RESUMEN
Germline mutations in the mismatch repair (MMR) genes MSH2, MSH6, MLH1, and PMS2 are linked to cancer of the colon and other organs, characterized by microsatellite instability and a large increase in mutation frequency. Unexpectedly, mutations in EXO1, encoding the only exonuclease genetically implicated in MMR, are not linked to familial cancer and cause a substantially weaker mutator phenotype. This difference could be explained if eukaryotic cells possessed additional exonucleases redundant with EXO1. Analysis of the MLH1 interactome identified FANCD2-associated nuclease 1 (FAN1), a novel enzyme with biochemical properties resembling EXO1. We now show that FAN1 efficiently substitutes for EXO1 in MMR assays and that this functional complementation is modulated by its interaction with MLH1. FAN1 also contributes to MMR in vivo; cells lacking both EXO1 and FAN1 have an MMR defect and display resistance to N-methyl-N-nitrosourea (MNU) and 6-thioguanine (TG). Moreover, FAN1 loss amplifies the mutational profile of EXO1-deficient cells, suggesting that the two nucleases act redundantly in the same antimutagenic pathway. However, the increased drug resistance and mutator phenotype of FAN1/EXO1-deficient cells are less prominent than those seen in cells lacking MSH6 or MLH1. Eukaryotic cells thus apparently possess additional mechanisms that compensate for the loss of EXO1.
Asunto(s)
Proteínas Aviares/metabolismo , Reparación de la Incompatibilidad de ADN , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Enzimas Multifuncionales/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Pollos , Endodesoxirribonucleasas/química , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/deficiencia , Exodesoxirribonucleasas/genética , Guanosina/análogos & derivados , Células HEK293 , Humanos , Metilnitronitrosoguanidina , Enzimas Multifuncionales/química , Mutación/genética , TionucleósidosRESUMEN
FAN1, a DNA structure-specific nuclease, interacts with MLH1, but the repair pathways in which this complex acts are unknown. FAN1 processes DNA interstrand crosslinks (ICLs) and FAN1 variants are modifiers of the neurodegenerative Huntington's disease (HD), presumably by regulating HD-causing CAG repeat expansions. Here, we identify specific amino acid residues in two adjacent FAN1 motifs that are critical for MLH1 binding. Disruption of the FAN1-MLH1 interaction confers cellular hypersensitivity to ICL damage and defective repair of CAG/CTG slip-outs, intermediates of repeat expansion mutations. FAN1-S126 phosphorylation, which hinders FAN1-MLH1 association, is cell cycle-regulated by cyclin-dependent kinase activity and attenuated upon ICL induction. Our data highlight the FAN1-MLH1 complex as a phosphorylation-regulated determinant of ICL response and repeat stability, opening novel paths to modify cancer and neurodegeneration.
Asunto(s)
Endodesoxirribonucleasas , Exodesoxirribonucleasas , ADN , Daño del ADN , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Enzimas Multifuncionales/genéticaRESUMEN
Mutational signatures are imprints of pathophysiological processes arising through tumorigenesis. We generated isogenic CRISPR-Cas9 knockouts (Δ) of 43 genes in human induced pluripotent stem cells, cultured them in the absence of added DNA damage, and performed whole-genome sequencing of 173 subclones. ΔOGG1, ΔUNG, ΔEXO1, ΔRNF168, ΔMLH1, ΔMSH2, ΔMSH6, ΔPMS1, and ΔPMS2 produced marked mutational signatures indicative of being critical mitigators of endogenous DNA modifications. Detailed analyses revealed mutational mechanistic insights, including how 8-oxo-dG elimination is sequence-context-specific while uracil clearance is sequence-context-independent. Mismatch repair (MMR) deficiency signatures are engendered by oxidative damage (C>A transversions), differential misincorporation by replicative polymerases (T>C and C>T transitions), and we propose a 'reverse template slippage' model for T>A transversions. ΔMLH1, ΔMSH6, and ΔMSH2 signatures were similar to each other but distinct from ΔPMS2. Finally, we developed a classifier, MMRDetect, where application to 7,695 WGS cancers showed enhanced detection of MMR-deficient tumors, with implications for responsiveness to immunotherapies.
Asunto(s)
Neoplasias Colorrectales , Células Madre Pluripotentes Inducidas , Neoplasias Encefálicas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Neoplasias Colorrectales/genética , Daño del ADN/genética , Humanos , Mutación , Síndromes Neoplásicos HereditariosRESUMEN
Replication factor C (RFC), a heteropentamer of RFC1-5, loads PCNA onto DNA during replication and repair. Once DNA synthesis has ceased, PCNA must be unloaded. Recent findings assign the uloader role primarily to an RFC-like (RLC) complex, in which the largest RFC subunit, RFC1, has been replaced with ATAD5 (ELG1 in Saccharomyces cerevisiae). ATAD5-RLC appears to be indispensable, given that Atad5 knock-out leads to embryonic lethality. In order to learn how the retention of PCNA on DNA might interfere with normal DNA metabolism, we studied the response of ATAD5-depleted cells to several genotoxic agents. We show that ATAD5 deficiency leads to hypersensitivity to methyl methanesulphonate (MMS), camptothecin (CPT) and mitomycin C (MMC), agents that hinder the progression of replication forks. We further show that ATAD5-depleted cells are sensitive to poly(ADP)ribose polymerase (PARP) inhibitors and that the processing of spontaneous oxidative DNA damage contributes towards this sensitivity. We posit that PCNA molecules trapped on DNA interfere with the correct metabolism of arrested replication forks, phenotype reminiscent of defective homologous recombination (HR). As Atad5 heterozygous mice are cancer-prone and as ATAD5 mutations have been identified in breast and endometrial cancers, our finding may open a path towards the therapy of these tumours.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/genética , Antineoplásicos/farmacología , Daño del ADN , Proteínas de Unión al ADN/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Pollos , Cromatina/enzimología , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Inestabilidad Genómica , Mutágenos/toxicidad , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1/metabolismoRESUMEN
The mechanisms that underpin how insertions or deletions (indels) become fixed in DNA have primarily been ascribed to replication-related and/or double-strand break (DSB)-related processes. Here, we introduce a method to evaluate indels, orientating them relative to gene transcription. In so doing, we reveal a number of surprising findings: First, there is a transcriptional strand asymmetry in the distribution of mononucleotide repeat tracts in the reference human genome. Second, there is a strong transcriptional strand asymmetry of indels across 2,575 whole genome sequenced human cancers. We suggest that this is due to the activity of transcription-coupled nucleotide excision repair (TC-NER). Furthermore, TC-NER interacts with mismatch repair (MMR) under physiological conditions to produce strand bias. Finally, we show how insertions and deletions differ in their dependencies on these repair pathways. Our analytical approach reveals insights into the contribution of DNA repair towards indel mutagenesis in human cells.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Neoplasias/genética , Secuencias Repetitivas de Ácidos Nucleicos , Secuencias de Aminoácidos , Biología Computacional , Análisis Mutacional de ADN , Replicación del ADN , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Variación Genética , Genoma Humano , Genómica , Humanos , Mutación INDEL , Mutagénesis , Neoplasias/metabolismo , Polinucleótidos/genética , Análisis de Secuencia de ARN , Transcripción GenéticaRESUMEN
The enhancer/promoter of the vitellogenin II gene (VTG) has been extensively studied as a model system of vertebrate transcriptional control. While deletion mutagenesis and in vivo footprinting identified the transcription factor (TF) binding sites governing its tissue specificity, DNase hypersensitivity and DNA methylation studies revealed the epigenetic changes accompanying its hormone-dependent activation. Moreover, upon induction with estrogen (E2), the region flanking the estrogen-responsive element (ERE) was reported to undergo active DNA demethylation. We now show that although the VTG ERE is methylated in embryonic chicken liver and in LMH/2A hepatocytes, its induction by E2 was not accompanied by extensive demethylation. In contrast, E2 failed to activate a VTG enhancer/promoter-controlled luciferase reporter gene methylated by SssI. Surprisingly, this inducibility difference could be traced not to the ERE but rather to a single CpG in an E-box (CACGTG) sequence upstream of the VTG TATA box, which is unmethylated in vivo but methylated by SssI. We demonstrate that this E-box binds the upstream stimulating factor USF1/2. Selective methylation of the CpG within this binding site with an E-box-specific DNA methyltransferase, Eco72IM, was sufficient to attenuate USF1/2 binding in vitro and abolish the hormone-induced transcription of the VTG gene in the reporter system.
Asunto(s)
Expresión Génica Ectópica/genética , Receptor alfa de Estrógeno/genética , Vitelogeninas/genética , Animales , Sitios de Unión , Línea Celular , Embrión de Pollo , Islas de CpG/genética , Metilación de ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN-Citosina Metilasas/metabolismo , Expresión Génica Ectópica/efectos de los fármacos , Receptor alfa de Estrógeno/metabolismo , Estrógenos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/metabolismo , Vitelogeninas/metabolismoRESUMEN
Poly(ADP-ribose) polymerases (PARPs) facilitate the repair of DNA single-strand breaks (SSBs). When PARPs are inhibited, unrepaired SSBs colliding with replication forks give rise to cytotoxic double-strand breaks. These are normally rescued by homologous recombination (HR), but, in cells with suboptimal HR, PARP inhibition leads to genomic instability and cell death, a phenomenon currently exploited in the therapy of ovarian cancers in BRCA1/2 mutation carriers. In spite of their promise, resistance to PARP inhibitors (PARPis) has already emerged. In order to identify the possible underlying causes of the resistance, we set out to identify the endogenous source of DNA damage that activates PARPs. We argued that if the toxicity of PARPis is indeed caused by unrepaired SSBs, these breaks must arise spontaneously, because PARPis are used as single agents. We now show that a significant contributor to PARPi toxicity is oxygen metabolism. While BRCA1-depleted or -mutated cells were hypersensitive to the clinically approved PARPi olaparib, its toxicity was significantly attenuated by depletion of OGG1 or MYH DNA glycosylases, as well as by treatment with reactive oxygen species scavengers, growth under hypoxic conditions or chemical OGG1 inhibition. Thus, clinical resistance to PARPi therapy may emerge simply through reduced efficiency of oxidative damage repair.
Asunto(s)
Proteína BRCA1/genética , ADN Glicosilasas/genética , Neoplasias Ováricas/tratamiento farmacológico , Poli(ADP-Ribosa) Polimerasas/genética , Línea Celular Tumoral , Roturas del ADN de Cadena Simple/efectos de los fármacos , Daño del ADN/efectos de los fármacos , ADN Glicosilasas/antagonistas & inhibidores , Resistencia a Antineoplásicos/genética , Femenino , Recombinación Homóloga/efectos de los fármacos , Recombinación Homóloga/genética , Humanos , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Oxidación-Reducción/efectos de los fármacos , Ftalazinas/efectos adversos , Ftalazinas/farmacología , Piperazinas/efectos adversos , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Mutaciones Letales Sintéticas/genéticaRESUMEN
The financial support for this Article was not fully acknowledged. The Acknowledgements should have included the following: This study was in part supported by the Swiss National Foundation Grant No.: 31003A-156023 to Alessandro Sartori.
RESUMEN
Interstrand cross-link (ICL) hypersensitivity is a characteristic trait of Fanconi anemia (FA). Although FANCD2-associated nuclease 1 (FAN1) contributes to ICL repair, FAN1 mutations predispose to karyomegalic interstitial nephritis (KIN) and cancer rather than to FA. Thus, the biological role of FAN1 remains unclear. Because fork stalling in FAN1-deficient cells causes chromosomal instability, we reasoned that the key function of FAN1 might lie in the processing of halted replication forks. Here, we show that FAN1 contains a previously-uncharacterized PCNA interacting peptide (PIP) motif that, together with its ubiquitin-binding zinc finger (UBZ) domain, helps recruit FAN1 to ubiquitylated PCNA accumulated at stalled forks. This prevents replication fork collapse and controls their progression. Furthermore, we show that FAN1 preserves replication fork integrity by a mechanism that is distinct from BRCA2-dependent homologous recombination. Thus, targeting FAN1 activities and its interaction with ubiquitylated PCNA may offer therapeutic opportunities for treatment of BRCA-deficient tumors.
Asunto(s)
Proteína BRCA2/metabolismo , Exodesoxirribonucleasas/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína BRCA2/genética , Línea Celular Tumoral , Reparación del ADN/genética , Reparación del ADN/fisiología , Replicación del ADN/genética , Replicación del ADN/fisiología , Endodesoxirribonucleasas , Exodesoxirribonucleasas/genética , Humanos , Enzimas Multifuncionales , Antígeno Nuclear de Célula en Proliferación/genética , Unión Proteica/genética , Unión Proteica/fisiología , Ubiquitinación/genética , Ubiquitinación/fisiologíaAsunto(s)
Distinciones y Premios , Oncología Médica/normas , Animales , Historia del Siglo XX , HumanosRESUMEN
DNA mismatch repair (MMR) is an evolutionarily-conserved process responsible for the repair of replication errors. In Escherichia coli, MMR is initiated by MutS and MutL, which activate MutH to incise transiently-hemimethylated GATC sites. MMR efficiency depends on the distribution of these GATC sites. To understand which molecular events determine repair efficiency, we quantitatively studied the effect of strand incision on unwinding and excision activity. The distance between mismatch and GATC site did not influence the strand incision rate, and an increase in the number of sites enhanced incision only to a minor extent. Two GATC sites were incised by the same activated MMR complex in a processive manner, with MutS, the closed form of MutL and MutH displaying different roles. Unwinding and strand excision were more efficient on a substrate with two nicks flanking the mismatch, as compared to substrates containing a single nick or two nicks on the same side of the mismatch. Introduction of multiple nicks by the human MutLα endonuclease also contributed to increased repair efficiency. Our data support a general model of prokaryotic and eukaryotic MMR in which, despite mechanistic differences, mismatch-activated complexes facilitate efficient repair by creating multiple daughter strand nicks.
Asunto(s)
Reparación de la Incompatibilidad de ADN , Replicación del ADN , Disparidad de Par Base/genética , Secuencia de Bases , Metilación de ADN/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Conformación ProteicaRESUMEN
During class switch recombination (CSR), antigen-stimulated B-cells rearrange their immunoglobulin constant heavy chain (CH) loci to generate antibodies with different effector functions. CSR is initiated by activation-induced deaminase (AID), which converts cytosines in switch (S) regions, repetitive sequences flanking the CH loci, to uracils. Although U/G mispairs arising in this way are generally efficiently repaired to C/Gs by uracil DNA glycosylase (UNG)-initiated base excision repair (BER), uracil processing in S-regions of activated B-cells occasionally gives rise to double strand breaks (DSBs), which trigger CSR. Surprisingly, genetic experiments revealed that CSR is dependent not only on AID and UNG, but also on mismatch repair (MMR). To elucidate the role of MMR in CSR, we studied the processing of uracil-containing DNA substrates in extracts of MMR-proficient and -deficient human cells, as well as in a system reconstituted from recombinant BER and MMR proteins. Here, we show that the interplay of these repair systems gives rise to DSBs in vitro and to genomic deletions and mutations in vivo, particularly in an S-region sequence. Our findings further suggest that MMR affects pathway choice in DSB repair. Given its amenability to manipulation, our system represents a powerful tool for the molecular dissection of CSR.
Asunto(s)
Linfocitos B/metabolismo , Reparación de la Incompatibilidad de ADN/inmunología , ADN/genética , Cambio de Clase de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/genética , Uracilo/metabolismo , Linfocitos B/citología , Linfocitos B/inmunología , Línea Celular Tumoral , Citidina Desaminasa/genética , Citidina Desaminasa/inmunología , Citosina/inmunología , Citosina/metabolismo , ADN/inmunología , Roturas del ADN de Doble Cadena , Regulación de la Expresión Génica , Células HEK293 , Humanos , Transducción de Señal , Uracilo/inmunología , Uracil-ADN Glicosidasa/genética , Uracil-ADN Glicosidasa/inmunologíaRESUMEN
RUVBL1 (RuvB-like1) and RUVBL2 (RuvB-like 2) are integral components of multisubunit protein complexes involved in processes ranging from cellular metabolism, transcription and chromatin remodeling to DNA repair. Here, we show that although RUVBL1 and RUVBL2 are known to form heterodimeric complexes in which they stabilize each other, the subunits separate during cytokinesis. In anaphase-to-telophase transition, RUVBL1 localizes to structures of the mitotic spindle apparatus, where it partially co-localizes with polo-like kinase 1 (PLK1). The ability of PLK1 to phosphorylate RUVBL1-but not RUVBL2-in vitro and their physical association in vivo suggest that this kinase differentially regulates the function of the RuvB-like proteins during mitosis. We further show that siRNA-mediated knock-down of RuvB-like proteins causes severe defects in chromosome alignment and segregation. In addition, we show that the ATPase activity of RUVBL1 is indispensable for cell proliferation. Our data thus demonstrate that RUVBL1 is essential for efficient mitosis and proliferation.
Asunto(s)
Proteínas Portadoras/metabolismo , Segregación Cromosómica/fisiología , Citocinesis/fisiología , ADN Helicasas/metabolismo , Mitosis/fisiología , ATPasas Asociadas con Actividades Celulares Diversas , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN Helicasas/genética , Reparación del ADN , Células HeLa , Humanos , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Huso Acromático/genética , Huso Acromático/metabolismo , Quinasa Tipo Polo 1RESUMEN
Cisplatin and its derivatives, nitrogen mustards and mitomycin C, are used widely in cancer chemotherapy. Their efficacy is linked primarily to their ability to generate DNA interstrand cross-links (ICLs), which effectively block the progression of transcription and replication machineries. Release of this block, referred to as unhooking, has been postulated to require endonucleases that incise one strand of the duplex on either side of the ICL. Here we investigated how the 5' flap nucleases FANCD2-associated nuclease 1 (FAN1), exonuclease 1 (EXO1), and flap endonuclease 1 (FEN1) process a substrate reminiscent of a replication fork arrested at an ICL. We now show that EXO1 and FEN1 cleaved the substrate at the boundary between the single-stranded 5' flap and the duplex, whereas FAN1 incised it three to four nucleotides in the double-stranded region. This affected the outcome of processing of a substrate containing a nitrogen mustard-like ICL two nucleotides in the duplex region because FAN1, unlike EXO1 and FEN1, incised the substrate predominantly beyond the ICL and, therefore, failed to release the 5' flap. We also show that FAN1 was able to degrade a linear ICL substrate. This ability of FAN1 to traverse ICLs in DNA could help to elucidate its biological function, which is currently unknown.
Asunto(s)
Enzimas Reparadoras del ADN/química , ADN/química , Exodesoxirribonucleasas/química , Endonucleasas de ADN Solapado/química , Línea Celular , ADN/genética , ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Endodesoxirribonucleasas , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , Humanos , Enzimas Multifuncionales , Especificidad por SustratoRESUMEN
The cytotoxicity of SN1-type alkylating agents such as N-methyl-N'-nitrosourea (MNU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), or the cancer chemotherapeutics temozolomide, dacarbazine and streptozotocin has been ascribed to the persistence of O(6)-methylguanine ((me)G) in genomic DNA. One hypothesis posits that (me)G toxicity is caused by futile attempts of the mismatch repair (MMR) system to process (me)G/C or (me)G/T mispairs arising during replication, while an alternative proposal suggests that the latter lesions activate DNA damage signaling, cell cycle arrest and apoptosis directly. Attempts to elucidate the molecular mechanism of (me)G-induced cell killing in vivo have been hampered by the fact that the above reagents induce several types of modifications in genomic DNA, which are processed by different repair pathways. In contrast, defined substrates studied in vitro did not undergo replication. We set out to re-examine this phenomenon in replication-competent Xenopus laevis egg extracts, using either phagemid substrates containing a single (me)G residue, or methylated sperm chromatin. Our findings provide further support for the futile cycling hypothesis.
Asunto(s)
Daño del ADN , Reparación de la Incompatibilidad de ADN/fisiología , ADN/metabolismo , Guanina/análogos & derivados , Animales , Extractos Celulares , ADN/química , Replicación del ADN , Guanina/metabolismo , Óvulo/metabolismo , Xenopus laevisRESUMEN
Replicative DNA polymerases are high fidelity enzymes that misincorporate nucleotides into nascent DNA with a frequency lower than [1/10(5)], and this precision is improved to about [1/10(7)] by their proofreading activity. Because this fidelity is insufficient to replicate most genomes without error, nature evolved postreplicative mismatch repair (MMR), which improves the fidelity of DNA replication by up to 3 orders of magnitude through correcting biosynthetic errors that escaped proofreading. MMR must be able to recognize non-Watson-Crick base pairs and excise the misincorporated nucleotides from the nascent DNA strand, which carries by definition the erroneous genetic information. In eukaryotes, MMR is believed to be directed to the nascent strand by preexisting discontinuities such as gaps between Okazaki fragments in the lagging strand or breaks in the leading strand generated by the mismatch-activated endonuclease of the MutL homologs PMS1 in yeast and PMS2 in vertebrates. We recently demonstrated that the eukaryotic MMR machinery can make use also of strand breaks arising during excision of uracils or ribonucleotides from DNA. We now show that intermediates of MutY homolog-dependent excision of adenines mispaired with 8-oxoguanine (G(O)) also act as MMR initiation sites in extracts of human cells or Xenopus laevis eggs. Unexpectedly, G(O)/C pairs were not processed in these extracts and failed to affect MMR directionality, but extracts supplemented with exogenous 8-oxoguanine DNA glycosylase (OGG1) did so. Because OGG1-mediated excision of G(O) might misdirect MMR to the template strand, our findings suggest that OGG1 activity might be inhibited during MMR.
Asunto(s)
Disparidad de Par Base , ADN Glicosilasas/genética , Reparación de la Incompatibilidad de ADN , Guanina/análogos & derivados , Purinas/metabolismo , Xenopus laevis/genética , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , ADN/química , ADN/metabolismo , Daño del ADN , ADN Glicosilasas/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Femenino , Guanina/metabolismo , Guanina/farmacología , Células HCT116 , Humanos , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto , Datos de Secuencia Molecular , Proteínas MutL , Oocitos/citología , Oocitos/metabolismo , Oxidación-Reducción , Purinas/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Xenopus laevis/metabolismoRESUMEN
Next-generation sequencing has revolutionized the search for disease-causing genetic alterations. Unfortunately, the task of distinguishing the handful of causative mutations from rare variants remains daunting. We now describe an assay that permits the analysis of all types of mutations in any gene of choice through the generation of stable human cell lines, in which the endogenous protein has been inducibly replaced with its genetic variant. Here we studied the phenotype of variants of the essential replicative polymerase-δ carrying missense mutations in its active site, similar to those recently identified in familial colon cancer patients. We show that expression of the mutants but not the wild-type protein endows the engineered cells with a mutator phenotype and that the mutations affect the fidelity and/or the exonuclease activity of the isolated enzyme in vitro. This proof-of-principle study demonstrates the general applicability of this experimental approach in the study of genotype-phenotype correlations.
Asunto(s)
ADN Polimerasa III/genética , Mutación Missense , Secuencia de Aminoácidos , Secuencia de Bases , Dominio Catalítico , Ciclo Celular , Línea Celular , Línea Celular Tumoral , Neoplasias del Colon/genética , ADN Complementario/metabolismo , Citometría de Flujo , Estudios de Asociación Genética , Vectores Genéticos , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta , Fenotipo , Interferencia de ARN , TransfecciónRESUMEN
OBJECTIVES: The outcome of patients with primary melanoma (PM) cannot be completely explained based on currently adopted clinical-histopathologic criteria. In this study, we evaluated the potential prognostic value of mismatch repair protein expression in PMs. METHODS: We examined the immunohistochemical staining of mismatch repair proteins in 18 benign nevi and 101 stage I to III PMs and investigated their association with tumor clinicopathologic variables and melanoma mortality. RESULTS: Expression of MSH2, MLH1, and PMS2 was high in benign nevi and reduced in a subset of PMs. Conversely, MSH6 expression was absent or extremely low in benign nevi and increased in a subset of PMs. In the multivariate analysis, including sex, age, Breslow thickness, and ulceration, high MSH6 expression in PMs (ie, immunostaining in >20% of tumor cells) was significantly associated with an increased risk of melanoma mortality (relative risk, 3.76; 95% confidence interval, 1.12-12.70). CONCLUSIONS: MSH6 protein expression can be a valuable marker to improve prognosis assessment in PMs.
Asunto(s)
Reparación de la Incompatibilidad de ADN , Proteínas de Unión al ADN/metabolismo , Melanoma/metabolismo , Nevo/metabolismo , Neoplasias Cutáneas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/metabolismo , Adulto , Anciano , Enzimas Reparadoras del ADN/metabolismo , Femenino , Humanos , Masculino , Melanoma/mortalidad , Persona de Mediana Edad , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Nucleares/metabolismo , Pronóstico , Neoplasias Cutáneas/mortalidad , Tasa de SupervivenciaRESUMEN
Mutations in the mismatch repair (MMR) genes MSH2, MSH6, MLH1 and PMS2 are associated with Lynch Syndrome (LS), a familial predisposition to early-onset cancer of the colon and other organs. Because not all LS families carry mutations in these four genes, the search for cancer-associated mutations was extended to genes encoding other members of the mismatch repairosome. This effort identified mutations in EXO1, which encodes the sole exonuclease implicated in MMR. One of these mutations, E109K, was reported to abrogate the catalytic activity of the enzyme, yet, in the crystal structure of the EXO1/DNA complex, this glutamate is far away from both DNA and the catalytic site of the enzyme. In an attempt to elucidate the reason underlying the putative loss of function of this variant, we expressed it in Escherichia coli, and tested its activity in a series of biochemical assays. We now report that, contrary to earlier reports, and unlike the catalytic site mutant D173A, the EXO1 E109K variant resembled the wild-type (wt) enzyme on all tested substrates. In the light of our findings, we attempt here to reinterpret the results of the phenotypic characterization of a knock-in mouse carrying the E109K mutation and cells derived from it.