The UvrD helicase and its modulation by the mismatch repair protein MutL

UvrD is a superfamily I DNA helicase with well documented roles in excision repair and methyl-directed mismatch repair (MMR) in addition to poorly understood roles in replication and recombination. The MutL protein is a homodimeric DNA-stimulated ATPase that plays a central role in MMR in Escherichia coli. This protein has been characterized as the master regulator of mismatch repair since it interacts with and modulates the activity of several other proteins involved in the mismatch repair pathway including MutS, MutH and UvrD. Here we present a brief summary of recent studies directed toward arriving at a better understanding of the interaction between MutL and UvrD, and the impact of this interaction on the activity of UvrD and its role in mismatch repair.     

Stoichiometry of MutS and MutL at unrepaired mismatches in vivo suggests a mechanism of repair

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.     

The coordinated functions of the E. coli MutS and MutL proteins in mismatch repair

The Escherichia coli MutS and MutL proteins have been conserved throughout evolution, although their combined functions in mismatch repair (MMR) are poorly understood. We have used biochemical and genetic studies to ascertain a physiologically relevant mechanism for MMR. The MutS protein functions as a regional lesion sensor. ADP-bound MutS specifically recognizes a mismatch. Repetitive rounds of mismatch-provoked ADP-->ATP exchange results in the loading of multiple MutS hydrolysis-independent sliding clamps onto the adjoining duplex DNA. MutL can only associate with ATP-bound MutS sliding clamps. Interaction of the MutS-MutL sliding clamp complex with MutH triggers ATP binding by MutL that enhances the endonuclease activity of MutH. Additionally, MutL promotes ATP binding-independent turnover of idle MutS sliding clamps. These results support a model of MMR that relies on two dynamic and redundant ATP-regulated molecular switches.     

DNA mismatch repair and mutation avoidance pathways

Unpaired and mispaired bases in DNA can arise by replication errors, spontaneous or induced base modifications, and during recombination. The major pathway for correction of mismatches arising during replication is the MutHLS pathway of Escherichia coli and related pathways in other organisms. MutS initiates repair by binding to the mismatch, and activates together with MutL the MutH endonuclease, which incises at hemimethylated dam sites and thereby mediates strand discrimination. Multiple MutS and MutL homologues exist in eukaryotes, which play different roles in the mismatch repair (MMR) pathway or in recombination. No MutH homologues have been identified in eukaryotes, suggesting that strand discrimination is different to E. coli. Repair can be initiated by the heterodimers MSH2-MSH6 (MutSalpha) and MSH2-MSH3 (MutSbeta). Interestingly, MSH3 (and thus MutSbeta) is missing in some genomes, as for example in Drosophila, or is present as in Schizosaccharomyces pombe but appears to play no role in MMR. MLH1-PMS1 (MutLalpha) is the major MutL homologous heterodimer. Again some, but not all, eukaryotes have additional MutL homologues, which all form a heterodimer with MLH1 and which play a minor role in MMR. Additional factors with a possible function in eukaryotic MMR are PCNA, EXO1, and the DNA polymerases delta and epsilon. MMR-independent pathways or factors that can process some types of mismatches in DNA are nucleotide-excision repair (NER), some base excision repair (BER) glycosylases, and the flap endonuclease FEN-1. A pathway has been identified in Saccharomyces cerevisiae and human that corrects loops with about 16 to several hundreds of unpaired nucleotides. Such large loops cannot be processed by MMR.     

Trapping and visualizing intermediate steps in the mismatch repair pathway in vivo

During mismatch repair, MutS is responsible for mismatch detection and the recruitment of MutL to the mismatch through a mechanism that is unknown in most organisms. Here, we identified a discrete site on MutS that is occupied by MutL in Bacillus subtilis. The MutL binding site is composed of two adjacent phenylalanine residues located laterally in an exposed loop of MutS. Disruption of this site renders MutS defective in binding MutL in vitro and in vivo, while also eliminating mismatch repair. Analysis of MutS repair complexes in vivo shows that MutS mutants defective in interaction with MutL are ‘trapped’ in a repetitive loading response. Furthermore, these mutant MutS repair complexes persist on DNA away from the DNA polymerase, suggesting that MutS remains loaded on mismatch proximal DNA awaiting arrival of MutL. We also provide evidence that MutS and MutL interact independent of mismatch binding by MutS in vivo and in vitro, suggesting that MutL can transiently probe MutS to determine if MutS is mismatch bound. Together, these data provide insights into the mechanism that MutS employs to recruit MutL, and the consequences that ensue when MutL recruitment is blocked.     

Spatial coupling between DNA replication and mismatch repair in Caulobacter crescentus

The DNA mismatch repair (MMR) process detects and corrects replication errors in organisms ranging from bacteria to humans. In most bacteria, it is initiated by MutS detecting mismatches and MutL nicking the mismatch-containing DNA strand. Here, we show that MMR reduces the appearance of rifampicin resistances more than a 100-fold in the Caulobacter crescentus Alphaproteobacterium. Using fluorescently-tagged and functional MutS and MutL proteins, live cell microscopy experiments showed that MutS is usually associated with the replisome during the whole S-phase of the C. crescentus cell cycle, while MutL molecules may display a more dynamic association with the replisome. Thus, MMR components appear to use a 1D-scanning mode to search for rare mismatches, although the spatial association between MutS and the replisome is dispensible under standard growth conditions. Conversely, the spatial association of MutL with the replisome appears as critical for MMR in C. crescentus, suggesting a model where the β-sliding clamp licences the endonuclease activity of MutL right behind the replication fork where mismatches are generated. The spatial association between MMR and replisome components may also play a role in speeding up MMR and/or in recognizing which strand needs to be repaired in a variety of Alphaproteobacteria.     

Biochemical characterization of mismatch-binding protein MutS1 and nicking endonuclease MutL from a euryarchaeon Methanosaeta thermophila

In eukaryotes and most bacteria, the MutS1/MutL-dependent mismatch repair system (MMR) corrects DNA mismatches that arise as replication errors. MutS1 recognizes mismatched DNA and stimulates the nicking endonuclease activity of MutL to incise mismatch-containing DNA. In archaea, there has been no experimental evidence to support the existence of the MutS1/MutL-dependent MMR. Instead, it was revealed that a large part of archaea possess mismatch-specific endonuclease EndoMS, indicating that the EndoMS-dependent MMR is widely adopted in archaea. However, some archaeal genomes encode MutS1 and MutL homologs, and their molecular functions have not been revealed. In this study, we purified and characterized recombinant MutS1 and the C-terminal endonuclease domain of MutL from a methanogenic archaeon Methanosaeta thermophila (mtMutS1 and the mtMutL CTD, respectively). mtMutS1 bound to mismatched DNAs with a higher affinity than to perfectly-matched and other structured DNAs, which resembles the DNA-binding specificities of eukaryotic and bacterial MutS1 homologs. The mtMutL CTD showed a Mn²⁺/Ni²⁺/Co²⁺-dependent nicking endonuclease activity that introduces single-strand breaks into a circular double-stranded DNA. The nicking endonuclease activity of the mtMutL CTD was impaired by mutagenizing the metal-binding motif that is identical to those of eukaryotic and bacterial MutL endonucleases. These results raise the possibility that not only the EndoMS-dependent MMR but also the traditional MutS1/MutL-dependent MMR exist in archaea.     

Functions of the Mismatch Repair Gene mutS from Acinetobacter sp. Strain ADP1

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.     

The Escherichia coli Mismatch Repair Protein MutL Recruits the Vsr and MutH Endonucleases in Response to DNA Damage

The activities of the Vsr and MutH endonucleases of Escherichia coli are stimulated by MutL. The interaction of MutL with each enzyme is enhanced in vivo by 2-aminopurine treatment and by inactivation of the mutY gene. We hypothesize that MutL recruits the endonucleases to sites of DNA damage.The Escherichia coli Dcm protein methylates the second C of CCWGG sites (W = A or T). Deamination of 5-methylcytosine converts CG base pairs to T/G mismatches, causing CCWGG-to-CTWGG transition mutations. Very-short-patch (VSP) repair minimizes these mutations (2). Repair is initiated by a sequence- and mismatch-specific endonuclease, Vsr, which cleaves the DNA 5′ of the T. DNA polymerase I removes the T along with a few 3′ nucleotides and resynthesizes the missing bases, restoring the CG base pair. Vsr is both necessary and sufficient for initiating VSP repair. However, two other proteins, MutS and MutL, enhance VSP repair of deamination damage (1).MutS and MutL are best known for their roles in postreplication mismatch repair (MMR) (9, 11). MutL couples mismatch recognition by MutS to the activation of MutH, an endonuclease that cleaves the unmethylated strand of GATC sequences that are transiently hemimethylated following DNA replication. The nicked strand, containing the erroneous base, is removed by the UvrD helicase and one of several exonucleases to beyond the mismatch and then resynthesized by DNA polymerase III.MutL stimulates the endonuclease activities of both Vsr and MutH in vitro (8, 17). The requirements for stimulation are the same: a mismatch, MutS, and ATP hydrolysis by MutL (8, 8a). Cross-linking studies showed that MutH and Vsr interact with the same region in the N-terminal domain of MutL (Heinze et al., submitted). Competition of Vsr with MutH for access to MutL explains the ability of Vsr to inactivate MMR in vivo when overexpressed (6, 13). Thus, the interactions of the two repair endonucleases with MutL are structurally and functionally very similar.In contrast to MMR, where the cleavage site for MutH may be several kilobases away from the mismatch, VSP repair requires that mismatch recognition and endonucleolytic cleavage occur at the same C(T/G)WGG site. How MutS and MutL stimulate VSP repair if MutS and Vsr compete for the same mismatch remains unknown (2, 12). We hypothesized that MutS binds the mismatch first and that a MutS-MutL complex then recruits Vsr. If so, then the MMR proteins would initially mask the mismatch, making the interaction of Vsr with MutL independent of lesion identity.To test this hypothesis, we studied the interaction of MutL with Vsr and with MutH in response to two types of mismatch by using a bacterial two-hybrid assay (10). This assay detects all known interactions among the Mut proteins: homodimerization of MutS and MutL, interaction of MutL with MutS and with MutH, and interaction of Vsr with the N-terminal domain of MutL (15). We found no false positives or false negatives. Furthermore, since the assay relies on reconstitution of a soluble protein (adenylate cyclase), the DNA repair proteins are free to interact with the DNA (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Known interactions among repair proteins as detected by the bacterial two-hybrid assay. The T18 and T25 subunits of CyaA are fused to any two repair proteins (illustrated here by MutL and Vsr), allowing measurement of all pairwise interactions as units of β-galactosidase (β-gal). T25 fusions are repair proficient. CRP, cyclic AMP (cAMP) receptor protein; P, lac operon promoter; RNAP, RNA polymerase.2-Aminopurine (2AP) mispairs with C during DNA replication, causing transition and frameshift mutations (5). The transitions are due primarily to the mismatch itself; the frameshifts are due to saturation of MMR, which leaves slipped-strand intermediates caused by DNA replication errors unrepaired (19). MutS and MutL bind to 2AP/C lesions (22), although the lesions may not be subject to MMR (19). As shown in Fig. ​Fig.2,2, treatment with 2AP causes a dose-dependent increase in the interaction of MutL with both Vsr and MutH; dimerization of MutL and interaction of MutL with MutS are somewhat increased.Open in a separate windowFIG. 2.Effect of 2AP treatment on protein-protein interactions in the bacterial two-hybrid assay. Results in units of β-galactosidase ± standard errors of the means (n = 9) are shown for BTH101(F galE15 ga1K16 rpsL1 hsdR2 mcrA1 mcrB1 cyaA-99) cells treated with 2AP as described previously (5, 19). Cells were cotransformed with pT18 and pT25 vectors (light gray bars), pT18-mutS and pT25-mutL (white bars), pT18-vsr and pT25-mutL (gray bars), pT18-mutH and pT25-mutL (black bars), or pT18-mutL and pT25-mutL (mottled bars). (NB: The dose-response curve for the pT18-mutS pT25-mutS transformants is similar to that of the pT18-mutL pT25-mutL transformants; it has been omitted for graphical clarity since the MutS-MutS interaction gives very high units of β-galactosidase activity [15]).The MutY adenine glycosylase removes A''s which have mispaired with oxidized guanine (8-oxoG) during DNA replication. Cells with a deletion of mutY have an elevated frequency of CG-to-AT transversion mutations (18); these are reduced by excess MutS, suggesting that 8-oxoG/A mismatches are also subject to MMR (23). As shown in Fig. ​Fig.3,3, the interactions between Vsr and MutL and between MutH and MutL increase in a mutY cell (stippled bars). Other interactions, such as MutS dimerization, are unaffected (not shown).Open in a separate windowFIG. 3.Effects of mutY and mutT deletions on protein-protein interactions in the bacterial two-hybrid assay. Results are in units of β-galactosidase, relative to the level in the wild type, in mutT (solid) and mutY (stippled) derivatives of BTH101 cotransformed with pT18 and pT25 vectors, pT18-mutH and pT25-mutL, pT18-vsr and pT25-mutL, or pT18-mutS and pT25-mutS (n = 3).8-OxoG/A mismatches also arise by incorporation of oxidized dGTP opposite A during DNA replication. The MutT nuclease minimizes this by removing oxidized dGTP from the nucleotide pool. The high frequency of AT-to-CG mutations in mutT strains is unaffected by the status of the MMR system (7, 21, 23), possibly because these 8-oxoG/A mispairs are in a conformation that MutS does not recognize. As shown in Fig. ​Fig.3,3, neither the interaction between MutL and Vsr nor that between MutL and MutH is elevated in a mutT strain (solid bars).These data show that mismatches which attract MutS and MutL increase the interaction of MutL with MutH in vivo. Although these mismatches are not subject to VSP repair, they also increase the interaction between MutL and Vsr. The simplest interpretation is that a MutS-MutL complex recruits MutH and Vsr to the DNA independent of the identity of the mismatch. MutS and MutL could then clear the mismatch, delivering the (activated) endonuclease to its specific target site, no matter how far away it is.Interaction of MutL with MutH, leading to MMR, is probably the default option. However, the MutS-MutL complex may recruit other repair proteins, such as Vsr or UvrB (20), to lesions that are poorly processed by MMR. The T/G mismatch in hemimethylated CTWGG sequences may be one such site. Vsr is expressed at very low levels in growing cells (14), so this recruitment would enhance VSP repair. However, recruitment of Vsr to other lesions would reduce VSP repair. For example, recruitment of Vsr by MutL to 2AP/C lesions (Fig. ​(Fig.2)2) could explain why CCWGG sites are hotspots for 2AP-induced mutations (4, 19).We have argued that Vsr is kept at low levels while DNA is replicating to avoid interference with MMR (14). However, if, as we suggest here, MutS and MutL are needed to recruit scarce Vsr to its target sequence, this argument loses its merit. It seems more likely that Vsr levels are kept low to avoid CTWGG-to-CCWGG mutations; Vsr creates these mutations by converting T/G mismatches formed at CTAGG sites by errors in DNA replication to CG (3, 6, 16). Vsr levels rise in nongrowing cells (14), when mutagenesis is no longer a risk. Under these circumstances, it is likely that MutS and MutL are no longer required for efficient VSP repair.     

In vitro studies of DNA mismatch repair proteins

The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells is important for understanding mechanisms involved in mutagenesis and tumorigenesis. Since mismatch repair proteins recognize mismatches containing both normal and chemically altered or damaged bases, in vitro assays must accommodate a variety of mismatches in different sequence contexts. Here we describe the construction of DNA mismatch substrates containing G:T or O⁶meG:T mismatches, the purification of recombinant native human MutSα (MSH2–MSH6) and MutLα (MLH1–PMS2) proteins, and in vitro mismatch repair and excision assays that can be adapted to study mismatch repair in nuclear extracts from mismatch repair proficient and deficient cells.     

DNA错配修复蛋白MutS和MutL的相互作用研究

MutL 和 MutS 是DNA错配修复系统中起关键作用的修复蛋白. 利用基因融合技术高效表达了MutL 和 MutS融合蛋白,并利用它们发展了一种研究二者相互作用的简便方法. 融合蛋白MutL-GFP (Trx-His6-GFP-(Ser-Gly)6-MutL),MutL-Strep tagⅡ (Trx-His6-(Ser-Gly)6-Strep tagⅡ-(Ser-Gly)6-MutL) 和 MutS (Trx-His6-(Ser-Gly)6-MutS) 被构建并在大肠杆菌中高效表达. 收集菌体细胞、超声波破碎后离心取上清进行SDS-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 分析,结果表明有与预期分子质量相应的诱导表达条带出现,其表达量约占全细胞蛋白的30％且以可溶形式存在. 利用固定化金属离子配体亲和层析柱分别纯化融合蛋白,其纯度达到90％. 通过将MutS蛋白固定的方法研究两种MutL融合蛋白分别与MutS之间的相互作用. 结果表明：只有MutS蛋白与含有错配碱基DNA分子结合后才与MutL蛋白发生相互作用. 通过检测MutL融合蛋白标记的绿色荧光信号或酶学显色信号来鉴定相互作用的发生. 建立的融合分子系统方法也为研究其他的蛋白质或生物大分子之间的相互作用提供了一个技术平台.     

Physical and functional interactions between Escherichia coli MutL and the Vsr repair endonuclease

DNA mismatch repair (MMR) and very-short patch (VSP) repair are two pathways involved in the repair of T:G mismatches. To learn about competition and cooperation between these two repair pathways, we analyzed the physical and functional interaction between MutL and Vsr using biophysical and biochemical methods. Analytical ultracentrifugation reveals a nucleotide-dependent interaction between Vsr and the N-terminal domain of MutL. Using chemical crosslinking, we mapped the interaction site of MutL for Vsr to a region between the N-terminal domains similar to that described before for the interaction between MutL and the strand discrimination endonuclease MutH of the MMR system. Competition between MutH and Vsr for binding to MutL resulted in inhibition of the mismatch-provoked MutS- and MutL-dependent activation of MutH, which explains the mutagenic effect of Vsr overexpression. Cooperation between MMR and VSP repair was demonstrated by the stimulation of the Vsr endonuclease in a MutS-, MutL- and ATP-hydrolysis-dependent manner, in agreement with the enhancement of VSP repair by MutS and MutL in vivo. These data suggest a mobile MutS–MutL complex in MMR signalling, that leaves the DNA mismatch prior to, or at the time of, activation of downstream effector molecules such as Vsr or MutH.     

Structure and function of mismatch repair proteins

DNA mismatch repair is required for maintaining genomic stability and is highly conserved from prokaryotes to eukaryotes. Errors made during DNA replication, such as deletions, insertions and mismatched basepairs, are substrates for mismatch repair. Mismatch repair is strand-specific and targets only the newly synthesized daughter strand. To initiate mismatch repair in Escherichia coli, three proteins are essential, MutS, for mismatch recognition, MutH, for introduction of a nick in the target strand, and MutL, for mediating the interactions between MutH and MutS. Homologues of MutS and MutL important for mismatch repair have been found in nearly all organisms. Mutations in MutS and MutL homologues have been linked to increased cancer susceptibility in both mice and humans. Here, we review the crystal structures of the MutH endonuclease, a conserved ATPase fragment of MutL (LN40), and complexes of LN40 with various nucleotides. Based on the crystal structure, the active site of MutH has been identified and an evolutionary relationship between MutH and type II restriction endonucleases established. Recent crystallographic and biochemical studies have revealed that MutL operates as a molecular switch with its interactions with MutH and MutS regulated by ATP binding and hydrolysis. These crystal structures also shed light on the general mechanism of mismatch repair and the roles of Mut proteins in preventing mutagenesis.     

Mispair-bound human MutS–MutL complex triggers DNA incisions and activates mismatch repair

DNA mismatch repair (MMR) relies on MutS and MutL ATPases for mismatch recognition and strand-specific nuclease recruitment to remove mispaired bases in daughter strands. However, whether the MutS–MutL complex coordinates MMR by ATP-dependent sliding on DNA or protein–protein interactions between the mismatch and strand discrimination signal is ambiguous. Using functional MMR assays and systems preventing proteins from sliding, we show that sliding of human MutSα is required not for MMR initiation, but for final mismatch removal. MutSα recruits MutLα to form a mismatch-bound complex, which initiates MMR by nicking the daughter strand 5′ to the mismatch. Exonuclease 1 (Exo1) is then recruited to the nick and conducts 5′ → 3′ excision. ATP-dependent MutSα dissociation from the mismatch is necessary for Exo1 to remove the mispaired base when the excision reaches the mismatch. Therefore, our study has resolved a long-standing puzzle, and provided new insights into the mechanism of MMR initiation and mispair removal.Subject terms: Molecular biology     

Is Thymidine Glycol Containing DNA a Substrate of E. coli DNA Mismatch Repair System?

The DNA mismatch repair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for E. coli MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system) to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET) and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the E. coli MMR system. Probably, the MutS transformation to active “sliding clamp” conformation on Tg-DNA is problematic.     

Proteomic Analysis Reveals a Novel Mutator S (MutS) Partner Involved in Mismatch Repair Pathway

The mismatch repair (MMR) family is a highly conserved group of proteins that function in correcting base–base and insertion–deletion mismatches generated during DNA replication. Disruption of this process results in characteristic microsatellite instability (MSI), repair defects, and susceptibility to cancer. However, a significant fraction of MSI-positive cancers express MMR genes at normal levels and do not carry detectable mutation in known MMR genes, suggesting that additional factors and/or mechanisms may exist to explain these MSI phenotypes in patients. To systematically investigate the MMR pathway, we conducted a proteomic analysis and identified MMR-associated protein complexes using tandem-affinity purification coupled with mass spectrometry (TAP-MS) method. The mass spectrometry data have been deposited to the ProteomeXchange with identifier PXD003014 and DOI 10.6019/PXD003014. We identified 230 high-confidence candidate interaction proteins (HCIPs). We subsequently focused on MSH2, an essential component of the MMR pathway and uncovered a novel MSH2-binding partner, WDHD1. We further demonstrated that WDHD1 forms a stable complex with MSH2 and MSH3 or MSH6, i.e. the MutS complexes. The specific MSH2/WDHD1 interaction is mediated by the second lever domain of MSH2 and Ala¹¹²³ site of WDHD1. Moreover, we showed that, just like MSH2-deficient cells, depletion of WDHD1 also led to 6-thioguanine (6-TG) resistance, indicating that WDHD1 likely contributes to the MMR pathway. Taken together, our study uncovers new components involved in the MMR pathway, which provides candidate genes that may be responsible for the development of MSI-positive cancers.Cells are equipped with a number of repair mechanisms to correct various types of DNA lesions. At least five major complimentary, but partially overlapping, multistep damage repair pathways are known to operate in mammals: mismatch repair (MMR)¹, nucleotide excision repair, base excision repair, and double-strand break repair, which includes both homologous recombination repair and nonhomologous end joining (see review: (1, 2)). In particular, MMR is a major repair pathway that prevents both base substitution and insertion–deletion mismatches due to replication errors (3–5).MMR is a highly conserved biological pathway that exists from bacteria to mammals. MMR process can be divided into three key steps: mismatch recognition, excision, and resynthesis (5, 6). The initial mismatch recognition step is fulfilled by MutS protein complexes, either MutSα (the MSH2-MSH6 heterodimer) or MutSβ (the MSH2-MSH3 heterodimer). The MutSα is primarily responsible for repairing base–base mismatches and small insertion–deletion loops of 1–2 nucleotides (7–9), while MutSβ preferentially recognizes larger insertion–deletion loops containing up to 14 extra nucleotides (10–12). Binding to mispaired DNA primes MutS to undergo a conformational change and recruitment of MutL to form an ATP-dependent ternary complex (13). Three different MutL heterodimeric complexes, MutLα, MutLβ, and MutLγ have been identified in the mammalian system. MLH1 heterodimerizes with PMS2, PMS1, or MLH3 to form MutLα, MutLβ, or MutLγ, respectively. MutLα plays a crucial role in MMR, as cells that lack either protein inactivate MMR in human cells, while loss of MutLβ or MutLγ heterodimers leads to minor defects in MMR. MutL is able to recognize and excise the lagging strand from the mismatch both distally and proximally (14, 15). Moreover, MutL interacts physically with MutS, enhances mismatch recognition, and recruits and activates exonuclease1 (EXO1) (16, 17). Exonuclease1 (EXO1) is the only enzyme with capabilities to excise nucleotide in 5′-3′ direction (18). In the case of 3′ excision, proliferating cell nuclear antigen (PCNA)/replication factor C-dependent endonuclease activity plays a critical role in 3′-5′ excision involving EXO1. EXO1 then excises nascent DNA from the nick toward and beyond the mismatch to generate a single-strand gap, which is filled by DNA polymerases δ (lagging strand) or ε (leading strand) using the parental DNA strand as a template. Finally, the nick is sealed by DNA ligase I (19, 20). In addition, two MutS homologues, MSH4 and MSH5, share similar structure and sequence features with the other members of the MutS family. Recent evidence suggests that they function beyond MMR and are involved in processes such as recombinant repair, DNA damage signaling, and immunoglobulin class switch recombination (21, 22).It has been well documented that impairment of MMR genes, especially MSH2 and MLH1, cause susceptibility to certain types of cancer, including human nonpolyposis colorectal cancer. At the cellular level, deficient MMR results in a strong mutator phenotype known as microsatellite instability (MSI), which is a hallmark of MMR deficiency (3–5). However, a significant fraction of MSI-positive colorectal cancers express MMR genes at normal levels and do not carry detectable mutation or hypermethylation in known MMR genes (23). Similarly, certain noncolorectal cancer cells with MSI also appear to have normal expression of known MMR protein (24, 25). These observations suggest that additional factors and/or mechanisms may exist to explain these MSI phenotypes in patients.To address this question, we performed tandem affinity purification coupled with mass spectrometry analysis (TAP-MS) to uncover MMR-associated protein complexes. Our proteomics study of the MMR family led to the discovery of many novel MMR-associated proteins, and gene ontology analysis expanded the roles of MMR in multiple biological processes. Specifically for MSH2, we uncovered a novel MutS binding partner WDHD1, which associates with both MutSα (MSH2-MSH6 heterodimer) and MutSβ (MSH2-MSH3 heterodimer). We provide additional evidence suggesting that WDHD1 is involved in the MMR pathway, which can be used as potential biomarker for MSI phenotypes in cancer patients.     

Interaction studies of muts and mutl with DNA containing the major cisplatin lesion and its mismatched counterpart under equilibrium and nonequilibrium conditions

The DNA mismatch repair (MMR) system participates in cis‐diamminedichloroplatinum (II) (cisplatin) cytotoxicity through signaling of cisplatin DNA lesions by yet unknown molecular mechanisms. It is thus of great interest to determine whether specialized function of MMR proteins could be associated with cisplatin DNA damage. The major cisplatin 1,2‐d(GpG) intrastrand crosslink and compound lesions arising from misincorporation of a mispaired base opposite either platinated guanine of the 1,2‐d(GpG) adduct are thought to be critical lesions for MMR signaling. Previously, we have shown that cisplatin compound lesion with a mispaired thymine opposite the 3′ platinated guanine triggers new Escherichia coli MutS ATP‐dependent biochemical activities distinguishable from those encountered with DNA mismatch consistent with a role of this lesion in MMR‐dependent signaling mechanism. In this report, we show that the major cisplatin 1,2‐d(GpG) intrastrand crosslink does not confer novel MutS postrecognition biochemical activity as studied by surface plasmon resonance spectroscopy. A fast rate of MutS ATP‐dependent dissociation prevents MutL recruitment to the major cisplatin lesion in contrast to cisplatin compound lesion which authorized MutS‐dependent recruitment of MutL with a dynamic of ternary complex formation distinguishable from that encountered with DNA mismatch substrate. We conclude that the mode of cisplatin DNA damage recognition by MutS and the nature of MMR post‐recognition events are lesion‐dependent and suggest that MMR signaling through the major cisplatin lesion is unlikely to occur. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 636–647, 2013.     

A novel interaction between human DNA polymerase η and MutLα

Human DNA polymerase η (Polη) is the gene product underlying xeroderma pigmentosum variant, and plays principal roles in translesion DNA synthesis. Here, we identified human MLH1, an essential component of mismatch repair (MMR), as a Polη-interacting protein. The middle area residues, which include the little finger domain, of Polη are important for the interaction with MLH1. Polη also interacts with the MLH1/PMS2 heterodimer (MutLα). Co-immunoprecipitation analyses revealed that MutLα, and also MSH2 and MSH6, components of the MutSα heterodimer, form complexes with Polη in human cells. Although MutSα had been reported to interact with C-terminal residues of Polη, MutLα and MutSα co-precipitated with C-terminally truncated Polη, suggesting that MutSα can interact with Polη through MutLα. MMR proteins were more abundant in the Polη complex on the chromatin of S phase-synchronized cells than of asynchronous cells, suggesting that the interaction between Polη and MLH1 is involved in DNA replication.     

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.     

Yeast mutator phenotype enforced by Arabidopsis PMS1 expression

The DNA mismatch repair (MMR) system is a major DNA repair pathway whose function is critical for the correction of DNA biosynthetic errors. MMR is initiated by the binding of MutS proteins to mismatches and unpaired nucleotides followed by the recruitment of MutL proteins. The major MutL activity in eukaryotes is performed by MutLα, the heterocomplex of MLH1-PMS1 in yeast and plants and MLH1-PMS2 in humans. We here report the effect the expression of Arabidopsis PMS1 protein exerts on Saccharomyces cerevisiae genomic stability. A strain carrying specific microsatellite instability reporter systems was chosen for the study. The plant protein failed to complement the hypermutator phenotype of a pms1 deficient strain but increased approximately 14-fold and 2,000-fold the mutation rates of his7-2 and lys2::InsE-A ₁₄ loci of MMR proficient strains when compared to wild-type strains, respectively. Overexpressing AtMLH1 in the AtPMS1-overproducing strain generated an increase in mutation rate comparable to that of AtPMS1 expression alone. Deletion of the C-terminal residues implicated in protein–protein interaction and including the putative endonuclease sequence of AtPMS1 completely eliminated the mutator phenotype. Taken together, these results indicate that the plant proteins affect yeast genomic stability, very possibly altering protein–protein interactions that are necessary to complete repair.