 2). Although the extent of the mutation rate increase was slightly larger in indels, a correlation analysis of mutation compositions between the wild-type and dnaE (D647G) strains showed good correlation (Supplementary Figure S9, r = 0.85). Especially with respect to BPSs, the correlation between the mutational composition of the two strains was high (Supplementary Figure S9, r = 0.96). These results indicate that the D647G mutation in dnaE successfully reduced replication fidelity to a similar extent in most BPS types. The observed ratio of BPSs in coding versus non-coding regions was 5.6 (395/71), which was not significantly different from the expected value of 6.6 (chi-squared test 2 = 0.7, P = 0.41). However, we detected significant differences between observed and expected ratios of nonsynonymous to synonymous nucleotide changes (265/130 [nonsynonymous/synonymous], chi-squared test 2 = 7, P = 0.008), implying that we could not exclude the possibility of some selection pressure during culture for ∼170 generations. This was probably due to the high mutation rate, which caused one mutation to accumulate per every six generations, suggesting that the dnaE (D647G) strain would be an appropriate model to reduce the time to accumulate natural mutations in our culture conditions. Disruption of endoMS in the wild-type background increased the transition mutation rate more than 50-fold, but the effect on transversions and indels was not obvious (Table 2). As a result, almost all mutations detected were transitions (526/530). Although a 2-to 3-fold increase was observed for some transversions, the number of detected mutations was very small and statistical power was insufficient to obtain a precise mutation rate for such mutations (Table  1).
When endoMS was disrupted in the background of dnaE (D647G), the rate of transitions increased 5-to 31-fold. As a result, the overall transition mutation rate in the endoMS-dnaE (D647G) strain was 3.8 × 10 −7 per nucleotide per generation, which was ∼4.9-fold higher than the sum of the mutation rates in the endoMS and dnaE (D647G) strains (4.6 × 10 −8 and 3.3 × 10 −8 per nucleotide per generation, respectively; Figure 3B). Thus, by focusing on the preferred target for the EndoMS-dependent pathway, the synergistic effects of replication fidelity reduction and endoMS gene disruption were more obvious. In contrast to transition mutations, the mutation rates of A:T ↔ C:G transversions and indels were not affected by endoMS. However, although the numbers of mutations detected were small (Table 1), the rates of A ↔ T and G ↔ C transversions tended to increase slightly with the loss of endoMS ( Table 2).

Effects of local sequence context on the occurrence of mutations
To verify the influence of flanking nucleotide sequences on the occurrence of BPSs, we focused on nucleotides immediately 5 (upstream) or 3 (downstream) to the BPS site on the leading strand (Supplementary Figure S10). When the nucleotides upstream of the BPS site were evaluated, 67% (161/240) and 69% (365/530) were G or C in the wild-type and endoMS, respectively (Supplementary Figure S10A and S10B). Similarly, the base pairs immediately down-  Table 2). Overall transition mutation rates are shown. Average and standard errors for all lines are presented.   Figure S10). Such a bias toward GC base pairing in neigh-boring nucleotides was also observed in the dnaE background (D647G); however, the extent of the bias was slightly decreased (Supplementary Figure S10). The fractions of BPSs that immediately flanked the GC base pair site were 84% (392/464) in dnaE (D647G) and 83% (2242/2677) in endoMS-dnaE (D647G). No indels were detected in MA assay of endoMS and the column for endoMS is not shown.
When 1-base pair indels were considered, 91% (20/22) were insertions or deletions in single mononucleotide repeat sites of more than four bases in the wild type and 68% (15/22) were located at five to seven base repeat sites (Table  3). These results were consistent with previous observations that indels are caused by slippage of the DNA polymerase at mononucleotide repeat sites (33,34). In the dnaE (D647G) strain, the occurrence of indels at shorter mononucleotide repeat sites increased. These results indicate that the D647G mutation in dnaE increased the frequency of slippage at short mononucleotide repeat sites. Interestingly, although the mutation rate of indels at mononucleotide repeat sites in the endoMS-dnaE (D647G) strain was comparable to that in the dnaE (D647G) strain, indels at no repeat sites increased 4.5-fold with the endoMS disruption (Table 3). These observations are consistent with the results of the in vitro cleavage assay of EndoMS Cg , in which an indel was cleaved in non-repeat sequences in the presence of DnaN Cg , but not in repeat sequences (Supplementary Figure S11).

DISCUSSION
Very recently, disruption of the endoMS/nucS gene in M. smegmatis and S. coelicolor was reported to considerably increase the spontaneous mutation rate of these bacteria (12); however, a biochemical analysis of mycobacterial EndoMS/NucS failed to detect DNA cleavage activity. In contrast, here, we showed that purified C. glutamicum EndoMS/NucS was able to cleave dsDNA containing mismatches by itself ( Figure 1B). A mutation in the RecB nuclease motif resulted in the loss of both nuclease activity and the ability to complement endoMS gene disruption in C. glutamicum ( Figure 1A and B). We also showed that overexpression of M. tuberculosis endoMS/nucS complemented the loss of endoMS Cg in C. glutamicum. However, mutation in the RecB nuclease motif completely diminished the complementation ability of endoMS/nucS Mt ( Figure 1A). Based on these observations, we suggest that mycobacterial EndoMS/NucS also has mismatch-specific nuclease activity and that some experimental differences may have impaired the ability to detect this activity in the previous study.
In addition to demonstrating EndoMS nuclease activity, we revealed that direct interaction of EndoMS with DnaN was essential to maintaining a lower mutation rate ( Figure  2, Supplementary Figures S5 and S6). We also succeeded in demonstrating DnaN-dependent enhancement of cleav-age efficiency of EndoMS. Then, how DnaN clamp enhance the EndoMS cleavage efficiency? One possibility is that DnaN interaction with EndoMS allosterically activates the EndoMS enzymatic activity. In the case of MutL of B. subtilis, it has been suggested that DnaN-MutL interaction, following MutL recruitment to the mismatch site by MutS-MutL interaction, coordinately causes large conformational change of MutL dimer, licensing the endonuclease activity of MutL (35). In the case of human flap endonuclease 1 (FEN1) which functions in both replication and repair, it has been reported that sliding clamp alters both Km and Vmax of FEN1 nuclease activity. The authors suggested that in addition to large decrease in Km by tethering the enzyme near the cleavage site, sliding clamp alters the conformation or orientation of FEN1, resulting in a small improvement of catalysis (36). Thus, there are various mechanisms in activation of nuclease by sliding clamp. Another possibility is that DnaN tethers EndoMS to the zone at where replication, and mismatches caused thereby, occurs and this tethering enhances the mismatch detection efficiency. In B. subtilis, it has been reported that elimination of DnaN-MutS interaction by deleting MutS C-terminal clamp binding region almost completely disrupted in vivo MMR and that the interaction is required for coupling of MutS with replisome prior to mismatch recognition (37,38). The authors also demonstrated that overexpression of this DnaN-interaction defective MutS mutant restored MMR, indicating MutS-DnaN interaction is required to increase accessibility of MutS to mismatch (37). Here, we demonstrated that overexpression of EndoMS Cg -Cdel mutant in endoMS Cg could not restore the mutation rate at all (Supplementary Figure S6A), suggesting that facilitation of mismatch detection is not the only work of EndoMS-DnaN interaction. Therefore, DnaN interaction with EndoMS may cause some conformational change of EndoMS to facilitate the cleavage activity and the allosteric activation of En-doMS may be the essential work of DnaN-EndoMS interaction in EndoMS-type MMR system. More detailed biochemical, structural and imaging analyses will uncover the role of DnaN in EndoMS-type MMR system.
Together with results showing that disruption of endoMS and a mutation in DNA polymerase DnaE had synergistic effects on the mutation rate (Figure 3), we conclude that En-doMS functions in cooperation with DNA polymerase to correct replication errors. These observations clearly indi-cated that the EndoMS-dependent MMR system conserved in organisms lacking mutS and mutL genes is the counterpart of the MutS-MutL-dependent canonical MMR system. Since the first report of the absence of mutS and mutL genes in M. tuberculosis (39,40), as well as the demonstration of comparable mutation rates between M. tuberculosis and other organisms with canonical MMR systems (41), the gap between the absence of a canonical MMR system and the presence of comparable mutation rate in Actinobacteria and organisms equipped with canonical MMR system has been an intriguing area for inquiry. Here, we established the first evidence that EndoMS plays the central role in the repair of replication errors, the most important task of a canonical MMR system, addressed the gap that has existed for two decades.
Interestingly, archaeal endoMS (endoMS Tk ) was able to partially complement the loss of endoMS in C. glutamicum in C-terminal sliding clamp binding motif-dependent manner (Supplementary Figure S6A). These results suggest that EndoMS Tk is able to interact with the bacterial sliding clamp as bacterial EndoMS does. We also observed that EndoMS Cg cleaved both strands of DNA in miamatch-dependent manner as observed in archaeal En-doMS. These observations strongly suggest that the bacterial and archaeal EndoMS-dependent MMR pathways utilize the same repair system after mismatch DNA cleavage. Because archaeal EndoMS can cause mismatch-dependent double-stranded breaks in vitro, together with the observation that T. kodakarensis genome is polyploid (42), homologous recombination repair has been postulated to be the downstream repair pathway (11,43). Alternatively, interaction with sliding clamp may provide strand information for EndoMS and the interaction may inhibit the cleavage of template strand as observed in eukaryotic MMR system (44). Although the possibility that sliding clamp-dependent strand discrimination should be tested as performed previously (44), we favor the homologous recombination hypothesis, because of the advantage of using homologous recombination is that there is no need to discriminate the nascent strand. Although, polyploidy has advantages in utilizing homologous recombination as a pathway to repair double strand breaks, it is well known that non-polyploid bacteria such as E. coli utilize homologous recombination to repair double strand breaks, suggesting that polyploidy is not essential to repair double strand breaks by homologous recombination. It is also interesting that, in some species harboring canonical MMR systems, the mechanism by which these systems discriminates the nascent strand has not been uncovered to date (45). For example, reconstitution experiments of a Thermus thermophilus MMR system revealed that MutL of this species can cleave both strands of mismatched DNA and that this activity is not affected by the sliding clamp (46). From these observations, together with previous and present findings in non-canonical MMR systems, it seems reasonable to consider homologous recombination as the alternative pathway to complete MMR without strand discrimination.
Application of a mutation accumulation assay revealed the following points. First and most importantly, a synergistic effect was observed with the combination of endoMS and the D647G mutation in dnaE, confirming that EndoMS is involved in the repair of replication errors ( Figure 3B).
Second, the types of mutations that increased with the en-doMS deletion were consistent with the substrate specificity of EndoMS. As shown in Figure 2F and Supplementary Figure S7C, EndoMS was able to cleave mismatches leading to transitions (G/T mismatch) and A ↔ T and G ↔ C transversions (T/T and G/G mismatches, respectively), but not those leading to A:T ↔ C:G transversions. In the case of indels, EndoMS Cg activated by DnaN Cg was able to cleave DNA substrates containing a 1-base indel in neighboring DNA in a nucleotide-dependent manner (Supplementary Figure S11). These results are consistent with the in vivo mutation spectrum (Tables 1-3). These observations strongly indicate that the biochemical properties of EndoMS Cg observed in this study reflect the in vivo function of the system.
Third, an MA assay of the endoMS strain of C. glutamicum revealed that most BPSs occurred at the site immediately flanking a GC base pair. These cases are very similar to those reported previously, in which E. coli, Pseudomonas fluorescens, and B. subtilis strains lacking canonical MMR system were analyzed (6,32,47). This consistency suggests that the basis for these mismatches is the same in each of these organisms. In addition, the fact that the mechanisms underlying proofreading differs between these organisms suggests that pre-proofreading mismatch generation mechanisms such as new nucleotide incorporation may be similar between these organisms. Unlike E. coli, in which the presence of an MMR system affects the occurrence of mutations in specific local sequence contexts (6), the presence of endoMS had no effects on the sequences neighboring mutations detected in C. glutamicum (Supplementary Figure  S10A and B). These results suggest that, whereas a MutSdependent canonical MMR system has a preference for a local sequence context and efficiently corrects mismatches to the sides of a GC base pair, a EndoMS-dependent system has no such preference for nucleotides immediately flanking the mismatch. These observations were consistent with our biochemical results that neighboring sequences did not affect the mismatch-type specificity of EndoMS Cg ( Figure  2F and Supplementary Figure S7C). These results are also consistent with the biochemical properties of T. kodakarensis EndoMS, whose efficiency in cleaving G/T mismatches is not affected by neighboring sequences (11,43).
It is noteworthy that unlike canonical MMR system in which broad types of mismatches are targeted (3)(4)(5), the En-doMS enzyme showed high specificity for G/T mismatches; therefore, we were able to discriminate whether C/A or G/T mismatches were likely to form during replication ( Figure  4). For example, when the origin of C→T transition in the leading strand is considered, there are two possibilities; C/A mismatch generated during lagging strand synthesis and G/T mismatch generated during leading strand synthesis. Because EndoMS is able to cleave only G/T mismatch, C→T transition mutation increased by disruption of en-doMS gene reflects the frequency of G/T mismatch occurrence during leading strand synthesis (Figure 4  fectly and is able to correct all of the G/T mismatches, the rate of transition mutations in the wild-type is expected to correspond to the frequency of replication errors that generates C/A mismatches. Inversely, a lower efficiency of the EndoMS-dependent pathway means a lower frequency of C/A mismatches. With respect to the C/A mismatch elimination pathway, although the effect on the mutation rate is much smaller than that of the MutS-dependent MMR system, the MutY N-glycosylase/AP (apurinic/apyrimidinic) endonuclease eliminates C/A mismatches in the absence of a MutS-dependent MMR system in E. coli (48,49). In C. glutamicum, we evaluated the effect of the mutY gene on the mutation rate and revealed that the mutY gene disruption only had a minor effect (Supplementary Figure S12). Similarly, in M. smegmatis, the lack of a mutY gene only slightly increased the rate of AT → GC transitions, suggesting the successful correction of C/A mismatches by MutY, the extent of increase was much smaller than the disruption of the EndoMS-dependent pathway (12,50). Although we are unable to rule out the possibility that some other mechanism functioned to eliminate C/A mismatches, our data, together with previous reports, indicate that the dnaEtype DNA polymerase is prone to generating many more G/T mismatches than C/A mismatches. Thus, by analyzing a G/T mismatch-specific MMR system, we succeeded in determining fundamental properties of DNA replication in vivo. Application of this non-canonical and asymmetric MMR system in other species will be required to demonstrate the generality of our hypothesis that G/T mismatches are much more likely to occur in vivo. Elimination of frequent G/T mismatches might be the most important task for the MMR system, regardless of the types of proteins in the system. The higher binding specificity of MutS for G/T mismatches than for other mismatches (3) seems to be an important property that allows this system to address the high frequency of G/T mismatches. Indeed, it has been reported that G/T mismatches are the most stable of all mismatched bases (51). Also, a biochemical analysis of the E. coli PolII ␣ subunit without the proofreading ε subunit showed that the misincorporations that generate G/T mismatches are about 3-fold more frequent than the misincorporations that generate C/A mismatches (52). In addition to such fundamental properties of the polymerase, some other factors such as local concentrations of nucleotide substrates might lead to higher frequencies of G/T mismatches (53)(54)(55).
The results of the MA assay of the endoMS strain, in which EndoMS-dependent post replication repair is absent, showed that the number of C → T transitions detected was significantly more than that of G → A transitions in the leading strand (Figure 4, upper panel and Table 1, 185 versus 105, chi-squared test 2 = 11.2, P < 0.001). Because G → A mutations in the leading strand correspond to C → T mutations in the lagging strand, C → T mutations are more likely to occur in the leading strand than in the lagging strand in the endoMS strain (Figure 4, upper panel). Together with the fact that EndoMS is able to cleave G/T mismatches but not C/A mismatches (Figure 4, green box) and that these mutations arise mainly because of the loss of the EndoMS-dependent pathway, the frequency of misincorporation of T at the template G site is expected to be higher in the leading strand than in the lagging strand. Similarly, although there was no statistical significance, A → G transitions seemed to be more likely to occur than T → C transi-tions in the leading strand ( Figure 4, lower panel, and Table  1, 137 versus 99, chi-squared test 2 = 3.1, P = 0.08), suggesting that the frequency of misincorporation of G at template T site may be higher in the leading strand than in the lagging strand. In the presence of intact endoMS, a similar preference for C → T and A → G mutations in the leading strand was observed, although the extent of this preference was slightly different from that in the endoMS strain. In the wild-type, the preference for A → G to T → C mutation was statistically significant (48 versus 13, chi-squared test 2 = 10.9, P < 0.001), but that for C → T to G → A was not (67 versus 40, chi-squared test 2 = 3.5, P = 0.06). In the case of AT ↔ CG transversions, no bias was observed. Comparison of the mutation rates in all BPS types revealed that the mutational pattern would likely produce more Ts and Gs than As and Cs, respectively, in the leading strand (Table 2 and Figure 4). This bias is consistent with the composition of nucleotides in the C. glutamicum genome and might be contributes to the formation of GC skew. These results are similar to those of E. coli and S. cerevisiae, in which the patterns of neutral mutations occurring in synonymous sites and intergenic regions are consistent with the nucleotide compositions of the genomes (6,56). Interestingly, in contrast to previous reports, our results enabled us to speculate about the origin of this mutational skew. When the misincorporation frequency was increased with the D647G mutation of dnaE, although the preference for C → T and A → G mutations in the leading strand was observed as in the wild-type, disruption of endoMS completely abolished such a strand-specific preference in these mutations (Tables 1 and  2). These observations suggest that a strand-specific preference in mutations is based on the combination of biases in nucleotide misincorporation and in the mismatch repair by the EndoMS pathway. Together with these observations, future analyses such as experimental evolution of these strains or precise measurement of strand-specific mutation rates will lead to new insights into the relationship between GC skewing and the strand-specific mutational spectrum.

DATA AVAILABILITY
Sequence data obtained for the MA lines were archived in DDBJ under BioProject PRJDB6606.