RESUMO
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.
Assuntos
Glutaminase , Células-Tronco Pluripotentes Induzidas , Expansão das Repetições de Trinucleotídeos , Humanos , Glutaminase/genética , Glutaminase/metabolismo , Expansão das Repetições de Trinucleotídeos/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas MutL/genética , Proteínas MutL/metabolismo , Sistemas CRISPR-CasRESUMO
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.
Assuntos
Reparo de Erro de Pareamento de DNA , Proteína Huntingtina , Doença de Huntington , Células-Tronco Pluripotentes Induzidas , Expansão das Repetições de Trinucleotídeos , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Reparo de Erro de Pareamento de DNA/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Expansão das Repetições de Trinucleotídeos/genética , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Genes Modificadores , Proteína 3 Homóloga a MutS/genética , Proteína 3 Homóloga a MutS/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas MutL/genética , Proteínas MutL/metabolismo , Sistemas CRISPR-Cas , Estudo de Associação Genômica AmplaRESUMO
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 â¼130-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.
Assuntos
Reparo de Erro de Pareamento de DNA , Proteínas MutL , Mutação , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas MutL/metabolismo , Proteínas MutL/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Instabilidade Genômica , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologiaRESUMO
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.
Assuntos
Proteínas de Ciclo Celular , Troca Genética , Meiose , Proteína 1 Homóloga a MutL , Proteínas MutL , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas MutL/metabolismo , Proteínas MutL/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Ligação ProteicaRESUMO
UvrD is a helicase vital for DNA replication and quality control processes. In its monomeric state, UvrD exhibits limited helicase activity, necessitating either dimerization or assistance from an accessory protein to efficiently unwind DNA. Within the DNA mismatch repair pathway, MutL plays a pivotal role in relaying the repair signal, enabling UvrD to unwind DNA from the strand incision site up to and beyond the mismatch. Although this interdependence is well-established, the precise mechanism of activation and the specific MutL-UvrD interactions that trigger helicase activity remain elusive. To address these questions, we employed site-specific crosslinking techniques using single-cysteine variants of MutL and UvrD followed by functional assays. Our investigation unveils that the C-terminal domain of MutL not only engages with UvrD but also acts as a self-sufficient activator of UvrD helicase activity on DNA substrates with 3'-single-stranded tails. Especially when MutL is covalently attached to the 2B or 1B domain the tail length can be reduced to a minimal substrate of 5 nucleotides without affecting unwinding efficiency.
Assuntos
DNA Helicases , Proteínas MutL , DNA/química , DNA Helicases/química , DNA Helicases/genética , Proteínas MutL/química , Proteínas MutL/genética , Ligação Proteica , Domínios Proteicos , Mesilatos/química , Reagentes de Ligações Cruzadas/químicaRESUMO
DNA mismatch repair endonuclease MutL is a member of GHKL ATPase superfamily. Mutations of MutL homologs are causative of a hereditary cancer, Lynch syndrome. We characterized MutL homologs from human and a hyperthermophile, Aquifex aeolicus, (aqMutL) to reveal the catalytic mechanism for the ATPase activity. Although involvement of a basic residue had not been conceived in the catalytic mechanism, analysis of the pH dependence of the aqMutL ATPase activity revealed that the reaction is catalyzed by a residue with an alkaline pKa. Analyses of mutant aqMutLs showed that Lys79 is the catalytic residue, and the corresponding residues were confirmed to be critical for activities of human MutL homologs, on the basis of which a catalytic mechanism for MutL ATPase is proposed. These and other results described here would contribute to evaluating the pathogenicity of Lynch syndrome-associated missense mutations. Furthermore, it was confirmed that the catalytic lysine residue is conserved among DNA gyrases and microrchidia ATPases, other members of GHKL ATPases, indicating that the catalytic mechanism proposed here is applicable to these members of the superfamily.
Assuntos
Adenosina Trifosfatases , DNA Girase , Proteínas de Ligação a DNA , Lisina , Proteínas MutL , Fatores de Transcrição , Humanos , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Sequência Conservada , Concentração de Íons de Hidrogênio , Lisina/química , Lisina/genética , Proteínas MutL/química , Proteínas MutL/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , DNA Girase/química , DNA Girase/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genéticaRESUMO
Huntington's disease (HD) is a dominant neurological disorder caused by an expanded HTT exon 1 CAG repeat that lengthens huntingtin's polyglutamine tract. Lowering mutant huntingtin has been proposed for treating HD, but genetic modifiers implicate somatic CAG repeat expansion as the driver of onset. We find that branaplam and risdiplam, small molecule splice modulators that lower huntingtin by promoting HTT pseudoexon inclusion, also decrease expansion of an unstable HTT exon 1 CAG repeat in an engineered cell model. Targeted CRISPR-Cas9 editing shows this effect is not due to huntingtin lowering, pointing instead to pseudoexon inclusion in PMS1. Homozygous but not heterozygous inactivation of PMS1 also reduces CAG repeat expansion, supporting PMS1 as a genetic modifier of HD and a potential target for therapeutic intervention. Although splice modulation provides one strategy, genome-wide transcriptomics also emphasize consideration of cell-type specific effects and polymorphic variation at both target and off-target sites.
Assuntos
Doença de Huntington , Humanos , Doença de Huntington/genética , Éxons/genética , Perfilação da Expressão Gênica , Heterozigoto , Homozigoto , Proteínas MutL , Proteínas de NeoplasiasRESUMO
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.
Assuntos
Reparo de Erro de Pareamento de DNA , DNA , Humanos , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Endonuclease PMS2 de Reparo de Erro de Pareamento/metabolismo , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Fosforilação , Domínios Proteicos , DNA/metabolismo , Proteínas MutL/genética , Proteínas MutL/metabolismoRESUMO
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.
Assuntos
Clivagem do DNA , Eucariotos , Proteínas MutL/química , Proteínas MutL/metabolismo , Eucariotos/genética , Eucariotos/metabolismo , Cisteína , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , DNA/genética , Endonucleases/metabolismoRESUMO
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.
Assuntos
Reparo de Erro de Pareamento de DNA , Proteínas de Escherichia coli , Trifosfato de Adenosina/metabolismo , DNA/metabolismo , Reparo do DNA , Endonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas MutL/genética , Proteínas MutL/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Nucleotídeos , Antígeno Nuclear de Célula em Proliferação/metabolismoRESUMO
Eukaryotic DNA mismatch repair (MMR) depends on recruitment of the Mlh1-Pms1 endonuclease (human MLH1-PMS2) to mispaired DNA. Both Mlh1 and Pms1 contain a long unstructured linker that connects the N- and carboxyl-terminal domains. Here, we demonstrated the Mlh1 linker contains a conserved motif (Saccharomyces cerevisiae residues 391-415) required for MMR. The Mlh1-R401A,D403A-Pms1 linker motif mutant protein was defective for MMR and endonuclease activity in vitro, even though the conserved motif could be >750 Å from the carboxyl-terminal endonuclease active site or the N-terminal adenosine triphosphate (ATP)-binding site. Peptides encoding this motif inhibited wild-type Mlh1-Pms1 endonuclease activity. The motif functioned in vivo at different sites within the Mlh1 linker and within the Pms1 linker. Motif mutations in human cancers caused a loss-of-function phenotype when modeled in S. cerevisiae. These results suggest that the Mlh1 motif promotes the PCNA-activated endonuclease activity of Mlh1-Pms1 via interactions with DNA, PCNA, RFC, or other domains of the Mlh1-Pms1 complex.
Assuntos
Neoplasias , Proteínas de Saccharomyces cerevisiae , Trifosfato de Adenosina/metabolismo , DNA/metabolismo , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Humanos , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Proteínas MutL , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Mutantes/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
Reparo de Erro de Pareamento de DNA , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatases/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA , Endonucleases/metabolismo , Humanos , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Proteínas MutL/metabolismo , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Wild rice (Oryza rufipogon) has a lower panicle seed setting rate (PSSR) and gamete fertility than domesticated rice (Oryza sativa), but the genetic mechanisms of this phenomenon remain unknown. Here, we cloned a null allele of OsMLH1, an ortholog of MutL-homolog 1 to yeast and mammals, from wild rice O. rufipogon W1943 and revealed a 5.4-kb retrotransposon insertion in OsMLH1 is responsible for the low PSSR in wild rice. In contrast to the wild-type, a near isogenic line NIL-mlh1 exhibits defective crossover (CO) formation during meiosis, resulting in reduced pollen viability, partial embryo lethality, and low PSSR. Except for the mutant of mismatch repair gene postmeiotic segregation 1 (Ospms1), all other MutL mutants from O. sativa indica subspecies displayed male and female semi-sterility similar to NIL-mlh1, but less severe than those from O. sativa japonica subspecies. MLH1 and MLH3 did not contribute in an additive fashion to fertility. Two types of MutL heterodimers, MLH1-PMS1 and MLH1-MLH3, were identified in rice, but only the latter functions in promoting meiotic CO formation. Compared to japonica varieties, indica cultivars had greater numbers of CO events per meiosis. Our results suggest that low fertility in wild rice may be caused by different gene defects, and indica and japonica subspecies have substantially different CO rates responsible for the discrepancy between the fertility of mlh1 and mlh3 mutants.
Assuntos
Oryza , Proteínas de Saccharomyces cerevisiae , Animais , Oryza/genética , Retroelementos/genética , Saccharomyces cerevisiae/genética , Sementes/genética , Meiose/genética , Mamíferos/genética , Proteínas MutL/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: It is likely that additional genes for hereditary breast cancer can be identified using a discordant sib pair design. Using this design we identified individuals harboring a rare PMS1 c.605G>A variant previously predicted to result in loss of function. OBJECTIVES: A family-based design and predictive algorithms were used to prioritize candidate variants possibly associated with an increased risk of hereditary breast cancer. Functional analyses were performed for one of the candidate variants, PMS1 c.605G>A. METHODS: 1) 14 discordant sister-pairs from hereditary breast cancer families were identified. 2) Whole exome sequencing was performed and candidate risk variants identified. 3) A rare PMS variant was identified in 2 unrelated affected sisters but no unaffected siblings. 4) Functional analysis of this variant was carried out using targeted mRNA sequencing. RESULTS: Genotype-phenotype correlation did not demonstrate tracking of the variant with cancer in the family. Functional analysis revealed no difference in exon 6 incorporation, which was validated by analyzing PMS1 allele specific expression. CONCLUSIONS: The PMS1 c.605G>A variant did not segregate with disease, and there was no variant-dependent impact on PMS1 exon 6 splicing, supporting this variant is likely benign. Functional analyses are imperative to understanding the clinical significance of predictive algorithms.
Assuntos
Neoplasias da Mama/genética , Sequenciamento do Exoma/métodos , Perfilação da Expressão Gênica/métodos , Proteínas MutL/genética , Proteínas de Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Adulto , Algoritmos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Mutação com Perda de Função , Pessoa de Meia-Idade , Linhagem , Análise de Sequência de RNA , IrmãosRESUMO
BACKGROUND: Microsatellites are short repeated DNA sequences normally found in the human genome. Following specific mutations, microsatellites can vary in the number of repeats thus making the DNA unstable. Microsatellite instability (MSI) is responsible for approximately 20% of rectal cancers, while the remaining 80% are caused by chromosomal instability. One of the following genes, MLH1, MLH2, MLH 6, and PMS2, is inactivated, leading to MSI colorectal cancers. AIM: This study aimed to analyze the expression of some MMR system genes presenting mutations in mucinous rectal cancer and their correlations with clinical data. METHODS: A retrospective study was performed on patients with rectal mucinous adenocarcinoma who underwent surgery between January 2000 and January 2017. We collected a total of 42 patients and analyzed the demographic data, histopathological results and MMR system genes mentioned above. RESULTS: Almost 93% of the cases analyzed had MSI-H and only 7% were MSI-L. For MLH1, 50% of stage T2 and 50% of stage T4 had weak expression, while in stage T3, 42.50% had moderate expression. Regarding the N stage, we found that 66.67% of the patients with moderate gene expression (2+) were N2, while 42% of the patients with weak expression were N0. For MSH2, the majority of patients with strong gene expression were in stage T3 (27%). Weak expression was found in 50% of the patients in stage T2, 35% of the patients in stage T3, and 33.3% in T4. In 44.44% of the weak expression was N2, while for strong expression, there was an equivalent percentage of 33.33% in stages N1 and N2. Describing the MSH6 gene, we found that the most heterogeneous results were in stage T3. Weak expression was observed in 38.46% of the patients, while moderate and strong expression was observed in 30.77% and 11.54% respectively. Analysis of PMS2 revealed that 66.67% of the patients in stage T4 had a weak expression of the gene, while the same expression was found in 38.46% of the patients in stage T3. A total of 23.08% of patients in stage T3 had strong gene expression. We also analyzed the overall gene expression. Thus, we found that three patients (7.14%) had only 1, three genes were expressed, nine (21.42%) had two genes and the remaining 27 patients had all 4. The 1-year survival rate in the analyzed lot was 75%, decreasing to 60% in the second year and 35% in the 3rd. There were no statistically significant differences in survival data between the stages or gene expression. CONCLUSIONS: Our study showed no statistical difference regarding the survival on different gene expression or staging, consistent with studies that found that mucin expression does not have a significant impact on local recurrence, nor does it affect nodal down staging. KEY WORDS: Mucinous adenocarcinoma, Microsatelites instability.
Assuntos
Adenocarcinoma Mucinoso , Neoplasias Retais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma Mucinoso/genética , Reparo de Erro de Pareamento de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Instabilidade de Microssatélites , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Proteína 1 Homóloga a MutL/genética , Proteínas MutL/genética , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Proteínas de Neoplasias/genética , Neoplasias Retais/genética , Neoplasias Retais/cirurgia , Estudos RetrospectivosRESUMO
Breast cancer is the most common cancer in women worldwide. Detection of breast cancer susceptibility genes is an important issue. Also, MLH3 is a DNA mismatch repair gene and mutation in this gene is harmful in different cancers. This study aimed to use exome sequencing to uncover previously undetected breast cancer-predisposing variants. Also, we investigated the MLH3 gene expression of breast cancer patients which can be a breast cancer susceptibility gene. A total of 80 samples including 40 paired normal and cancer tissue samples were collected at Zheen International Hospital, Erbil, Iraq. Exome sequencing was used to identify mutations. Different in silico tools were used to predict the effect of mutation on the structural features or protein function. Real-time PCR was used for assessing the expression of MLH3 in breast cancer patients. We identified 26 variants in breast cancer patients, 22 inherited variants were found in MLH3, CHECK2, BRCA1, BRCA2, BLM, TP53, MSH6, NBN and PTEN genes and 4 somatic variants were found in PALB2, RAD50 and RBM10 genes. It was found that the expression of the MLH3 gene in tumor samples was significantly down-regulated compared with normal tissues. Statistically, high significance was found. The decreased expression of MLH3 was significant in all ranges of ages and all breast cancer types. Also, the expression of MLH3 decreased significantly in patients with breast cancer grades of II and III. In conclusion, MLH3 can be used as a susceptibility gene especially in grades II and III of breast cancer.
Assuntos
Neoplasias da Mama/genética , Sequenciamento do Exoma/métodos , Predisposição Genética para Doença/genética , Proteínas MutL/genética , Mutação , Adulto , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/patologia , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , RecQ Helicases/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
Assuntos
Reparo de Erro de Pareamento de DNA , Exodesoxirribonucleases/metabolismo , Endonucleases Flap/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , DNA Ligases/metabolismo , DNA Fúngico/metabolismo , Exodesoxirribonucleases/genética , Endonucleases Flap/genética , Proteínas MutL/genética , Proteínas MutL/metabolismo , Mutação , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The DNA mismatch repair (MMR) factor Mlh1-Pms1 contains long intrinsically disordered regions (IDRs) whose exact functions remain elusive. We performed cross-linking mass spectrometry to identify interactions within Mlh1-Pms1 and used this information to insert FRB and FKBP dimerization domains into their IDRs. Baker's yeast strains bearing these constructs were grown with rapamycin to induce dimerization. A strain containing FRB and FKBP domains in the Mlh1 IDR displayed a complete defect in MMR when grown with rapamycin. but removing rapamycin restored MMR functions. Strains in which FRB was inserted into the IDR of one MLH subunit and FKBP into the other subunit were also MMR defective. The MLH complex containing FRB and FKBP domains in the Mlh1 IDR displayed a rapamycin-dependent defect in Mlh1-Pms1 endonuclease activity. In contrast, linking the Mlh1 and Pms1 IDRs through FRB-FKBP dimerization inappropriately activated Mlh1-Pms1 endonuclease activity. We conclude that dynamic and coordinated rearrangements of the MLH IDRs both positively and negatively regulate how the MLH complex acts in MMR. The application of the FRB-FKBP dimerization system to interrogate in vivo functions of a critical repair complex will be useful for probing IDRs in diverse enzymes and to probe transient loss of MMR on demand.
Assuntos
Reparo de Erro de Pareamento de DNA/genética , Proteínas Intrinsicamente Desordenadas/genética , Proteína 1 Homóloga a MutL/genética , Proteínas MutL/genética , Proteínas de Saccharomyces cerevisiae/genética , Domínios Proteicos/genética , Multimerização Proteica/genética , Saccharomyces cerevisiae/genética , Sirolimo/farmacologia , Proteínas de Ligação a Tacrolimo/genéticaRESUMO
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.
Assuntos
Aborto Espontâneo/genética , Aneuploidia , Perda do Embrião/genética , Proteína 1 Homóloga a MutL/genética , Proteínas MutL/genética , Aborto Espontâneo/metabolismo , Aborto Espontâneo/fisiopatologia , Alelos , Animais , Troca Genética , Reparo de Erro de Pareamento de DNA , Perda do Embrião/fisiopatologia , Feminino , Recombinação Homóloga , Humanos , Tamanho da Ninhada de Vivíparos , Masculino , Meiose , Camundongos , Proteína 1 Homóloga a MutL/metabolismo , Proteínas MutL/metabolismo , Gravidez , Reprodução , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
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.