Stabilised DNA secondary structures with increasing transcription localise hypermutable bases for somatic hypermutation in IGHV3-23

Abstract

Somatic hypermutation (SHM) mediated by activation-induced cytidine deaminase (AID) is a transcription-coupled mechanism most responsible for generating high affinity antibodies. An issue remaining enigmatic in SHM is how AID is preferentially targeted during transcription to hypermutable bases in its substrates (WRC motifs) on both DNA strands. AID targets only single stranded DNA. By modelling the dynamical behaviour of IGHV3-23 DNA, a commonly used human variable gene segment, we observed that hypermutable bases on the non-transcribed strand are paired whereas those on transcribed strand are mostly unpaired. Hypermutable bases (both paired and unpaired) are made accessible to AID in stabilised secondary structures formed with increasing transcription levels. This observation provides a rationale for the hypermutable bases on both the strands of DNA being targeted to a similar extent despite having differences in unpairedness. We propose that increasing transcription and RNAP II stalling resulting in the formation and stabilisation of stem-loop structures with AID hotspots in negatively supercoiled region can localise the hypermutable bases of both strands of DNA, to AID-mediated SHM.

Gene Conversion-Like Events in the Diversification of Human Rearranged IGHV3-23*01 Gene Sequences

“…Our previous studies (Duvvuri et al, 2011, 2012) revealed SHM patterns suggestive of defects in DNA repair mechanisms in some CVID patients. Given the role of DNA repair processes in GCV mechanisms, we compared the occurrence of GCV-like events between two cohorts, CVID pts and healthy individuals.…”

“…In our previous studies (Duvvuri et al, 2011, 2012) we provided evidence for defective DNA repair mechanisms in a subgroup of CVID patients. These patients had functional AID as evidenced by the presence of SHM and by a switch in immunoglobulin class of their immunoglobulin genes.…”

“…Detailed analysis of IGHV3-23 * 01 sequence data from CVID patients and healthy individuals is in Duvvuri et al (2011, 2012). …”

Activation‐induced cytidine deaminase can target multiple topologies of double‐stranded DNA in a transcription‐independent manner

“…84 Another body of evidence indicates that the choice of AID toward either strand is mainly dependent on the sequence and/or structure of the target gene. 82,105,113 Examining the distribution of substrates with C → T and G → A mutations confirmed that AID can indeed mutate both DNA strands during the process of transcription. There was an approximate 70%/30% distribution of substrates with C → T and G → A mutations in both the fast and medium transcription rates, and approximately 80%/20% in the slow transcription rate, again showing a preference for AID targeting the top (nontranscribed) strand.…”

“…It is thought that the purpose of transcription in AID targeting is to directly generate ssDNA by the traversing RNAP or lead to changes in supercoiling of duplex DNA in the wake of the polymerase, which encourages transient ssDNA in the form of DNA secondary structures. 41,77,82,83,105 Therefore, we sought to compare AID activity in the presence of transcription, to its transcription-independent activity levels on duplex DNA. To gain context for the efficacy of transcription-independent targeting of duplex DNA by AID, we aimed to attain the highest possible level of AID targeting in the presence of transcription in our system, and then, compare this level to that in the absence of transcription.…”