Abstract
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.
Highlights
AID1 is required for the secondary Ig gene diversification processes, somatic hypermutation (SHM) and class-switch recognition (CSR) (1)
A mutational spectrum generated during transcription of closed circular double-stranded DNA indicates that wild type activation-induced cytidine deaminase (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
Spectral and Clonal Analysis of Wild Type AID-catalyzed C Deamination Specificity on ssDNA—The WRC hot spot and SYC cold spot distinctions are not absolute because some WRC sites are more reactive than others, even those having identical WR bases preceding a target C residue, and non-hot spot C residues are occasionally deaminated in preference to WRC sites (14)
Summary
Construction and Purification of AID Mutants and DNA Substrates— Mutant AID proteins (R35E, R24E, R35E/R36D, and a 10-amino acid C-terminal deletion) were constructed by site-directed mutagenesis (QuikChange site-directed mutagenesis kit, Stratagene) using the pAcG2T-AID vector (13, 14) as the template. Mutation Analysis of AID-targeted C Deamination in Vitro—Deamination specificities of wt and mutant AID were measured under the following reaction conditions: 30-l volume, 50 mM HEPES (pH 7.5), 1 mM dithiothreitol, 10 mM MgCl2, 500 ng of gapped DNA (ϭ100 fmol), 0.2 g of RNase A, and 25–200 ng of wt or mutant AID (ϭ0.5– 4 pmol). Transcription-dependent AID deamination specificity was measured using a covalently closed circular dsDNA M13mp2T7 substrate, undergoing active transcription by T7 RNA polymerase. In a typical 30-l reaction, RNase-activated GST-AID attached to 25 l of glutathione-Sepharose beads (13), 50 ng of dsDNA M13mp2T7 substrate, and 1 l of T7 RNA polymerase in a reaction buffer containing 50 mM HEPES (pH 7.5), 1 mM dithiothreitol, 10 mM MgCl2 containing 250 M rNTPs was incubated at 37 °C for 30 min.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
