Biochemical analysis of hypermutational targeting by wild type and mutant activation-induced cytidine deaminase.
The synthesis of high affinity antibodies requires activation-induced cytidine deaminase (AID) to initiate somatic hypermutation and class-switch recombination. Here we investigate AID-catalyzed deamination of C --> U on single-stranded DNA and on actively transcribed closed circular double-stranded DNA. Mutations are initially favored at canonical WRC (W = A or T, R = A or G) somatic hypermutation hot spot motifs, but over time mutations at neighboring non-hot spot sites increase creating random clusters of mutated regions in a seemingly processive manner. N-terminal AID mutants R35E and R35E/R36D appear less processive and have altered mutational specificity compared with wild type AID. In contrast, a C-terminal deletion mutant defective in CSR in vivo closely resembles wild type AID. A mutational spectrum generated during transcription of closed circular double-stranded DNA indicates that wild type AID retains its specificity for WRC hot spot motifs within the confines of a moving transcription bubble while introducing clusters of multiple deaminations predominantly on the nontranscribed strand.
Bransteitter
Ronda
R
Department of Biological Sciences, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, California 90089-1340, USA.
Pham
Phuong
P
Calabrese
Peter
P
Goodman
Myron F
MF
D020816:Amino Acid Motifs; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D003564:Cytidine Deaminase; D043525:Cytosine Deaminase / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004252:DNA Mutational Analysis; D004277:DNA, Single-Stranded / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007313:Insecta; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D014158:Transcription, Genetic; D014764:Viral Proteins; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D004277:DNA, Single-Stranded; D011994:Recombinant Proteins; D014764:Viral Proteins; D004247:DNA; D005982:Glutathione Transferase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D043525:Cytosine Deaminase; D003564:Cytidine Deaminase