The mutation spectrum of purified AID is similar to the mutability index in Ramos cells and in ung(-/-)msh2(-/-) mice.
Somatic hypermutation and class switch recombination are initiated by the enzyme activation-induced cytidine deaminase (AID). Although other models exist for AID function, one model suggests that AID initiates these processes by deaminating cytidines within DNA, thereby initiating mutagenic repair pathways that involve either UNG or Msh2. Recent work shows that GST-hAID prefers to mutate WRC motifs, a motif frequently mutated in vivo. Because this is a strong argument in favor of the DNA deamination model, we sought to extend this analysis by examining the activity of purified AID with a small polyhistidine tag (His-hAID) on all 16 trinucleotide combinations (i.e., NNC). Here we show that purified His-hAID preferentially mutated cytidines within WRC (i.e., A/T, A/G, C) motifs, but poorly mutated cytidines within GYC (G, C/T, C) motifs. We next compared this mutability preference with those in hypermutating Ramos cells and in msh2(-/-)ung(-/-) mice, since both are reduced or deficient in UNG- and/or Msh2-induced mutations and are thus likely to reflect the sequence specificity of the mutator in vivo. Indeed, the mutation spectrums of purified His-hAID and GST-hAID matched the trinucleotide mutability indexes in Ramos cells and in msh2(-/-)ung(-/-) mice. Thus, the activity of AID on single-stranded DNA produces the same mutation pattern as double-stranded DNA in hypermutating cells. These data lend support to the DNA deamination model and indicate that AID does not require co-factors for its WRC specificity.
Larijani
Mani
M
Department of Immunology, Medical Sciences Bldg. 5265, University of Toronto, Toronto, M5S 1A8, Canada. alberto.martin@utoronto.ca
Frieder
Darina
D
Basit
Wajiha
W
Martin
Alberto
A
D000818:Animals; D001483:Base Sequence; D003564:Cytidine Deaminase / Q000235:genetics*; D045647:DNA Glycosylases / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D051718:MutS Homolog 2 Protein; D009154:Mutation; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; C492570:CCNO protein, human; D045647:DNA Glycosylases; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase; C497173:Msh2 protein, mouse; D051718:MutS Homolog 2 Protein