Negative Supercoiling Creates Single-Stranded Patches of DNA That Are Substrates for AID–Mediated Mutagenesis

Abstract

Antibody diversification necessitates targeted mutation of regions within the immunoglobulin locus by activation-induced cytidine deaminase (AID). While AID is known to act on single-stranded DNA (ssDNA), the source, structure, and distribution of these substrates in vivo remain unclear. Using the technique of in situ bisulfite treatment, we characterized these substrates—which we found to be unique to actively transcribed genes—as short ssDNA regions, that are equally distributed on both DNA strands. We found that the frequencies of these ssDNA patches act as accurate predictors of AID activity at reporter genes in hypermutating and class switching B cells as well as in Escherichia coli. Importantly, these ssDNA patches rely on transcription, and we report that transcription-induced negative supercoiling enhances both ssDNA tract formation and AID mutagenesis. In addition, RNaseH1 expression does not impact the formation of these ssDNA tracts indicating that these structures are distinct from R-loops. These data emphasize the notion that these transcription-generated ssDNA tracts are one of many in vivo substrates for AID.

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

“…DRIP analysis demonstrated that the expression of RNase HI reduced R‐loop formation in IGVL to near background levels (Fig 4C). However, it did not affect Ig diversification (Fig 4D), consistent with the observations of Martin and colleagues in hypermutating human B cells (Parsa et al , 2012). Thus, while H3.3 promotes the formation of R‐loops in IGVL, R‐loops are not necessary intermediates in AID‐dependent Ig diversification.…”

“…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.…”

“…Bisulphite deaminates dC present in single‐stranded DNA at the time of treatment, which can be detected by subsequent sequencing, the resulting dU being read as dT. Using this approach, short patches of single‐stranded DNA (ssDNA) have previously been reported on both strands of the Ig V genes in a hypermutating human B‐cell line and primary murine B cells (Ronai et al , 2007; Parsa et al , 2012). Applying this technique to DT40 cells also revealed tracts of ssDNA throughout IGVL R on both strands (Fig 5A).…”

“…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).…”

AID targeting: old mysteries and new challenges

“…These DNA lesions are then converted into point mutations during SHM and into DNA double-stranded breaks (DSBs) during CSR or aberrant chromosomal translocations. AID shows selectivity and deaminates single-stranded DNA (ssDNA) or supercoiled DNA that creates single-stranded patches of DNA [9, 10], but does not deaminate double-stranded DNA (dsDNA) or DNA:RNA hybrids [11, 12]. During SHM, point mutations and sometimes insertions and deletions, are introduced at a very high rate into the recombined variable region exons encoding IgH and IgL, which in some instances will generate B cells with increased antigen affinity.…”