Targeting of the Activation-Induced Cytosine Deaminase Is Strongly Influenced by the Sequence and Structure of the Targeted DNA

Abstract

Activation-induced deaminase (AID) initiates immunoglobulin somatic hypermutation (SHM). Since in vitroAID was shown to deaminate cytosines on single-stranded DNA or the nontranscribed strand, it remained a puzzle how in vivo AID targets both DNA strands equally. Here we investigate the roles of transcription and DNA sequence in cytosine deamination. Strikingly different results are found with different substrates. Depending on the target sequence, the transcribed DNA strand is targeted as well as or better than the nontranscribed strand. The preferential targeting is not related to the frequency of AID hot spots. Comparison of cytosine deamination by AID and bisulfite shows different targeting patterns suggesting that AID may locally unwind the DNA. We conclude that somatic hypermutation on both DNA strands is the natural outcome of AID action on a transcribed gene; furthermore, the DNA sequence or structure and topology play major roles in targeting AID in vitro and in vivo. On the other hand, the lack of mutations in the first ϳ100 nucleotides and beyond about 1 to 2 kb from the promoter of immunoglobulin genes during SHM must be due to special conditions of transcription and chromatin in vivo.The variable regions of immunoglobulin (Ig) genes encode the antigen binding sites of antibodies for estimated billions of different antigenic determinants. Thousands to millions of different antibody binding sites are created when the hundred or so variable, diversity, and joining genes for Ig heavy and light chains are recombined and diversified by nucleotide deletions and insertions at the V(D)J joints in developing B lymphocytes. In mature B lymphocytes, the rearranged V(D)J sequences are extensively further diversified by somatic hypermutation (SHM) during exposure to antigens that react with the specific Igs comprising the B-cell receptors.

Somatic Hypermutation and Class Switch Recombination in Msh6−/−Ung−/− Double-Knockout Mice

“…It is thus very unlikely that AID targets these regions. This is strikingly different from the result of the cell-free study that showed that AID could access cytidines immediately downstream of the promoter in naked DNAs (44). Therefore, the interesting questions that remain are whether in vivo AID may be associated with transcription complexes only in the ϳϩ100 nt to ϳϩ2 kb region of Ig genes, whether it can only access this region even without association with the transcription complex, or whether it is present in complexes with DNA in the 5Ј and 3Ј regions but cannot act.…”

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.…”

Interference of mismatch and base excision repair during the processing of adjacent U/G mispairs may play a key role in somatic hypermutation

“…However, available evidence suggests that AID catalyses several deaminations per molecule (17)(18)(19), which might give rise to several U/G mispairs in close proximity. We argued that in such a scenario, the picture might change.…”