RESUMO
BACKGROUND: Lynch syndrome (LS) is an autosomal dominant cancer predisposition condition caused by germline heterozygous mutations in mismatch repair (MMR) genes. However, as one of the MMR genes, PMS2 mutation-induced LS-associated ovarian cancer (LSAOC) has rarely been reported. METHODS: Next-generation sequencing (NGS) or Sanger sequencing was used to detect the genetic status of one family including four generations with 16 members. Then, quantitative real-time PCR (qPCR), western blotting, immunohistochemistry (IHC) staining, and Swiss-Model software were used to identify the function of the PMS2 mutation. RESULTS: Five individuals [I-1, II-1, II-2, II-4, and III-2 (proband)] suffered from LS-associated cancers, for example, colon cancer, gastric cancer, and ovarian cancer, with the age of onset ranging from 39 to 70 years old. A PMS2 germline heterozygous mutation (c.943C>T) was confirmed in three members [II-9, III-2, and IV-1] by gene sequencing. In addition, this PMS2 mutation was verified by qPCR, western blotting, and IHC, and a dramatic change with partial loss of the C-terminal domain in an α-helix might be exhibited. CONCLUSION: Carrying PMS2 germline mutations (c.943C>T) confers an extremely high susceptibility of suffering from LS-associated cancers. Thus, close clinical monitoring and prophylactic surgery is highly recommended to help reduce the morbidity and mortality of LS-associated cancers.
Assuntos
Carcinoma/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Mutação em Linhagem Germinativa , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Neoplasias Ovarianas/genética , Adulto , Carcinoma/patologia , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Feminino , Humanos , Endonuclease PMS2 de Reparo de Erro de Pareamento/química , Neoplasias Ovarianas/patologia , LinhagemRESUMO
Lynch syndrome (LS) is an autosomal dominant inherited disorder that is associated with an increased predisposition to certain cancers caused by loss-of-function mutations in one of four DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6, or PMS2). The diagnosis of LS is often challenged by the identification of missense mutations where the functional effects are not known. These are termed variants of uncertain significance (VUSs) and account for 20%-30% of noncoding and missense mutations. VUSs cause ambiguity during clinical diagnosis and hinder implementation of appropriate medical management. In the current study, we focus on the functional and biological consequences of two nonsynonymous VUSs in PMS2. These variants, c.620G>A and c.123_131delGTTAGTAGA, result in the alteration of glycine 207 to glutamate (p.Gly207Glu) and the deletion of amino acid residues 42-44 (p.Leu42_Glu44del), respectively. While the PMS2 p.Gly207Glu variant retains in vitro MMR and ATPase activities, PMS2 p.Leu42_Glu44del appears to lack such capabilities. Structural and biophysical characterization using circular dichroism, small-angle X-ray scattering, and X-ray crystallography of the N-terminal domain of the PMS2 variants indicate that the p.Gly207Glu variant is properly folded similar to the wild-type enzyme, whereas p.Leu42_Glu44del is disordered and prone to aggregation.
Assuntos
Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Alelos , Substituição de Aminoácidos , Biomarcadores , Linhagem Celular , Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA , Frequência do Gene , Humanos , Endonuclease PMS2 de Reparo de Erro de Pareamento/química , Mutação , Linhagem , Conformação Proteica , Medição de Risco , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Caseína Quinase II/metabolismo , Proteína 1 Homóloga a MutL/química , Proteína 1 Homóloga a MutL/metabolismo , Proteínas MutL/metabolismo , Animais , Ciclo Celular , Linhagem Celular Tumoral , Reparo de Erro de Pareamento de DNA , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Espectrometria de Massas , Endonuclease PMS2 de Reparo de Erro de Pareamento/química , Endonuclease PMS2 de Reparo de Erro de Pareamento/metabolismo , Modelos Moleculares , Proteínas MutL/química , Fosforilação , Processamento de Proteína Pós-Traducional , Serina/metabolismo , Células Sf9RESUMO
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.
Assuntos
Núcleo Celular/metabolismo , Reparo de Erro de Pareamento de DNA , Endonuclease PMS2 de Reparo de Erro de Pareamento/metabolismo , Proteína 1 Homóloga a MutL/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Humanos , Carioferinas/metabolismo , Camundongos , Endonuclease PMS2 de Reparo de Erro de Pareamento/química , Modelos Moleculares , Proteína 1 Homóloga a MutL/química , Conformação ProteicaRESUMO
DNA repair pathways are essential for cellular survival as our DNA is constantly under assault from both exogenous and endogenous DNA damaging agents. Five major mammalian DNA repair pathways exist within a cell to maintain genomic integrity. Of these, the DNA mismatch repair (MMR) pathway is highly conserved among species and is well documented in bacteria. In humans, the importance of MMR is underscored by the discovery that a single mutation in any 1 of 4 genes within the MMR pathway (MLH1, MSH2, MSH6 and PMS2) results in Lynch syndrome (LS). LS is a autosomal dominant condition that predisposes individuals to a higher incidence of many malignancies including colorectal, endometrial, ovarian, and gastric cancers. In this review, we discuss the role of PMS2 in the MMR pathway, the evolving testing criteria used to identify variants in the PMS2 gene, the LS phenotype as well as the autosomal recessive condition called constitutional mismatch repair deficiency syndrome, and current methods used to elucidate the clinical impact of PMS2 mutations.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Neoplasias Colorretais Hereditárias sem Polipose/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Mutação , Alelos , Reparo de Erro de Pareamento de DNA , Testes Genéticos/métodos , Humanos , Endonuclease PMS2 de Reparo de Erro de Pareamento/química , Fenótipo , Pseudogenes , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
DNA Cruciforme/metabolismo , Endonuclease PMS2 de Reparo de Erro de Pareamento/metabolismo , Proteína 1 Homóloga a MutL/metabolismo , Proteínas MutL/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Substituição de Aminoácidos , Quebras de DNA de Cadeia Dupla , DNA Circular/química , DNA Circular/metabolismo , DNA Cruciforme/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Humanos , Hidrólise , Endonuclease PMS2 de Reparo de Erro de Pareamento/química , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Peso Molecular , Proteína 1 Homóloga a MutL/química , Proteína 1 Homóloga a MutL/genética , Proteínas MutL/química , Proteínas MutL/genética , Proteína 2 Homóloga a MutS/química , Proteína 2 Homóloga a MutS/genética , Proteína 3 Homóloga a MutS , Mutação , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por SubstratoRESUMO
Eukaryotic MutLα (mammalian MLH1-PMS2 heterodimer; MLH1-PMS1 in yeast) functions in early steps of mismatch repair as a latent endonuclease that requires a mismatch, MutSα/ß, and DNA-loaded proliferating cell nuclear antigen (PCNA) for activation. We show here that human PCNA and MutLα interact specifically but weakly in solution to form a complex of approximately 1:1 stoichiometry that depends on PCNA interaction with the C-terminal endonuclease domain of the MutLα PMS2 subunit. Amino acid substitution mutations within a PMS2 C-terminal 721QRLIAP motif attenuate or abolish human MutLα interaction with PCNA, as well as PCNA-dependent activation of MutLα endonuclease, PCNA- and DNA-dependent activation of MutLα ATPase, and MutLα function in in vitro mismatch repair. Amino acid substitution mutations within the corresponding yeast PMS1 motif (723QKLIIP) reduce or abolish mismatch repair in vivo. Coupling of a weak allele within this motif (723AKLIIP) with an exo1Δ null mutation, which individually confer only weak mutator phenotypes, inactivates mismatch repair in the yeast cell.