RESUMEN
Proliferating cell nuclear antigen (PCNA) is a DNA polymerase cofactor and regulator of replication-linked functions. Upon DNA damage, yeast and vertebrate PCNA is modified at the conserved lysine K164 by ubiquitin, which mediates error-prone replication across lesions via translesion polymerases. We investigated the role of PCNA ubiquitination in variants of the DT40 B cell line that are mutant in K164 of PCNA or in Rad18, which is involved in PCNA ubiquitination. Remarkably, the PCNA(K164R) mutation not only renders cells sensitive to DNA-damaging agents, but also strongly reduces activation induced deaminase-dependent single-nucleotide substitutions in the immunoglobulin light-chain locus. This is the first evidence, to our knowledge, that vertebrates exploit the PCNA-ubiquitin pathway for immunoglobulin hypermutation, most likely through the recruitment of error-prone DNA polymerases.
Asunto(s)
Cadenas lambda de Inmunoglobulina/genética , Mutación , Antígeno Nuclear de Célula en Proliferación/genética , Ubiquitina/metabolismo , Animales , Secuencia de Bases , Línea Celular/efectos de los fármacos , Línea Celular/metabolismo , Línea Celular/patología , Supervivencia Celular/efectos de los fármacos , Pollos , Daño del ADN , Humanos , Cadenas lambda de Inmunoglobulina/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutágenos/farmacología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Enzimas Ubiquitina-ConjugadorasRESUMEN
Ig gene conversion is most likely initiated by activation-induced cytidine deaminase-mediated cytosine deamination. If the resulting uracils need to be further processed by uracil DNA glycosylase (UNG), UNG inactivation should block gene conversion and induce transition mutations. In this study, we report that this is indeed the phenotype in the B cell line DT40. Ig gene conversion is almost completely extinguished in the UNG-deficient mutant and large numbers of transition mutations at C/G bases accumulate within the rearranged Ig L chain gene (IgL). The mutation rate of UNG-deficient cells is about seven times higher than that of pseudo V gene-deleted (psiV-) cells in which mutations arise presumably after uracil excision. In addition, UNG-deficient cells show relatively more mutations upstream and downstream of the VJ segment. This suggests that hypermutating B cells process activation-induced cytidine deaminase-induced uracils with approximately one-seventh of uracils giving rise to mutations depending on their position.