All three MutL complexes are required for repeat expansion in a human stem cell model of CAG-repeat expansion mediated glutaminase deficiency.

The Repeat Expansion Diseases (REDs) arise from the expansion of a disease-specific short tandem repeat (STR). Different REDs differ with respect to the repeat involved, the cells that are most expansion prone and the extent of expansion. Furthermore, whether these diseases share a common expansion mechanism is unclear. To date, expansion has only been studied in a limited number of REDs. Here we report the first studies of the expansion mechanism in induced pluripotent stem cells derived from a patient with a form of the glutaminase deficiency disorder known as Global Developmental Delay, Progressive Ataxia, And Elevated Glutamine (GDPAG; OMIM# 618412) caused by the expansion of a CAG-STR in the 5' UTR of the glutaminase (GLS) gene. We show that alleles with as few as ~ 120 repeats show detectable expansions in culture despite relatively low levels of R-loops formed at this locus. Additionally, using a CRISPR-Cas9 knockout approach we show that PMS2 and MLH3, the constituents of MutLα and MutLγ, the 2 mammalian MutL complexes known to be involved in mismatch repair (MMR), are essential for expansion. Furthermore, PMS1, a component of a less well understood MutL complex, MutLß, is also important, if not essential, for repeat expansion in these cells. Our results provide insights into the factors important for expansion and lend weight to the idea that, despite some differences, the same mechanism is responsible for expansion in many, if not all, REDs.

MutLα suppresses error-prone DNA mismatch repair and preferentially protects noncoding DNA from mutations.

The DNA mismatch repair (MMR) system promotes genome stability and protects humans from certain types of cancer. Its primary function is the correction of DNA polymerase errors. MutLα is an important eukaryotic MMR factor. We have examined the contributions of MutLα to maintaining genome stability. We show here that loss of MutLα in yeast increases the genome-wide mutation rate by ∼130-fold and generates a genome-wide mutation spectrum that consists of small indels and base substitutions. We also show that loss of yeast MutLα leads to error-prone MMR that produces T > C base substitutions in 5'-ATA-3' sequences. In agreement with this finding, our examination of human whole-genome DNA sequencing data has revealed that loss of MutLα in induced pluripotent stem cells triggers error-prone MMR that leads to the formation of T > C mutations in 5'-NTN-3' sequences. Our further analysis has shown that MutLα-independent MMR plays a role in suppressing base substitutions in N3 homopolymeric runs. In addition, we describe that MutLα preferentially protects noncoding DNA from mutations. Our study defines the contributions of MutLα-dependent and independent mechanisms to genome-wide MMR.

Therapeutic validation of MMR-associated genetic modifiers in a human ex vivo model of Huntington disease.

The pathological huntingtin (HTT) trinucleotide repeat underlying Huntington disease (HD) continues to expand throughout life. Repeat length correlates both with earlier age at onset (AaO) and faster progression, making slowing its expansion an attractive therapeutic approach. Genome-wide association studies have identified candidate variants associated with altered AaO and progression, with many found in DNA mismatch repair (MMR)-associated genes. We examine whether lowering expression of these genes affects the rate of repeat expansion in human ex vivo models using HD iPSCs and HD iPSC-derived striatal medium spiny neuron-enriched cultures. We have generated a stable CRISPR interference HD iPSC line in which we can specifically and efficiently lower gene expression from a donor carrying over 125 CAG repeats. Lowering expression of each member of the MMR complexes MutS (MSH2, MSH3, and MSH6), MutL (MLH1, PMS1, PMS2, and MLH3), and LIG1 resulted in characteristic MMR deficiencies. Reduced MSH2, MSH3, and MLH1 slowed repeat expansion to the largest degree, while lowering either PMS1, PMS2, or MLH3 slowed it to a lesser degree. These effects were recapitulated in iPSC-derived striatal cultures where MutL factor expression was lowered. CRISPRi-mediated lowering of key MMR factor expression to levels feasibly achievable by current therapeutic approaches was able to effectively slow the expansion of the HTT CAG tract. We highlight members of the MutL family as potential targets to slow pathogenic repeat expansion with the aim to delay onset and progression of HD and potentially other repeat expansion disorders exhibiting somatic instability.

The Dmc1 recombinase physically interacts with and promotes the meiotic crossover functions of the Mlh1-Mlh3 endonuclease.

The accurate segregation of homologous chromosomes during the Meiosis I reductional division in most sexually reproducing eukaryotes requires crossing over between homologs. In baker's yeast approximately 80% of meiotic crossovers result from Mlh1-Mlh3 and Exo1 acting to resolve double-Holliday junction intermediates in a biased manner. Little is known about how Mlh1-Mlh3 is recruited to recombination intermediates to perform its role in crossover resolution. We performed a gene dosage screen in baker's yeast to identify novel genetic interactors with Mlh1-Mlh3. Specifically, we looked for genes whose lowered dosage reduced meiotic crossing over using sensitized mlh3 alleles that disrupt the stability of the Mlh1-Mlh3 complex and confer defects in mismatch repair but do not disrupt meiotic crossing over. To our surprise we identified genetic interactions between MLH3 and DMC1, the recombinase responsible for recombination between homologous chromosomes during meiosis. We then showed that Mlh3 physically interacts with Dmc1 in vitro and in vivo. Partial complementation of Mlh3 crossover functions was observed when MLH3 was expressed under the control of the CLB1 promoter (NDT80 regulon), suggesting that Mlh3 function can be provided late in meiotic prophase at some functional cost. A model for how Dmc1 could facilitate Mlh1-Mlh3's role in crossover resolution is presented.

Key role of phosphorylation sites in ATPase domain and Linker region of MLH1 for DNA binding and functionality of MutLα.

MutLα is essential for human DNA mismatch repair (MMR). It harbors a latent endonuclease, is responsible for recruitment of process associated proteins and is relevant for strand discrimination. Recently, we demonstrated that the MMR function of MutLα is regulated by phosphorylation of MLH1 at serine (S) 477. In the current study, we focused on S87 located in the ATPase domain of MLH1 and on S446, S456 and S477 located in its linker region. We analysed the phosphorylation-dependent impact of these amino acids on DNA binding, MMR ability and thermal stability of MutLα. We were able to demonstrate that phosphorylation at S87 of MLH1 inhibits DNA binding of MutLα. In addition, we detected that its MMR function seems to be regulated predominantly via phosphorylation of serines in the linker domain, which are also partially involved in the regulation of DNA binding. Furthermore, we found that the thermal stability of MutLα decreased in relation to its phosphorylation status implying that complete phosphorylation might lead to instability and degradation of MLH1. In summary, we showed here, for the first time, a phosphorylation-dependent regulation of DNA binding of MutLα and hypothesized that this might significantly impact its functional regulation during MMR in vivo.

Insights into DNA cleavage by MutL homologs from analysis of conserved motifs in eukaryotic Mlh1.

MutL family proteins contain an N-terminal ATPase domain (NTD), an unstructured interdomain linker, and a C-terminal domain (CTD), which mediates constitutive dimerization between subunits and often contains an endonuclease active site. Most MutL homologs direct strand-specific DNA mismatch repair by cleaving the error-containing daughter DNA strand. The strand cleavage reaction is poorly understood; however, the structure of the endonuclease active site is consistent with a two- or three-metal ion cleavage mechanism. A motif required for this endonuclease activity is present in the unstructured linker of Mlh1 and is conserved in all eukaryotic Mlh1 proteins, except those from metamonads, which also lack the almost absolutely conserved Mlh1 C-terminal phenylalanine-glutamate-arginine-cysteine (FERC) sequence. We hypothesize that the cysteine in the FERC sequence is autoinhibitory, as it sequesters the active site. We further hypothesize that the evolutionary co-occurrence of the conserved linker motif with the FERC sequence indicates a functional interaction, possibly by linker motif-mediated displacement of the inhibitory cysteine. This role is consistent with available data for interactions between the linker motif with DNA and the CTDs in the vicinity of the active site.

The mismatch repair endonuclease MutLα tethers duplex regions of DNA together and relieves DNA torsional tension.

In eukaryotic mismatch repair, MutS homologs recognize mismatches and recruit the MutLα endonuclease which introduces a nick in the newly replicated, error-containing DNA strand. The nick occurs in response to the mismatch, but at a site up to several hundred base pairs away. The MutLα nick promotes mismatch excision by an exonuclease (Exo1) or removal by the strand displacement activity of a DNA polymerase which may work in conjunction with a flap endonuclease. Models have suggested that MutL homolog endonucleases form oligomeric complexes which facilitate and are activated by strand capture mechanisms, although such models have never been explicitly tested. We present evidence that the mismatch repair MutLα endonuclease is activated by DNA-DNA associations and that it can use this property to overcome DNA torsional barriers. Using DNA ligation and pull-down experiments, we determined that the MutLα endonuclease associates two DNA duplexes. Using nuclease assays, we determined that this activity stimulates MutLα's endonuclease function. We also observe that MutLα enhances a topoisomerase without nicking the DNA itself. Our data provide a mechanistic explanation for how MutL proteins interact with DNA during mismatch repair, and how MutL homologs participate in other processes, such as recombination and trinucleotide repeat expansions.

MutS functions as a clamp loader by positioning MutL on the DNA during mismatch repair.

Highly conserved MutS and MutL homologs operate as protein dimers in mismatch repair (MMR). MutS recognizes mismatched nucleotides forming ATP-bound sliding clamps, which subsequently load MutL sliding clamps that coordinate MMR excision. Several MMR models envision static MutS-MutL complexes bound to mismatched DNA via a positively charged cleft (PCC) located on the MutL N-terminal domains (NTD). We show MutL-DNA binding is undetectable in physiological conditions. Instead, MutS sliding clamps exploit the PCC to position a MutL NTD on the DNA backbone, likely enabling diffusion-mediated wrapping of the remaining MutL domains around the DNA. The resulting MutL sliding clamp enhances MutH endonuclease and UvrD helicase activities on the DNA, which also engage the PCC during strand-specific incision/excision. These MutS clamp-loader progressions are significantly different from the replication clamp-loaders that attach the polymerase processivity factors ß-clamp/PCNA to DNA, highlighting the breadth of mechanisms for stably linking crucial genome maintenance proteins onto DNA.

Neisseria gonorrhoeae: DNA Repair Systems and Their Role in Pathogenesis.

Neisseria gonorrhoeae (a Gram-negative diplococcus) is a human pathogen and causative agent of gonorrhea, a sexually transmitted infection. The bacterium uses various approaches for adapting to environmental conditions and multiplying efficiently in the human body, such as regulation of expression of gene expression of surface proteins and lipooligosaccharides (e.g., expression of various forms of pilin). The systems of DNA repair play an important role in the bacterium ability to survive in the host body. This review describes DNA repair systems of N. gonorrhoeae and their role in the pathogenicity of this bacterium. A special attention is paid to the mismatch repair system (MMR) and functioning of the MutS and MutL proteins, as well as to the role of these proteins in regulation of the pilin antigenic variation of the N. gonorrhoeae pathogen.

Mlh1 interacts with both Msh2 and Msh6 for recruitment during mismatch repair.

Eukaryotic DNA mismatch repair (MMR) initiates through mispair recognition by the MutS homologs Msh2-Msh6 and Msh2-Msh3 and subsequent recruitment of the MutL homologs Mlh1-Pms1 (human MLH1-PMS2). In bacteria, MutL is recruited by interactions with the connector domain of one MutS subunit and the ATPase and core domains of the other MutS subunit. Analysis of the S. cerevisiae and human homologs have only identified an interaction between the Msh2 connector domain and Mlh1. Here we investigated whether a conserved Msh6 ATPase/core domain-Mlh1 interaction and an Msh2-Msh6 interaction with Pms1 also act in MMR. Mutations in MLH1 affecting interactions with both the Msh2 and Msh6 interfaces caused MMR defects, whereas equivalent pms1 mutations did not cause MMR defects. Mutant Mlh1-Pms1 complexes containing Mlh1 amino acid substitutions were defective for recruitment to mispaired DNA by Msh2-Msh6, did not support MMR in reconstituted Mlh1-Pms1-dependent MMR reactions in vitro, but were proficient in Msh2-Msh6-independent Mlh1-Pms1 endonuclease activity. These results indicate that Mlh1, the common subunit of the Mlh1-Pms1, Mlh1-Mlh2, and Mlh1-Mlh3 complexes, but not Pms1, is recruited by Msh2-Msh6 through interactions with both of its subunits.

Orc6 is a component of the replication fork and enables efficient mismatch repair.

In eukaryotes, the origin recognition complex (ORC) is required for the initiation of DNA replication. The smallest subunit of ORC, Orc6, is essential for prereplication complex (pre-RC) assembly and cell viability in yeast and for cytokinesis in metazoans. However, unlike other ORC components, the role of human Orc6 in replication remains to be resolved. Here, we identify an unexpected role for hOrc6, which is to promote S-phase progression after pre-RC assembly and DNA damage response. Orc6 localizes at the replication fork and is an accessory factor of the mismatch repair (MMR) complex. In response to oxidative damage during S phase, often repaired by MMR, Orc6 facilitates MMR complex assembly and activity, without which the checkpoint signaling is abrogated. Mechanistically, Orc6 directly binds to MutSα and enhances the chromatin-association of MutLα, thus enabling efficient MMR. Based on this, we conclude that hOrc6 plays a fundamental role in genome surveillance during S phase.

Rad27 and Exo1 function in different excision pathways for mismatch repair in Saccharomyces cerevisiae.

Eukaryotic DNA Mismatch Repair (MMR) involves redundant exonuclease 1 (Exo1)-dependent and Exo1-independent pathways, of which the Exo1-independent pathway(s) is not well understood. The exo1Δ440-702 mutation, which deletes the MutS Homolog 2 (Msh2) and MutL Homolog 1 (Mlh1) interacting peptides (SHIP and MIP boxes, respectively), eliminates the Exo1 MMR functions but is not lethal in combination with rad27Δ mutations. Analyzing the effect of different combinations of the exo1Δ440-702 mutation, a rad27Δ mutation and the pms1-A99V mutation, which inactivates an Exo1-independent MMR pathway, demonstrated that each of these mutations inactivates a different MMR pathway. Furthermore, it was possible to reconstitute a Rad27- and Msh2-Msh6-dependent MMR reaction in vitro using a mispaired DNA substrate and other MMR proteins. Our results demonstrate Rad27 defines an Exo1-independent eukaryotic MMR pathway that is redundant with at least two other MMR pathways.

Human MLH1/3 variants causing aneuploidy, pregnancy loss, and premature reproductive aging.

Embryonic aneuploidy from mis-segregation of chromosomes during meiosis causes pregnancy loss. Proper disjunction of homologous chromosomes requires the mismatch repair (MMR) genes MLH1 and MLH3, essential in mice for fertility. Variants in these genes can increase colorectal cancer risk, yet the reproductive impacts are unclear. To determine if MLH1/3 single nucleotide polymorphisms (SNPs) in human populations could cause reproductive abnormalities, we use computational predictions, yeast two-hybrid assays, and MMR and recombination assays in yeast, selecting nine MLH1 and MLH3 variants to model in mice via genome editing. We identify seven alleles causing reproductive defects in mice including female subfertility and male infertility. Remarkably, in females these alleles cause age-dependent decreases in litter size and increased embryo resorption, likely a consequence of fewer chiasmata that increase univalents at meiotic metaphase I. Our data suggest that hypomorphic alleles of meiotic recombination genes can predispose females to increased incidence of pregnancy loss from gamete aneuploidy.

OsMLH1 interacts with OsMLH3 to regulate synapsis and interference-sensitive crossover formation during meiosis in rice.

Meiotic recombination is essential for reciprocal exchange of genetic information between homologous chromosomes and their subsequent proper segregation in sexually reproducing organisms. MLH1 and MLH3 belong to meiosis-specific members of the MutL-homolog family, which are required for normal level of crossovers (COs) in some eukaryotes. However, their functions in plants need to be further elucidated. Here, we report the identification of OsMLH1 and reveal its functions during meiosis in rice. Using CRISPR-Cas9 approach, two independent mutants, Osmlh1-1 and Osmlh1-2, are generated and exhibited significantly reduced male fertility. In Osmlh1-1, the clearance of PAIR2 is delayed and partial ZEP1 proteins are not loaded into the chromosomes, which might be due to the deficient in resolution of interlocks at late zygotene. Thus, OsMLH1 is required for the assembly of synapsis complex. In Osmlh1-1, CO number is dropped by ~53% and the distribution of residual COs is consistent with predicted Poisson distribution, indicating that OsMLH1 is essential for the formation of interference-sensitive COs (class I COs). OsMLH1 interacts with OsMLH3 through their C-terminal domains. Mutation in OsMLH3 also affects the pollen fertility. Thus, our experiments reveal that the conserved heterodimer MutLγ (OsMLH1-OsMLH3) is essential for the formation of class I COs in rice.

Molecular basis of the dual role of the Mlh1-Mlh3 endonuclease in MMR and in meiotic crossover formation.

In budding yeast, the MutL homolog heterodimer Mlh1-Mlh3 (MutLγ) plays a central role in the formation of meiotic crossovers. It is also involved in the repair of a subset of mismatches besides the main mismatch repair (MMR) endonuclease Mlh1-Pms1 (MutLα). The heterodimer interface and endonuclease sites of MutLγ and MutLα are located in their C-terminal domain (CTD). The molecular basis of MutLγ's dual roles in MMR and meiosis is not known. To better understand the specificity of MutLγ, we characterized the crystal structure of Saccharomyces cerevisiae MutLγ(CTD). Although MutLγ(CTD) presents overall similarities with MutLα(CTD), it harbors some rearrangement of the surface surrounding the active site, which indicates altered substrate preference. The last amino acids of Mlh1 participate in the Mlh3 endonuclease site as previously reported for Pms1. We characterized mlh1 alleles and showed a critical role of this Mlh1 extreme C terminus both in MMR and in meiotic recombination. We showed that the MutLγ(CTD) preferentially binds Holliday junctions, contrary to MutLα(CTD). We characterized Mlh3 positions on the N-terminal domain (NTD) and CTD that could contribute to the positioning of the NTD close to the CTD in the context of the full-length MutLγ. Finally, crystal packing revealed an assembly of MutLγ(CTD) molecules in filament structures. Mutation at the corresponding interfaces reduced crossover formation, suggesting that these superstructures may contribute to the oligomer formation proposed for MutLγ. This study defines clear divergent features between the MutL homologs and identifies, at the molecular level, their specialization toward MMR or meiotic recombination functions.

Mismatch repair deficiency predicts response to HER2 blockade in HER2-negative breast cancer.

Resistance to endocrine treatment occurs in ~30% of ER+ breast cancer patients resulting in ~40,000 deaths/year in the USA. Preclinical studies strongly implicate activation of growth factor receptor, HER2 in endocrine treatment resistance. However, clinical trials of pan-HER inhibitors in ER+/HER2- patients have disappointed, likely due to a lack of predictive biomarkers. Here we demonstrate that loss of mismatch repair activates HER2 after endocrine treatment in ER+/HER2- breast cancer cells by protecting HER2 from protein trafficking. Additionally, HER2 activation is indispensable for endocrine treatment resistance in MutL- cells. Consequently, inhibiting HER2 restores sensitivity to endocrine treatment. Patient data from multiple clinical datasets supports an association between MutL loss, HER2 upregulation, and sensitivity to HER inhibitors in ER+/HER2- patients. These results provide strong rationale for MutL loss as a first-in-class predictive marker of sensitivity to combinatorial treatment with endocrine intervention and HER inhibitors in endocrine treatment-resistant ER+/HER2- breast cancer patients.

[MutL Protein from the Neisseria gonorrhoeae Mismatch Repair System: Interaction with ATP and DNA].

The mismatch repair system (MMR) ensures the stability of genetic information during DNA replication in almost all organisms. Mismatch repair is initiated after recognition of a non-canonical nucleotide pair by the MutS protein and the formation of a complex between MutS and MutL. Eukaryotic and most bacterial MutL homologs function as endonucleases that introduce a single-strand break in the daughter strand of the DNA, thus activating the repair process. However, many aspects of the functioning of this protein remain unknown. We studied the ATPase and DNA binding functions of the MutL protein from the pathogenic bacterium Neisseria gonorrhoeae (NgoMutL), which exhibits endonuclease activity. For the first time, the kinetic parameters of ATP hydrolysis by the full-length NgoMutL protein were determined. Its interactions with single- and double-stranded DNA fragments of various lengths were studied. NgoMutL was shown to be able to efficiently form complexes with DNA fragments that are longer than 40 nucleotides. Using modified DNA duplexes harboring a 2-pyridyldisulfide group on linkers of various lengths, we obtained NgoMutL conjugates with DNA for the first time. According to these results, the Cys residues of the wild-type protein are located at a distance of approximately 18-50 Šfrom the duplex. The efficiency of the affinity modification of Cys residues in NgoMutL with reactive DNAs was shown to decrease in the presence of ATP or its non-hydrolyzable analog, as well as ZnCl2, in the reaction mixture. We hypothesize that the conserved Cys residues of the C-terminal domain of NgoMutL, which are responsible for the coordination of metal ions in the active center of the protein, are involved in its interaction with DNA. This information may be useful in reconstruction of the main stages of MMR in prokaryotes that are different from γ-proteobacteria, as well as in the search for new targets for drugs against N. gonorrhoeae.

Experimental exchange of paralogous domains in the MLH family provides evidence of sub-functionalization after gene duplication.

Baker's yeast contains a large number of duplicated genes; some function redundantly, whereas others have more specialized roles. We used the MLH family of DNA mismatch repair (MMR) proteins as a model to better understand the steps that lead to gene specialization following a gene duplication event. We focused on two highly conserved yeast MLH proteins, Pms1 and Mlh3, with Pms1 having a major role in the repair of misincorporation events during DNA replication and Mlh3 acting to resolve recombination intermediates in meiosis to form crossovers. The baker's yeast Mlh3 and Pms1 proteins are significantly diverged (19% overall identity), suggesting that an extensive number of evolutionary steps, some major, others involving subtle refinements, took place to diversify the MLH proteins. Using phylogenetic and molecular approaches, we provide evidence that all three domains (N-terminal ATP binding, linker, C-terminal endonuclease/MLH interaction) in the MLH protein family are critical for conferring pathway specificity. Importantly, mlh3 alleles in the ATP binding and endonuclease domains improved MMR functions in strains lacking the Pms1 protein and did not disrupt Mlh3 meiotic functions. This ability for mlh3 alleles to complement the loss of Pms1 suggests that an ancestral Pms1/Mlh3 protein was capable of performing both MMR and crossover functions. Our strategy for analyzing MLH pathway specificity provides an approach to understand how paralogs have evolved to support distinct cellular processes.

Coordinated and Independent Roles for MLH Subunits in DNA Repair.

The MutL family of DNA mismatch repair proteins (MMR) acts to maintain genomic integrity in somatic and meiotic cells. In baker's yeast, the MutL homolog (MLH) MMR proteins form three heterodimeric complexes, MLH1-PMS1, MLH1-MLH2, and MLH1-MLH3. The recent discovery of human PMS2 (homolog of baker's yeast PMS1) and MLH3 acting independently of human MLH1 in the repair of somatic double-strand breaks questions the assumption that MLH1 is an obligate subunit for MLH function. Here we provide a summary of the canonical roles for MLH factors in DNA genomic maintenance and in meiotic crossover. We then present the phenotypes of cells lacking specific MLH subunits, particularly in meiotic recombination, and based on this analysis, propose a model for an independent early role for MLH3 in meiosis to promote the accurate segregation of homologous chromosomes in the meiosis I division.

The Pif1 helicase is actively inhibited during meiotic recombination which restrains gene conversion tract length.

Meiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversions events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, containing the Mer3 helicase and the MutLß heterodimer, to meiotic recombination intermediates. The molecular actors inhibited by this complex are elusive. The Pif1 DNA helicase is known to stimulate DNA polymerase delta (Pol δ) -mediated DNA synthesis from D-loops, allowing long synthesis required for break-induced replication. We show that Pif1 is also recruited genome wide to meiotic DNA double-strand break (DSB) sites. We further show that Pif1, through its interaction with PCNA, is required for the long gene conversions observed in the absence of MutLß recruitment to recombination sites. In vivo, Mer3 interacts with the PCNA clamp loader RFC, and in vitro, Mer3-MutLß ensemble inhibits Pif1-stimulated D-loop extension by Pol δ and RFC-PCNA. Mechanistically, our results suggest that Mer3-MutLß may compete with Pif1 for binding to RFC-PCNA. Taken together, our data show that Pif1's activity that promotes meiotic DNA repair synthesis is restrained by the Mer3-MutLß ensemble which in turn prevents long gene conversion tracts and possibly associated mutagenesis.