RESUMEN
Previous small scale sequencing studies have indicated that DNA polymerase ß (pol ß) variants are present on average in 30% of human tumors of varying tissue origin. Many of these variants have been shown to have aberrant enzyme function in vitro and to induce cellular transformation and/or genomic instability in vivo, suggesting that their presence is associated with tumorigenesis or its progression. In this study, the human POLB gene was sequenced in a collection of 134 human colorectal tumors and was found to contain coding region mutations in 40% of the samples. The variants map to many different sites of the pol ß protein and are not clustered. Many variants are nonsynonymous amino acid substitutions predicted to affect enzyme function. A subset of these variants was found to have reduced enzyme activity in vitro and failed to fully rescue pol ß-deficient cells from methylmethane sulfonate-induced cytotoxicity. Tumors harboring variants with reduced enzyme activity may have compromised base excision repair function, as evidenced by our methylmethane sulfonate sensitivity studies. Such compromised base excision repair may drive tumorigenesis by leading to an increase in mutagenesis or genomic instability.
Asunto(s)
Sustitución de Aminoácidos , Neoplasias Colorrectales/genética , ADN Polimerasa beta/genética , Mutación , Animales , Sitios de Unión/genética , Biocatálisis , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , ADN Polimerasa beta/química , ADN Polimerasa beta/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Prueba de Complementación Genética , Células HEK293 , Humanos , Cinética , Metilmetanosulfonato/toxicidad , Ratones , Ratones Noqueados , Modelos Moleculares , Mutágenos/toxicidad , Tasa de Mutación , Estadificación de Neoplasias , Estructura Terciaria de ProteínaRESUMEN
Studies show that 30% of 189 tumors sequenced to date express variants of the polymerase beta (pol beta) protein that are not present in normal tissue. This raises the possibility that variants of pol beta might be linked to the etiology of cancer. Here, we characterize the I260M prostate-cancer-associated variant of pol beta. Ile260 is a key residue of the hydrophobic hinge that is important for the closing of the polymerase. In this study, we demonstrate that the I260M variant is a sequence context-dependent mutator polymerase. Specifically, I260M is a mutator for misalignment-mediated errors in dipyrimidine sequences. I260M is also a low-fidelity polymerase with regard to the induction of transversions within specific sequence contexts. Our results suggest that the hinge influences the geometry of the DNA within the polymerase active site that is important for accurate DNA synthesis. Importantly, characterization of the I260M variant shows that it has a functional phenotype that could be linked to the etiology or malignant progression of human cancer.
Asunto(s)
Transformación Celular Neoplásica/genética , ADN Polimerasa beta/genética , Reparación del ADN/fisiología , Mutación/genética , Neoplasias de la Próstata/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Dicroismo Circular , ADN Polimerasa beta/metabolismo , ADN Circular , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Neoplasias de la Próstata/enzimología , Pliegue de Proteína , Moldes GenéticosRESUMEN
Thirty percent of the 189 tumors studied to date express DNA polymerase beta variants. One of these variants was identified in a prostate carcinoma and is altered from isoleucine to methionine at position 260, within the hydrophobic hinge region of the protein. Another variant was identified in a colon carcinoma and is altered at position 289 from lysine to methionine, within helix N of the protein. We have shown that the types of mutations induced by these cancer-associated variants are different from those induced by the wild-type enzyme. In this study, we show that expression of the I260M and K289M cancer-associated variants in mouse C127 cells results in a transformed phenotype in the great majority of cell clones tested, as assessed by focus formation and anchorage-independent growth. Strikingly, cellular transformation occurs after a variable number of passages in culture but, once established, does not require continuous expression of the polymerase beta variant proteins, implying that it has a mutational basis. Because DNA polymerase beta functions in base excision repair, our results suggest that mutations that arise during this process can lead to the onset or progression of cancer.