Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase

Abstract

The expression of activation-induced cytidine deaminase (AID) is prerequisite to a ''trifecta'' of key molecular events in B cells: class-switch recombination and somatic hypermutation in humans and mice and gene conversion in chickens. Although this critically important enzyme shares common sequence motifs with apolipoprotein B mRNA-editing enzyme, and exhibits deaminase activity on free deoxycytidine in solution, it has not been shown to act on either RNA or DNA. Recent mutagenesis data in Escherichia coli suggest that AID may deaminate dC on DNA, but its putative biochemical activities on either DNA or RNA remained a mystery. Here, we show that AID catalyzes deamination of dC residues on single-stranded DNA in vitro but not on double-stranded DNA, RNA-DNA hybrids, or RNA. Remarkably, it has no measurable deaminase activity on single-stranded DNA unless pretreated with RNase to remove inhibitory RNA bound to AID. AID catalyzes dC 3 dU deamination activity most avidly on double-stranded DNA substrates containing a small ''transcription-like'' single-stranded DNA bubble, suggesting a targeting mechanism for this enigmatic enzyme during somatic hypermutation.T he breakthrough discovery of activation-induced cytidine (CR) deaminase (AID) (1, 2) has opened a way to identify the biochemical reactions responsible for generating highaffinity antibodies. Although AID is normally expressed in germinal center B cells, its forced expression in B cells at the wrong stage of differentiation (3), in non-B cells (4, 5), and in Escherichia coli (6, 7) results in enhanced mutagenesis and mutational spectra mimicking somatic hypermutation (SHM). This suggests that AID can operate effectively out of its natural milieu and does not require either B cell-specific V-gene targeting elements or other B cell-specific proteins (8). Based on its similarity to apolipoprotein B mRNA-editing enzyme, which catalyzes the deamination of C on mRNA (9), it has been suggested that conversion of dC 3 dU on DNA, followed by the generation of an abasic site, might account for at least a subset of mutations targeted to SHM hot-spot sequences (6,8,10). Recent data showing that AID-and apolipoprotein B mRNAediting enzyme-generated hypermutation is enhanced in uracil-N-glycosylase-deficient strains of E. coli imply that both AID and apolipoprotein B mRNA-editing enzyme can act on DNA (7).Because previous studies have failed to detect AID activity on either DNA or RNA, the biologically relevant substrate for AID remains an important open question. In this article we identify and characterize AID activity on nucleic acid substrates and provide a surprising explanation for why its activity has been elusive. Materials and MethodsMaterials. Ultrapure dNTP, 2Ј,3Ј-dideoxynucleoside triphosphate, and T4 polynucleotide kinase were purchased from Amersham Pharmacia; RNaseA, CR, deoxycytidine (CdR), uridine, and deoxyuridine were from Sigma; T7 sequenase (version 2.0) and RNase inhibitor were from United States Biochemical; and uracil DNA glycosylase (UDG) and apu...

Histone H3.3 promotes IgV gene diversification by enhancing formation of AID ‐accessible single‐stranded DNA

“…R‐loops levels thus provide an indirect indication of the single strandedness of a locus. It has also been proposed that R‐loops may facilitate Ig diversification: although AID is unable to access DNA when in a DNA–RNA hybrid (Bransteitter et al , 2003), R‐loops may help to stabilise ssDNA allowing AID access to the opposite strand (Gómez‐González & Aguilera, 2007). To assess the formation of R‐loops in IGVL R , we employed DNA–RNA hybrid immunoprecipitation (DRIP) with the S9.6 antibody (Boguslawski et al , 1986).…”

“…Activation‐induced deaminase acts only on single‐stranded DNA (ssDNA) (Bransteitter et al , 2003; Dickerson et al , 2003), and therefore, in vivo , IgV deamination requires the exposure of this substrate. Multiple lines of evidence have linked the generation of ssDNA and the recruitment of AID to the level of transcription (Peters & Storb, 1996; Fukita et al , 1998; Ramiro et al , 2003).…”

Human Lymphoid Translocation Fragile Zones Are Hypomethylated and Have Accessible Chromatin

“…We have established that AID is responsible for initiating most DNA lesions that lead to human B cell translocations (3,5,6,8,21,22). In order to deaminate C to U or methyl-C to T in these translocation fragile zones, these zones must be accessible to AID (26,27). The boundary match between fragile zones and hypomethylation zones provides possible support for such a hypothesis.…”

“…The total number of molecules in each condition is less than 32 due to exclusion of molecules that contain no accessible chromatin feature both inside and outside fragile zones. requires single-stranded DNA substrates (26), the DNA in the fragile zone must at least transiently become single stranded in a manner as yet unknown. (Single-strandedness might conceivably be triggered by the passage of a DNA polymerase through the region, or by some other local cause, and this is a point of future study.)…”

Forced expression of AID facilitates the isolation of class switch variants from hybridoma cells

“…These changes in isotype can be very important, since various isotypes are distributed differently in the body, have different half-lives in the circulation and carry out distinct subsets of effector functions (Stavnezer, 2000). Both SHM and CSR require activation induced cytidine deaminase (AID), which converts deoxycytidines in the V and switch regions to uracil and initiates both processes (Muramatsu et al, 1999;Di Noia et al, 2002;Manis et al, 2002;Bransteitter et al, 2003). Patients that are genetically deficient in AID make only low affinity IgM antibodies and die of infections if they are not treated with hyperimmune immunoglobulins (Quartier et al, 2004).…”