Suppressive cancer nonstop extension mutations increase C-terminal hydrophobicity and disrupt evolutionarily conserved amino acid patterns.

Nonstop extension mutations, a.k.a. stop-lost or stop-loss mutations, convert a stop codon into a sense codon resulting in translation into the 3' untranslated region until the next in-frame stop codon, thereby extending the C-terminus of a protein. In cancer, only nonstop mutations in SMAD4 have been functionally characterized, while the impact of other nonstop mutations remain unknown. Here, we exploit our pan-cancer NonStopDB dataset and test all 2335 C-terminal extensions arising from somatic nonstop mutations in cancer for their impact on protein expression. In a high-throughput screen, 56.1% of the extensions effectively reduce protein abundance. Extensions of multiple tumor suppressor genes like PTEN, APC, B2M, CASP8, CDKN1B and MLH1 are effective and validated for their suppressive impact. Importantly, the effective extensions possess a higher hydrophobicity than the neutral extensions linking C-terminal hydrophobicity with protein destabilization. Analyzing the proteomes of eleven different species reveals conserved patterns of amino acid distribution in the C-terminal regions of all proteins compared to the proteomes like an enrichment of lysine and arginine and a depletion of glycine, leucine, valine and isoleucine across species and kingdoms. These evolutionary selection patterns are disrupted in the cancer-derived effective nonstop extensions.

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.

Linker domain function predicts pathogenic MLH1 missense variants.

The pathogenic consequences of 369 unique human HsMLH1 missense variants has been hampered by the lack of a detailed function in mismatch repair (MMR). Here single-molecule images show that HsMSH2-HsMSH6 provides a platform for HsMLH1-HsPMS2 to form a stable sliding clamp on mismatched DNA. The mechanics of sliding clamp progression solves a significant operational puzzle in MMR and provides explicit predictions for the distribution of clinically relevant HsMLH1 missense mutations.

Regulation of the MLH1-MLH3 endonuclease in meiosis.

During prophase of the first meiotic division, cells deliberately break their DNA1. These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables the reciprocal exchange of DNA segments between homologous chromosomes2. A pathway that depends on the MLH1-MLH3 (MutLγ) nuclease has been implicated in the biased processing of meiotic recombination intermediates into crossovers by an unknown mechanism3-7. Here we have biochemically reconstituted key elements of this pro-crossover pathway. We show that human MSH4-MSH5 (MutSγ), which supports crossing over8, binds branched recombination intermediates and associates with MutLγ, stabilizing the ensemble at joint molecule structures and adjacent double-stranded DNA. MutSγ directly stimulates DNA cleavage by the MutLγ endonuclease. MutLγ activity is further stimulated by EXO1, but only when MutSγ is present. Replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) are additional components of the nuclease ensemble, thereby triggering crossing-over. Saccharomyces cerevisiae strains in which MutLγ cannot interact with PCNA present defects in forming crossovers. Finally, the MutLγ-MutSγ-EXO1-RFC-PCNA nuclease ensemble preferentially cleaves DNA with Holliday junctions, but shows no canonical resolvase activity. Instead, it probably processes meiotic recombination intermediates by nicking double-stranded DNA adjacent to the junction points9. As DNA nicking by MutLγ depends on its co-factors, the asymmetric distribution of MutSγ and RFC-PCNA on meiotic recombination intermediates may drive biased DNA cleavage. This mode of MutLγ nuclease activation might explain crossover-specific processing of Holliday junctions or their precursors in meiotic chromosomes4.

Identification of a novel pathogenic MLH1 mutation and recommended genetic screening strategy: An investigation of three Chinese Lynch syndrome pedigrees.

BACKGROUND: Lynch syndrome (LS) is an autosomal-dominant disorder that increases the risk of many cancers. The genetic basis of LS is germline mutations in DNA mismatch repair genes. METHODS: We performed next-generation sequencing on blood cells obtained from the members of three unrelated LS pedigrees. Immunohistochemistry staining was performed to analyze protein expression. RESULTS: Multigene panel screening revealed three mutL homolog 1 (MLH1) pathogenic mutations (c.199G>A, c.790 + 1G>A, and c.1557_1558 + 8delGGGTACGTAA, unreported) confirmed by Sanger sequencing. Immunohistochemistry showed a loss of MLH1 protein expression. We also confirmed that the unreported mutant allele was inherited for at least three generations. CONCLUSION: These results provide new insights into the molecular mechanisms underlying the pathogenicity of MLH1 mutations and reaffirm the importance of genetic screening for the early diagnosis of LS.

Human MutLγ, the MLH1-MLH3 heterodimer, is an endonuclease that promotes DNA expansion.

MutL proteins are ubiquitous and play important roles in DNA metabolism. MutLγ (MLH1-MLH3 heterodimer) is a poorly understood member of the eukaryotic family of MutL proteins that has been implicated in triplet repeat expansion, but its action in this deleterious process has remained unknown. In humans, triplet repeat expansion is the molecular basis for ∼40 neurological disorders. In addition to MutLγ, triplet repeat expansion involves the mismatch recognition factor MutSß (MSH2-MSH3 heterodimer). We show here that human MutLγ is an endonuclease that nicks DNA. Strikingly, incision of covalently closed, relaxed loop-containing DNA by human MutLγ is promoted by MutSß and targeted to the strand opposite the loop. The resulting strand break licenses downstream events that lead to a DNA expansion event in human cell extracts. Our data imply that the mammalian MutLγ is a unique endonuclease that can initiate triplet repeat DNA expansions.

Correlation of MLH1 polymorphisms, survival statistics, in silico assessment and gene downregulation with clinical outcomes among breast cancer cases.

This study aimed to investigate the role of MLH1 polymorphisms, respective protein structure prediction, survival analysis, related clinicopathological details and MLH1 expression in breast cancer (BC). Genotyping of selected SNPs in BC patients (493) and age matched controls (387) were performed by Tetra-ARMS PCR. Gene expression among breast tumors (127) and adjacent control tissues were analysed using reverse transcriptase PCR (RT-PCR) and immunohistochemistry. Statistical analysis was performed by SPSS and MedCalc. Conditional logistic regression analysis was applied to compute the odds ratio and confidence interval. Phyre2 and I-TASSER were used to generate MLH1 protein structures and verified by a variety of computational tools. Genotyping illustrated that MLH1 polymorphisms (rs63749795 and rs63749820) were significantly associated (P ≤ 0.05) with risk of developing BC. Down regulation of MLH1 gene expression/loss of the MLH1 protein (OR 12; CI 2.8-53.1) was observed in BC cases, illustrating its potential role in disease development. Moreover, loss of the MLH1 protein was found to be associated with higher grade cancer (P = 0.02) and lymph node positivity (P = 0.03), highlighting its essential role, as a component of the mismatch repair (MMR) machinery. Bioinformatics analysis confirmed that nonsense mutations produce a truncated MLH1 protein, causing a reduction in MMR efficiency. No association between MLH1 polymorphisms and overall and progression free survival statistics was observed among BC cases, possibly due to short follow-up study. Results at DNA, RNA and protein levels, along with in silico analysis, highlights the potential role of MLH1 in DNA repair mechanisms, within BC. Therefore, it was concluded that MLH1 may contribute towards BC development and progression.

Computational and cellular studies reveal structural destabilization and degradation of MLH1 variants in Lynch syndrome.

Defective mismatch repair leads to increased mutation rates, and germline loss-of-function variants in the repair component MLH1 cause the hereditary cancer predisposition disorder known as Lynch syndrome. Early diagnosis is important, but complicated by many variants being of unknown significance. Here we show that a majority of the disease-linked MLH1 variants we studied are present at reduced cellular levels. We show that destabilized MLH1 variants are targeted for chaperone-assisted proteasomal degradation, resulting also in degradation of co-factors PMS1 and PMS2. In silico saturation mutagenesis and computational predictions of thermodynamic stability of MLH1 missense variants revealed a correlation between structural destabilization, reduced steady-state levels and loss-of-function. Thus, we suggest that loss of stability and cellular degradation is an important mechanism underlying many MLH1 variants in Lynch syndrome. Combined with analyses of conservation, the thermodynamic stability predictions separate disease-linked from benign MLH1 variants, and therefore hold potential for Lynch syndrome diagnostics.

DNA mismatch repair activity of MutLα is regulated by CK2-dependent phosphorylation of MLH1 (S477).

MutLα, a heterodimer consisting of MLH1 and PMS2, is a key player of DNA mismatch repair (MMR), yet little is known about its regulation. In this study, we used mass spectrometry to identify phosphorylated residues within MLH1 and PMS2. The most frequently detected phosphorylated amino acid was serine 477 of MLH1. Pharmacological treatment indicates that Casein kinase II (CK2) could be responsible for the phosphorylation of MLH1 at serine 477 in vivo. In vitro kinase assay verified MLH1 as a substrate of CK2. Most importantly, using in vitro MMR assay we could demonstrate that p-MLH1S477 lost MMR activity. Moreover, we found that levels of p-MLH1S477 varied during the cell cycle. In summary, we identified that phosphorylation of MLH1 by CK2 at amino acid position 477 can switch off MMR activity in vitro. Since CK2 is overexpressed in many tumors and is able to inactivate MMR, the new mechanism here described could have an important impact on tumors overactive in CK2.

The MLH1 ATPase domain is needed for suppressing aberrant formation of interstitial telomeric sequences.

Genome instability gives rise to cancer. MLH1, commonly known for its important role in mismatch repair (MMR), DNA damage signaling and double-strand break (DSB) repair, safeguards genome stability. Recently we have reported a novel role of MLH1 in preventing aberrant formation of interstitial telomeric sequences (ITSs) at intra-chromosomal regions. Deficiency in MLH1, in particular its N-terminus, leads to an increase of ITSs. Here, we identify that the ATPase activity in the MLH1 N-terminal domain is important for suppressing the formation of ITSs. The ATPase activity is also needed for recruiting MLH1 to DSBs. Moreover, defective ATPase activity of MLH1 causes an increase in micronuclei formation. Our results highlight the crucial role of MLH1's ATPase domain in preventing the aberrant formation of telomeric sequences at the intra-chromosomal regions and preserving genome stability.

Evaluation of MLH1 variants of unclear significance.

Inactivating mutations in the MLH1 gene cause the cancer predisposition Lynch syndrome, but for small coding genetic variants it is mostly unclear if they are inactivating or not. Nine such MLH1 variants have been identified in South American colorectal cancer (CRC) patients (p.Tyr97Asp, p.His112Gln, p.Pro141Ala, p.Arg265Pro, p.Asn338Ser, p.Ile501del, p.Arg575Lys, p.Lys618del, p.Leu676Pro), and evidence of pathogenicity or neutrality was not available for the majority of these variants. We therefore performed biochemical laboratory testing of the variant proteins and compared the results to protein in silico predictions on structure and conservation. Additionally, we collected all available clinical information of the families to come to a conclusion concerning their pathogenic potential and facilitate clinical diagnosis in the affected families. We provide evidence that four of the alterations are causative for Lynch syndrome, four are likely neutral and one shows compromised activity which can currently not be classified with respect to its pathogenic potential. The work demonstrates that biochemical testing, corroborated by congruent evolutionary and structural information, can serve to reliably classify uncertain variants when other data are insufficient.

DNA mismatch repair proteins MLH1 and PMS2 can be imported to the nucleus by a classical nuclear import pathway.

MLH1 and PMS2 proteins form the MutLα heterodimer, which plays a major role in DNA mismatch repair (MMR) in humans. Mutations in MMR-related proteins are associated with cancer, especially with colon cancer. The N-terminal region of MutLα comprises the N-termini of PMS2 and MLH1 and, similarly, the C-terminal region of MutLα is composed by the C-termini of PMS2 and MLH1, and the two are connected by linker region. The nuclear localization sequences (NLSs) necessary for the nuclear transport of the two proteins are found in this linker region. However, the exact NLS sequences have been controversial, with different sequences reported, particularly for MLH1. The individual components are not imported efficiently, presumably due to their C-termini masking their NLSs. In order to gain insights into the nuclear transport of these proteins, we solved the crystal structures of importin-α bound to peptides corresponding to the supposed NLSs of MLH1 and PMS2 and performed isothermal titration calorimetry to study their binding affinities. Both putative MLH1 and PMS2 NLSs can bind to importin-α as monopartite NLSs, which is in agreement with some previous studies. However, MLH1-NLS has the highest affinity measured by a natural NLS peptide, suggesting a major role of MLH1 protein in nuclear import compared to PMS2. Finally, the role of MLH1 and PMS2 in the nuclear transport of the MutLα heterodimer is discussed.

Distinct DNA-binding surfaces in the ATPase and linker domains of MutLγ determine its substrate specificities and exert separable functions in meiotic recombination and mismatch repair.

Mlh1-Mlh3 (MutLγ) is a mismatch repair factor with a central role in formation of meiotic crossovers, presumably through resolution of double Holliday junctions. MutLγ has DNA-binding, nuclease, and ATPase activities, but how these relate to one another and to in vivo functions are unclear. Here, we combine biochemical and genetic analyses to characterize Saccharomyces cerevisiae MutLγ. Limited proteolysis and atomic force microscopy showed that purified recombinant MutLγ undergoes ATP-driven conformational changes. In vitro, MutLγ displayed separable DNA-binding activities toward Holliday junctions (HJ) and, surprisingly, single-stranded DNA (ssDNA), which was not predicted from current models. MutLγ bound DNA cooperatively, could bind multiple substrates simultaneously, and formed higher-order complexes. FeBABE hydroxyl radical footprinting indicated that the DNA-binding interfaces of MutLγ for ssDNA and HJ substrates only partially overlap. Most contacts with HJ substrates were located in the linker regions of MutLγ, whereas ssDNA contacts mapped within linker regions as well as the N-terminal ATPase domains. Using yeast genetic assays for mismatch repair and meiotic recombination, we found that mutations within different DNA-binding surfaces exert separable effects in vivo. For example, mutations within the Mlh1 linker conferred little or no meiotic phenotype but led to mismatch repair deficiency. Interestingly, mutations in the N-terminal domain of Mlh1 caused a stronger meiotic defect than mlh1Δ, suggesting that the mutant proteins retain an activity that interferes with alternative recombination pathways. Furthermore, mlh3Δ caused more chromosome missegregation than mlh1Δ, whereas mlh1Δ but not mlh3Δ partially alleviated meiotic defects of msh5Δ mutants. These findings illustrate functional differences between Mlh1 and Mlh3 during meiosis and suggest that their absence impinges on chromosome segregation not only via reduced formation of crossovers. Taken together, our results offer insights into the structure-function relationships of the MutLγ complex and reveal unanticipated genetic relationships between components of the meiotic recombination machinery.

The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans.

Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker's yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of Mlh1-Mlh3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the Mlh1-Mlh3 endonuclease is activated is unknown. Here, we provide evidence that Mlh1-Mlh3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient Mlh1-Mlh3. In addition, Mlh1-Mlh3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that Mlh1-Mlh3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which Mlh1-Mlh3 is loaded onto DNA to form an activated polymer that cleaves DNA.