Evolution of coordinated mutagenesis and somatic hypermutation in VH5

Abstract

The VH5 human antibody gene was analyzed using a computer program (mfg) which simulates transcription, to better understand transcription-driven mutagenesis events that occur during “phase 1” of somatic hypermutation. Results show that the great majority of mutations in the non-transcribed strand occur within loops of two predicted high-stability stem-loop structures, termed SLS 14.9 and SLS 13.9. In fact, 89% of the 2,505 mutations reported are within the encoded complementarity-determining region (CDR) and occur in loops of these high-stability structures. In vitro studies were also done and verified the existence of SLS 14.9. Following the formation of SLS 14.9 and SLS 13.9, a sustained period of transcriptional activity occurs within a window size of 60-70 nucleotides. During this period, the stability of these two SLSs does not change, and may provide the substrate for base exchanges and mutagenesis. The data suggest that many mutable bases are exposed simultaneously at pause sites, allowing for coordinated mutagenesis.

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

“…Briefly, S-IB defines the mechanism where the folding process in ssDNA pauses when encountering a paired base in the stable stem and backtracks to re-initiate the most stable fold at an unpaired base (Wright et al 2008a, b). Recently, Wright et al provided in vitro evidence for the predicted pause sites (Wright et al 2011) and the pauses are consistent with those predicted by backtracking analysis (Wright et al 2008a).…”

Kinetic models reveal the in vivo mechanisms of mutagenesis in microbes and man

“…2B and C) of three different-sized segments of VH 5 in the non-transcribed strand have verified the existence of the two major mfg -predicted 65 nt secondary structures seen in Fig. 4 (SLS 14.9) and (SLS 13.9) [19]. The present model of SHM accounts for all seven mutable sites in ssDNA loops in the variable region, and 89% of the 2,505 total mutations identified are accounted for in unpaired bases of SLSs 14.9 and 13.9.…”

“…The SLSs are from the non-transcribed strand, while experimental cleavage and pausing data were derived from the transcribed strand. Comparable analysis demonstrating the existence of predicted SLSs in p53 have been demonstrated for hypermutable codons 245, 248, 273, and 282 [10], and in somatic hypermutation [19] pausing analysis demonstrated the existence of three different sized segments of VH 5.…”

“…The role of activation-induced (cytidine) deaminase (AID) has not yet been entirely clarified, and we suggest that its activation may in part serve to redirect the fate of mutable Cs from T to U (Fig. 5G), leading to high frequency enzyme diversification compensated for by lower rates of intrinsic C-to-T mutations [19]. …”

“…The predictive value of our mfg SHM model [17–19] has also been demonstrated in several ways by: (a) the location of high mutation frequency bases in mfg -predicted highly exposed unpaired bases, (b) the fact that unpaired bases in SLS 14.9 are identical to those identified independently in ssDNA patches during transcription [41], (c) the fact that the great majority of mutations in the non-transcribed strand occur within loops of predicted high-stability SLSs, (d) the observation that 89% of the 2,505 mutations reported in SLS sites 1–7 of these regions are predicted by mfg to occur in identified loops of secondary structures in the encoded CDR, and (e) in vitro analyses that have verified the existence of SLS 14.9.…”