Immunoglobulin gene conversion in chicken DT40 cells largely proceeds through an abasic site intermediate generated by excision of the uracil produced by AID-mediated deoxycytidine deamination.
Diversification of the primary antibody repertoire in chickens is achieved by a gene conversion process that uses a set of immunoglobulin variable (IgV) pseudogenes as templates. Studies usingthe chicken DT40 B lymphoma cell line have shown that this gene conversion is dependent on activation-induced deaminase, which deaminates deoxycytidine to deoxyuridine in the IgV gene. The mechanism by which the resultant deoxyuridine/deoxyguanosine (dU/dG) mismatch acts to initiate the gene conversion process is unknown but likely involves either (i) recognition of the dU/dG pair by the mismatch repair complex or (ii) recognition of the dU itself by uracil-DNA glycosylase. To discriminate these possibilities, we have investigated the effects on IgV gene conversion of inhibiting uracil-DNA glycosylase. We find that such inhibition diminishes gene conversion, biasing instead towards point mutations. These results demonstrate that IgV gene conversion in DT40 cells is substantially dependent on uracil excision and implies that it proceeds by a pathway involving an abasic site, which could be acted upon by an apyrimidinic endonuclease to generate a DNA strand break facilitating the conversion process.
Di Noia
Javier M
JM
Medical Research Council Laboratory of Molecular Biology, Cambridge, GB.
Neuberger
Michael S
MS
D000818:Animals; D000916:Antibody Diversity / Q000235:genetics / Q000276:immunology; D020137:Base Pair Mismatch / Q000276:immunology; D045744:Cell Line, Tumor; D002645:Chickens / Q000235:genetics / Q000276:immunology*; D003564:Cytidine Deaminase / Q000378:metabolism*; D045647:DNA Glycosylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D003641:Deamination; D003841:Deoxycytidine / Q000378:metabolism; D003857:Deoxyuridine / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005785:Gene Conversion / Q000276:immunology*; D005803:Genes, Immunoglobulin / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D027041:Somatic Hypermutation, Immunoglobulin / Q000276:immunology; D051981:Uracil-DNA Glycosidase; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D004791:Enzyme Inhibitors; D007135:Immunoglobulin Variable Region; D003841:Deoxycytidine; D045647:DNA Glycosylases; D051981:Uracil-DNA Glycosidase; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase; D003857:Deoxyuridine