The cytidine deaminases AID and APOBEC-1 exhibit distinct functional properties in a novel yeast selectable system.
Activation-induced cytidine deaminase (AID) is indispensable for immunoglobulin maturation by somatic hypermutations and class switch recombination and is supposed to deaminate cytidines in DNA, while its homolog APOBEC-1 edits apolipoprotein (apo) B mRNA by cytidine deamination. We studied the editing activity of APOBEC-1 and AID in yeast using the selectable marker Gal4 linked to its specific inhibitor protein Gal80 via an apo B cassette (Gal4-C) or via the variable region of a mouse immunoglobulin heavy chain gene (Gal4-VH). Expression of APOBEC-1 induced C to U editing in up to 15% of the Gal4-C transcripts, while AID was inactive in this reaction even in the presence of the APOBEC-1 complementation factor. After expression of APOBEC-1 as well as AID approximately 10(-3) of yeast cells survived low stringency selection and expressed beta-galactosidase. Neither AID nor APOBEC-1 mutated the VH sequence of Gal4-VH, and consequently the yeast colonies did not escape high stringent selection. AID, however, induced frequent plasmid recombinations that were only rarely observed with APOBEC-1. In conclusion, AID cannot substitute APOBEC-1 to edit the apo B mRNA, and the expression of AID in yeast is not sufficient for the generation of point mutations in a highly transcribed Gal4-VH sequence. Cofactors for AID induced somatic hypermutations of immunoglobulin variable regions, that are present in B cells and a variety of non-B cells, appear to be missing in yeast. In contrast to APOBEC-1, AID alone does not exhibit an intrinsic specificity for its target sequences.
Krause
Kristina
K
Department of Clinical Research, University of Berne, Berne, Switzerland.
Marcu
Kenneth B
KB
Greeve
Jobst
J
D000071479:APOBEC-1 Deaminase; D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics; D003562:Cytidine / Q000378:metabolism; D003564:Cytidine Deaminase / Q000235:genetics / Q000502:physiology*; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins; D005786:Gene Expression Regulation; D005803:Genes, Immunoglobulin; D017930:Genes, Reporter; D005813:Genes, Synthetic; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D016296:Mutagenesis; D017354:Point Mutation; D017393:RNA Editing / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D027041:Somatic Hypermutation, Immunoglobulin / Q000235:genetics / Q000502:physiology*; D013379:Substrate Specificity; D014157:Transcription Factors / Q000235:genetics; D014498:Uracil / Q000378:metabolism; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D001055:Apolipoproteins B; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; C055002:GAL80 protein, S cerevisiae; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014498:Uracil; D003562:Cytidine; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D000071479:APOBEC-1 Deaminase; C000605090:Apobec1 protein, mouse; C000605091:Apobec1 protein, rat; D003564:Cytidine Deaminase