Activation-induced cytidine deaminase (AID) can target both DNA strands when the DNA is supercoiled

Abstract

The activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class-switch recombination of Ig genes. It has been shown that in vitro, AID protein deaminates C in single-stranded DNA or the coding-strand DNA that is being transcribed but not in double-stranded DNA. However, in vivo, both DNA strands are mutated equally during SHM. We show that AID efficiently deaminates C on both DNA strands of a supercoiled plasmid, acting preferentially on SHM hotspot motifs. However, this DNA is not targeted by AID when it is relaxed after treatment with topoisomerase I, and thus, supercoiling plays a crucial role for AID targeting to this DNA. Most of the mutations are in negatively supercoiled regions, suggesting a mechanism of AID targeting in vivo. During transcription the DNA sequences upstream of the elongating RNA polymerase are negatively supercoiled, and this transient change in DNA topology may allow AID to access both DNA strands.A major advance in the study of somatic hypermutation (SHM) and class-switch recombination (CSR) has been the discovery of the activation-induced cytidine deaminase (AID) (1-3), which appears to be the long-sought SHM mutator factor and inducer of CSR (4). Current in vitro data show that AID is a DNA-specific cytidine deaminase that preferentially removes the amino group of cytidine in single-stranded DNA and in the nontranscribed strand when transcription is active (5-11). These findings are consistent with previous experiments in which SHM is linked to transcription (12, 13). However, in vivo, both DNA strands are equally mutated (14). Furthermore, in ung Ϫ/Ϫ mice, where almost all of the C or G mutations are transitions due to unrepaired AID lesions, equal targeting of both strands is confirmed (15). Single-stranded DNA would also occur in vivo as a potential AID target during DNA replication, which would explain why both DNA strands are targeted during SHM and CSR. However, experiments with a cell line that undergoes SHM in culture support the conclusion that AID can act during the G 1 phase of the cell cycle, and therefore, is not restricted to the S phase (16) (S. Gasior and U.S., unpublished data). A nonexclusive third possibility is that DNA topology (e.g., supercoiling) creates an AID-accessible conformation. In vivo, Ig genes are associated in nucleosomes with histones and other chromatin proteins. These associations, as well as the process of transcription, affect the topology of DNA. To test the possibility that supercoiled DNA as it exists in vivo may be a target for AID, we carried out cytidine deamination assays in vitro with AID purified from insect cells (5). The supercoiled target DNA was an Escherichia coli plasmid that had been manipulated to allow bacterial resistance to carbenicillin only when the initiator AUG of an ampicillin resistance (Amp r ) gene was created by AID deamination of an ACG triplet. Materials and MethodsPlasmid Construction. The kanamycin-resistance (Kan r ) gene was inserted into the SacII site in pBluescript KS(II...

Activation-induced cytidine deaminase action is strongly stimulated by mutations of the THO complex

“…Analysis of the products of SHM in B cells, however, reveals that both DNA strands are mutated (13). Other in vitro studies have shown that AID can deaminate both strands within regions of supercoiled DNA (14) and that the ssDNA-binding replication protein A is required for deamination of SHM targets (15). All of these findings are consistent with the idea that transient formation of ssDNA during transcription facilitates AID action.…”

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

“…An alternative, but not mutually exclusive, model for generating AID‐accessible ssDNA is based on the formation of denaturation bubbles generated by transcription‐associated negative supercoiling (Shen & Storb, 2004; Shen et al , 2005; Parsa et al , 2012). In regions of very active transcription, topoisomerase‐mediated resolution of the negative supercoiling generated in the wake of RNAPII does not appear to be able to keep pace resulting in overall negative supercoiling upstream of RNAPII (Kouzine et al , 2008).…”

“…In regions of very active transcription, topoisomerase‐mediated resolution of the negative supercoiling generated in the wake of RNAPII does not appear to be able to keep pace resulting in overall negative supercoiling upstream of RNAPII (Kouzine et al , 2008). Negative supercoiling creates localised denaturation bubbles (Jeon et al , 2010), which have been shown to provide an ideal substrate for AID on both strands of a plasmid, even in the absence of transcription (Shen & Storb, 2004; Shen et al , 2005). Transcription‐associated negative supercoiling has also been proposed to explain the short patches of AID and bisulphite‐accessible ssDNA observed in the Ig genes of hypermutating Ramos cells and stimulated murine primary B cells (Ronai et al , 2007; Parsa et al , 2012), which are similar to the patches of ssDNA we observe in the diversifying IGVL R of DT40 and, importantly, show are sensitive to the absence of H3.3.…”

Antisense transcripts from immunoglobulin heavy-chain locus V(D)J and switch regions

“…In the latter context, it was reported that AID can target both DNA strands in the context of transcription with Escherichia coli RNA polymerase (28). Another possibility is that differences in chromatin structure or supercoiled DNA in vivo could allow AID to access both strands (29). Finally, it is conceivable that antisense transcription through variable regions and S regions could target AID to both strands (30).…”