RESUMEN
Activation-induced deaminase (AID) initiates diversity of immunoglobulin genes through deamination of cytosine to uracil. Two opposing models have been proposed for the deamination of DNA or RNA by AID. Although most data support DNA deamination, there is no physical evidence of uracil residues in immunoglobulin genes. Here we demonstrate their presence by determining the sensitivity of DNA to digestion with uracil DNA glycosylase (UNG) and abasic endonuclease. Using several methods of detection, we identified uracil residues in the variable and switch regions. Uracil residues were generated within 24 h of B cell stimulation, were present on both DNA strands and were found to replace mainly cytosine bases. Our data provide direct evidence for the model that AID functions by deaminating cytosine residues in DNA.
Asunto(s)
Linfocitos B/metabolismo , Citidina Desaminasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Uracil-ADN Glicosidasa/metabolismo , Animales , Variación Antigénica/genética , Linfocitos B/inmunología , Linfocitos B/patología , Células Cultivadas , Citidina Desaminasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Cambio de Clase de Inmunoglobulina , Región Variable de Inmunoglobulina , Interleucina-4/inmunología , Interleucina-4/metabolismo , Lipopolisacáridos/inmunología , Lipopolisacáridos/metabolismo , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Químicos , Bazo/patología , Uracilo/análisis , Uracil-ADN Glicosidasa/genéticaRESUMEN
The molecular features that allow activation-induced cytidine deaminase (AID) to target Ig and certain non-Ig genes are not understood, although transcription has been implicated as one important parameter. We explored this issue by testing the mutability of a non-Ig transcription cassette in Ig and non-Ig loci of the chicken B cell line DT40. The cassette did not act as a stable long term mutation target but was able to be mutated in an AID-dependent manner for a limited time post-integration. This indicates that newly integrated DNA has molecular characteristics that render it susceptible to modification by AID, with implications for how targeting and mis-targeting of AID occurs.