RESUMEN
Current methods to assess the drug response of individual human cancers are often inaccurate, costly, or slow. Functional approaches that rapidly and directly assess the response of patient cancer tissue to drugs or small molecules offer a promising way to improve drug testing, and have the potential to identify the best therapy for individual patients. We developed a digitally manufactured microfluidic platform for multiplexed drug testing of intact cancer slice cultures, and demonstrate the use of this platform to evaluate drug responses in slice cultures from human glioma xenografts and patient tumor biopsies. This approach retains much of the tissue microenvironment and can provide results rapidly enough, within days of surgery, to guide the choice of effective initial therapies. Our results establish a useful preclinical platform for cancer drug testing and development with the potential to improve cancer personalized medicine.
RESUMEN
Present approaches to assess cancer treatments are often inaccurate, costly, and/or cumbersome. Functional testing platforms that use live tumor cells are a promising tool both for drug development and for identifying the optimal therapy for a given patient, i.e. precision oncology. However, current methods that utilize patient-derived cells from dissociated tissue typically lack the microenvironment of the tumor tissue and/or cannot inform on a timescale rapid enough to guide decisions for patient-specific therapy. We have developed a microfluidic platform that allows for multiplexed drug testing of intact tumor slices cultured on a porous membrane. The device is digitally-manufactured in a biocompatible thermoplastic by laser-cutting and solvent bonding. Here we describe the fabrication process in detail, we characterize the fluidic performance of the device, and demonstrate on-device drug-response testing with tumor slices from xenografts and from a patient colorectal tumor.
Asunto(s)
Antineoplásicos/farmacología , Doxorrubicina/farmacología , Dispositivos Laboratorio en un Chip , Animales , Antineoplásicos/administración & dosificación , Dióxido de Carbono/química , Proliferación Celular/efectos de los fármacos , Difusión , Doxorrubicina/administración & dosificación , Humanos , Masculino , Ratones , Ratones Desnudos , Neoplasias Experimentales/diagnóstico por imagen , Neoplasias Experimentales/tratamiento farmacológico , Imagen Óptica , Células Tumorales CultivadasRESUMEN
The chromosome 8p11-12 Werner syndrome (WRN ) locus encodes a RecQ helicase protein of unknown function that possesses both 3' --> 5' helicase and 3' --> 5' exonuclease activities. We show that WRN cell lines display a marked reduction in cell proliferation following mitotic recombination, and generate few viable gene conversion-type recombinants. These findings indicate that WRN plays a role in mitotic recombination, and that a loss of WRN function may promote genetic instability and disease via recombination-initiated mitotic arrest, cell death, or gene rearrangement.
Asunto(s)
ADN Helicasas/genética , ADN Helicasas/fisiología , Mitosis , Recombinación Genética , Síndrome de Werner/genética , Muerte Celular , División Celular , Línea Celular , Cromosomas Humanos Par 8 , Exodesoxirribonucleasas , Fibroblastos/metabolismo , Humanos , Modelos Biológicos , Modelos Genéticos , Fenotipo , Plásmidos/metabolismo , RecQ Helicasas , Helicasa del Síndrome de WernerRESUMEN
Homing endonucleases, like restriction enzymes, cleave double-stranded DNA at specific target sites. The cleavage mechanism(s) utilized by LAGLIDADG endonucleases have been difficult to elucidate; their active sites are divergent, and only one low resolution cocrystal structure has been determined. Here we report two high resolution structures of the dimeric I-CreI homing endonuclease bound to DNA: a substrate complex with calcium and a product complex with magnesium. The bound metals in both complexes are verified by manganese anomalous difference maps. The active sites are positioned close together to facilitate cleavage across the DNA minor groove; each contains one metal ion bound between a conserved aspartate (Asp 20) and a single scissile phosphate. A third metal ion bridges the two active sites. This divalent cation is bound between aspartate residues from the active site of each subunit and is in simultaneous contact with the scissile phosphates of both DNA strands. A metal-bound water molecule acts as the nucleophile and is part of an extensive network of ordered water molecules that are positioned by enzyme side chains. These structures illustrate a unique variant of a two-metal endonuclease mechanism is employed by the highly divergent LAGLIDADG enzyme family.
Asunto(s)
Enzimas de Restricción del ADN/química , Enzimas de Restricción del ADN/metabolismo , Metales/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Calcio/metabolismo , Catálisis , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Dimerización , Magnesio/metabolismo , Modelos Moleculares , Conformación Proteica , Alineación de Secuencia , Solventes , Agua/química , Agua/metabolismoRESUMEN
The homing endonuclease I-PpoI severely bends its DNA target, resulting in significant deformations of the minor and major groove near the scissile phosphate groups. To study the role of conformational changes within the protein catalyst and the DNA substrate, we have determined the structure of the enzyme in the absence of bound DNA, performed gel retardation analyses of DNA binding and bending, and have mutagenized a leucine residue that contacts an adenine nucleotide at the site of cleavage. The structure of the L116A/DNA complex has been determined and the effects of the mutation on affinity and catalysis have been measured. The wild-type protein displays a rigid-body rotation of its individual subunits upon DNA binding. Homing site DNA is not detectably bent in the absence of protein, but is sharply bent in both the wild-type and L116A complexes. These results indicate that binding involves a large distortion of the DNA and a smaller change in protein conformation. Leucine 116 is critical for binding and catalysis: it appears to be important for forming a well-ordered protein-DNA complex at the cleavage site, for maximal deformation of the DNA, and for desolvation of the nucleotide bases that are partially unstacked in the enzyme complex.
Asunto(s)
Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/metabolismo , Leucina/metabolismo , Sustitución de Aminoácidos/genética , Secuencia de Bases , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Dimerización , Endodesoxirribonucleasas/genética , Leucina/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Rotación , Alineación de Secuencia , TermodinámicaRESUMEN
Eukaryotic chromosome segregation depends on the mitotic spindle apparatus, a bipolar array of microtubules nucleated from centrosomes. Centrosomal microtubule nucleation requires attachment of gamma-tubulin ring complexes to a salt-insoluble centrosomal core, but the factor(s) underlying this attachment remains unknown. In budding yeast, this attachment is provided by the coiled-coil protein Spc110p, which links the yeast gamma-tubulin complex to the core of the yeast centrosome. Here, we show that the large coiled-coil protein kendrin is a human orthologue of Spc110p. We identified kendrin by its C-terminal calmodulin-binding site, which shares homology with the Spc110p calmodulin-binding site. Kendrin localizes specifically to centrosomes throughout the cell cycle. N-terminal regions of kendrin share significant sequence homology with pericentrin, a previously identified murine centrosome component known to interact with gamma-tubulin. In mitotic human breast carcinoma cells containing abundant centrosome-like structures, kendrin is found only at centrosomes associated with spindle microtubules.
Asunto(s)
Antígenos/química , Proteínas de Unión a Calmodulina/aislamiento & purificación , Calmodulina/metabolismo , Centrosoma/metabolismo , Proteínas de Neoplasias/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de Unión a Calmodulina/química , Proteínas de Unión a Calmodulina/genética , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patología , Proteínas del Citoesqueleto , Fibroblastos/metabolismo , Proteínas Fúngicas/química , Biblioteca de Genes , Humanos , Microscopía Fluorescente , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas Nucleares/química , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Huso Acromático/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Werner syndrome (WRN) is an uncommon autosomal recessive disease in which progeroid features are associated with genetic instability and an elevated risk of neoplasia. We have used the glycophorin A (GPA) somatic cell mutation assay to analyze genetic instability in vivo in WRN patients and heterozygotes. GPA variant frequencies were determined for 11 WRN patients and for 10 heterozygous family members who collectively carry 10 different WRN mutations. Genetic instability as measured by GPA O/N allele loss variant frequency was significantly increased, and this increase was strongly age-dependent in WRN patients. GPA O/N allele loss variants were also significantly elevated in heterozygous family members, thus providing the first evidence for in vivo genetic instability in heterozygous carriers in an autosomal recessive genetic instability syndrome. Our results and comparable data on other human genetic instability syndromes allow an estimate of the level of genetic instability that increases the risk of human bone marrow dysfunction or neoplasia.
Asunto(s)
Enfermedades Hematológicas/genética , Heterocigoto , Síndrome de Werner/genética , Adolescente , Adulto , Factores de Edad , Anciano , Alelos , Estudios de Casos y Controles , ADN Helicasas/genética , Exodesoxirribonucleasas , Salud de la Familia , Femenino , Citometría de Flujo , Genotipo , Glicoforinas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Mutación , RecQ Helicasas , Factores de Riesgo , Helicasa del Síndrome de WernerRESUMEN
Mutations in the chromosome 8p WRN gene cause Werner syndrome (WRN), a human autosomal recessive disease that mimics premature aging and is associated with genetic instability and an increased risk of cancer. All of the WRN mutations identified in WRN patients are predicted to truncate the WRN protein with loss of a C-terminal nuclear localization signal. However, many of these truncated proteins would retain WRN helicase and/or nuclease functional domains. We have used a combination of immune blot and immune precipitation assays to quantify WRN protein and its associated 3'-->5' helicase activity in genetically characterized WRN patient cell lines. None of the cell lines from patients harboring four different WRN mutations contained detectable WRN protein or immune-precipitable WRN helicase activity. Cell lines from WRN heterozygous individuals contained reduced amounts of both WRN protein and helicase activity. Quantitative immune blot analyses indicate that both lymphoblastoid cell lines and fibroblasts contain approximately 6 x 10(4)WRN molecules/cell. Our results indicate that most WRN mutations result in functionally equivalent null alleles, that WRN heterozygote effects may result from haploinsufficiency and that successful modeling of WRN pathogenesis in the mouse or in other model systems will require the use of WRN mutations that eliminate WRN protein expression.
Asunto(s)
ADN Helicasas/metabolismo , Síndrome de Werner/enzimología , Animales , Western Blotting , Línea Celular Transformada , Exodesoxirribonucleasas , Heterocigoto , Humanos , Ratones , Plásmidos , Pruebas de Precipitina , RecQ Helicasas , Transfección , Síndrome de Werner/genética , Síndrome de Werner/patología , Helicasa del Síndrome de WernerRESUMEN
A novel mechanism of DNA endonucleolytic cleavage has been visualized for the homing endonuclease I-PpoI by trapping the uncleaved enzyme-substrate complex and comparing it to the previously visualized product complex. This enzyme employs a unique single metal mechanism. A magnesium ion is coordinated by an asparagine residue and two DNA oxygen atoms and stabilizes the phosphoanion transition state and the 3'oxygen leaving group. A hydrolytic water molecule is activated by a histidine residue for an in-line attack on the scissile phosphate. A strained enzyme-substrate-metal complex is formed before cleavage, then relaxed during the reaction.
Asunto(s)
Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/metabolismo , Physarum polycephalum/enzimología , Sustitución de Aminoácidos/genética , Animales , Sitios de Unión , Catálisis , Cationes/metabolismo , Cristalografía por Rayos X , ADN/química , ADN/genética , ADN/metabolismo , Electrones , Endodesoxirribonucleasas/genética , Análisis de Fourier , Magnesio/metabolismo , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Oxígeno/metabolismo , Fosfatos/metabolismo , Conformación Proteica , Sodio/metabolismo , Solventes , Relación Estructura-Actividad , Agua/química , Agua/metabolismoRESUMEN
We have shown that Werner syndrome (WRN) fibroblast cell lines are unusually sensitive to the DNA-damaging agent 4-nitroquinoline 1-oxide (4NQO), though not to gamma radiation or to hydrogen peroxide. The fusion of 4NQO-sensitive WRN and 4NQO-resistant control fibroblast cell lines generated proliferating WRN x control cell hybrids that expressed WRN protein and were 4NQO-resistant. These results establish the recessive nature of 4NQO sensitivity in WRN cell lines and provide a cellular assay for WRN protein function.
Asunto(s)
4-Nitroquinolina-1-Óxido/farmacología , Fusión Celular , Fibroblastos/efectos de los fármacos , Mutágenos/farmacología , Síndrome de Werner/genética , Alelos , Western Blotting , Línea Celular Transformada , Supervivencia Celular , Daño del ADN , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Resistencia a Medicamentos , Genotipo , Humanos , Fenotipo , TransfecciónRESUMEN
Aberrant hypoxanthine phosphoribosyltransferase (HUGO-approved gene symbol HPRT1; MIM# 308000) RNA splicing promoted by splice site mutation or loss is a common mechanism for loss of the purine salvage enzyme HPRT1 from human cells. We report here two in vivo somatic HPRT1 mutations in human kidney tubular epithelial cells that disrupt HPRT1 intron 1 splicing and lead to the inclusion of intron 1 sequence in mature mRNA. Analysis of these mutations and of 14 additional HPRT1 intron 1 inclusion mutations provides an explanation for use of a common, cryptic intron 1 splice donor site by all 16 mutations.
Asunto(s)
Mutación de Línea Germinal , Hipoxantina Fosforribosiltransferasa/genética , Túbulos Renales/enzimología , Mutación , Empalme del ARN , Humanos , Intrones/genética , Túbulos Renales/citología , ARN Mensajero/genéticaAsunto(s)
Neoplasias de la Tiroides/etnología , Síndrome de Werner/etnología , Análisis Mutacional de ADN , ADN de Neoplasias/análisis , Genotipo , Humanos , Japón , Fenotipo , Pronóstico , Grupos Raciales/genética , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Síndrome de Werner/complicaciones , Síndrome de Werner/genéticaRESUMEN
Aberrant hypoxanthine phosphoribosyltransferase (HUGO-approved gene symbol HPRT1; MIM# 308000) mRNA splicing, promoted by splice site mutation or loss, is a common mechanism for loss of the purine salvage enzyme HPRT1 from human cells. We report here two in vivo somatic HPRT1 mutations in human kidney tubular epithelial cells that disrupt HPRT1 intron 1 splicing and lead to the inclusion of intron 1 sequence. We propose an explanation for the use of a common, cryptic intron 1 splice donor site by these two mutations, and by 14 additional human HPRT1 mutations that lead to aberrant splicing with the incorporation of intron 1 sequence into mRNA.
Asunto(s)
Empalme Alternativo , Células Epiteliales/metabolismo , Mutación de Línea Germinal , Hipoxantina Fosforribosiltransferasa/genética , Intrones , Riñón/metabolismo , Mutación , HumanosRESUMEN
Werner syndrome (WS) is one of a group of human genetic diseases that have recently been linked to deficits in cellular helicase function. We review the spectrum of WS-associated WRN mutations, the organization and potential functions of the WRN protein, and potential mechanistic links between the loss of WRN function and pathogenesis of the WS clinical and cellular phenotypes.
Asunto(s)
ADN Helicasas/genética , Mutación , Síndrome de Werner/genética , Animales , Cromosomas Humanos Par 8 , ADN Helicasas/química , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Exodesoxirribonucleasas , Humanos , Ratones , Modelos Genéticos , Señales de Localización Nuclear , Polimorfismo Genético , Procesamiento Proteico-Postraduccional , RecQ Helicasas , Síndrome de Werner/diagnóstico , Helicasa del Síndrome de WernerRESUMEN
We have determined the ability of two well-characterized eukaryotic homing endonucleases, I-PpoI from the myxomycete Physarum polycephalum and I-CreI from the green alga Chlamydomonas reinhardtii, to generate site-specific DNA double-strand breaks in human cells. These 18-kDa proteins cleave highly conserved 15- or 24-bp rDNA homing sites in their respective hosts to generate homogeneous 4-base, 3' ends that initiate target intron transposition or "homing." We show that both endonucleases can be expressed in human cells and can generate site-specific DNA double-strand breaks in 28S rDNA and homing site plasmids. These endonuclease-induced breaks can be repaired in vivo, although break repair is mutagenic with the frequent generation of short deletions or insertions. I-PpoI and I-CreI should be useful for analyzing DNA double-strand break repair in human cells and rDNA.
Asunto(s)
Daño del ADN , Enzimas de Restricción del ADN/genética , Endodesoxirribonucleasas/genética , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Daño del ADN/genética , Reparación del ADN , Humanos , Datos de Secuencia Molecular , Plásmidos/genética , Alineación de SecuenciaRESUMEN
The structure of the LAGLIDADG intron-encoded homing endonuclease I-CreI bound to homing site DNA has been determined. The interface is formed by an extended, concave beta sheet from each enzyme monomer that contacts each DNA half-site, resulting in direct side-chain contacts to 18 of the 24 base pairs across the full-length homing site. The structure indicates that I-CreI is optimized to its role in genetic transposition by exhibiting long site-recognition while being able to cleave many closely related target sequences. DNA cleavage is mediated by a compact pair of active sites in the I-CreI homodimer, each of which contains a separate bound divalent cation.
Asunto(s)
Enzimas de Restricción del ADN/química , Enzimas de Restricción del ADN/metabolismo , Sitios de Unión/genética , Calcio/química , Calcio/metabolismo , Cristalografía , ADN/química , ADN/metabolismo , Enzimas de Restricción del ADN/genética , Intrones , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Homing endonucleases are a diverse collection of proteins that are encoded by genes with mobile, self-splicing introns. They have also been identified in self-splicing inteins (protein introns). These enzymes promote the movement of the DNA sequences that encode them from one chromosome location to another; they do this by making a site-specific double-strand break at a target site in an allele that lacks the corresponding mobile intron. The target sites recognized by these small endonucleases are generally long (14-44 base pairs). Four families of homing endonucleases have been identified, including the LAGLIDADG, the His-Cys box, the GIY-YIG and the H-N-H endonucleases. The first identified His-Cys box homing endonuclease was I-PpoI from the slime mould Physarum polycephalum. Its gene resides in one of only a few nuclear introns known to exhibit genetic mobility. Here we report the structure of the I-PpoI homing endonuclease bound to homing-site DNA determined to 1.8 A resolution. I-PpoI displays an elongated fold of dimensions 25 x 35 x 80 A, with mixed alpha/beta topology. Each I-PpoI monomer contains three antiparallel beta-sheets flanked by two long alpha-helices and a long carboxy-terminal tail, and is stabilized by two bound zinc ions 15 A apart. The enzyme possesses a new zinc-bound fold and endonuclease active site. The structure has been determined in both uncleaved substrate and cleaved product complexes.
Asunto(s)
ADN/metabolismo , Endodesoxirribonucleasas/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Proteínas de Unión al ADN , Endodesoxirribonucleasas/metabolismo , Intrones , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Alineación de Secuencia , Zinc/metabolismoRESUMEN
Plasmid libraries containing partially randomized cleavage sites for the eukaryotic homing endonucleases I-PpoI and I-CreI were constructed, and sites that could be cleaved by I-PpoI or I-CreI were selectively recovered by successive cycles of cleavage and gel separation followed by religation and growth in Escherichia coli. Twenty-one different I-PpoI-sensitive homing sites, including the native homing site, were isolated. These sites were identical at four nucleotide positions within the 15 bp homing site, had a restricted pattern of base substitutions at the remaining 11 positions and displayed a preference for purines flanking the top strand of the homing site sequence. Twenty-one different I-CreI-sensitive homing sites, including the native site, were isolated. Ten nucleotide positions were identical in homing site variants that were I-CreI-sensitive and required the addition of SDS for efficient cleavage product release. Four of these ten positions were identical in homing sites that did not require SDS for product release. There was a preference for pyrimidines flanking the top strand of the homing site sequence. Three of the 24 I-CreI homing site nucleotide positions apparently lacked informational content, i. e. were permissive of cleavage when occupied by any nucleotide. These results suggest that I-PpoI and I-CreI make a large number of DNA-protein contacts across their homing site sequences, and that different subsets of these contacts may be sufficient to maintain a high degree of sequence-specific homing site recognition and cleavage. The sequential enrichment protocol we used should be useful for defining the sequence degeneracy and informational content of other homing endonuclease target sites.
Asunto(s)
Enzimas de Restricción del ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Mutagénesis , Secuencia de Bases , Sitios de Unión , Enzimas de Restricción del ADN/genética , Endodesoxirribonucleasas/genética , Escherichia coli/genética , Intrones/genética , Datos de Secuencia Molecular , Plásmidos/genética , Alineación de Secuencia , Especificidad por SustratoRESUMEN
We have developed and used a genetic selection system in Escherichia coli to study functional requirements for homing site recognition and cleavage by a representative eukaryotic mobile intron endonuclease. The homing endonuclease, I-CreI, was originally isolated from the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. I-CreI homing site mutants contained base pair substitutions or single base deletions that altered the rate of homing site cleavage and/or product release. I-CreI endonuclease mutants fell into six phenotypic classes that differed in in vivo activity, toxicity or genetic dominance. Inactivating mutations clustered in the N-terminal 60% of the I-CreI amino acid sequence, and two frameshift mutations were isolated that resulted in premature translation termination though retained partial activity. These mutations indicate that the N-terminal two-thirds of the I-CreI endonuclease is sufficient for homing site recognition and cleavage. Substitution mutations altered in four potential active site residues were examined: D20N, Q47H or R70A substitutions inactivated endonuclease activity, whereas S22A did not. The genetic approach we have taken complements phylogenetic and structural studies of mobile intron endonucleases and has provided new information on the mechanistic basis of I-CreI homing site recognition and cleavage.
Asunto(s)
Chlamydomonas reinhardtii/enzimología , Enzimas de Restricción del ADN/metabolismo , Escherichia coli/genética , Intrones , Animales , Sitios de Unión , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/genética , Modelos Moleculares , Mutagénesis , Conformación ProteicaRESUMEN
Werner syndrome (WS) is an autosomal recessive disease, the phenotype of which is a caricature of premature aging. WS cells and cell lines display several types of genetic instability, and WS patients have an increased risk of developing cancer. The WS locus (WRN) encodes a protein that shows significant sequence homology to the RecQ family of DNA helicases. Because a DNA helicase may function in DNA mismatch repair, we examined extracts of WS cell lines for mismatch repair activity. Extracts from four different WS lymphoblastoid cell lines containing different WRN mutations and from three within-pedigree control cell lines were all proficient in mismatch repair. In marked contrast, extracts from three independent WS fibroblastoid cell lines were deficient in repair of base-base and insertion/deletion mismatches. Extracts of one of these lines restored activity to extracts of mismatch repair-deficient tumor cells with defined mutations in hMSH2, hMSH3, hMSH6, hMLH1, or hPMS2. This suggests that the WRN mutation in this fibroblast line is not a dominant negative inhibitor of mismatch repair activity and that the repair defect does not reside in these five known mismatch repair genes. Defective mismatch repair in fibroblastoid but not lymphoblastoid cells is consistent with the possibility that WRN protein could have a cell type- and/or tissue-specific role in mismatch repair. Alternatively, a mutation in WRN could predispose cells to mutations in other genes required for mismatch repair activity, at least one of which could be an unknown gene.