Mesoscale DNA feature in antibody-coding sequence facilitates somatic hypermutation

A high-throughput protocol for deamination of long single-stranded DNA and oligo pools containing complex sequences

“…Therefore, we first verified that the in vitro deamination profile of AID on a single long ssDNA substrate strongly correlates with the intrinsic SHM profile in vivo . 1 The expansion of substrates into oligo pools containing complex sequences allows the high-throughput assay of AID-initiated mutations in numerous antibody sequences or selected regions on genomic DNA. This assay allows rapid and sensitive evaluation of antibody sequence mutation profiles in vitro , which is useful for antibody sequence optimization, antibody sequence evolution studies, and validation of potential AID-initiated mutations in cancer genomes.…”

Dancing with DNA: AID embraces flexible partners

“…9 Notably, insertion of a synthetic mesoscale Py-Py dinucleotide-rich motif adjacent to one of several poorly targeted WRC motifs was sufficient to strongly increase mutation of the motif. 8 The findings of Wang et al solve an important mystery in the field of SHM and have broad implications for the evolution of IgV sequences, the off-target sites of action of AID, and our ability to engineer sequences with desired mutational profiles, for example, to create broadly neutralizing antibodies for treatment or prevention of viral infections, as noted by the authors. 8 In addition, their results have the intriguing mechanistic implication that AID's in vivo DNA substrate is a long (> 20 nt) stretch of ssDNA, which is notable given that the ssDNA region in the elongating Pol2 complex has been reported in many studies to be only 10-20 nt long.…”

“…8 The findings of Wang et al solve an important mystery in the field of SHM and have broad implications for the evolution of IgV sequences, the off-target sites of action of AID, and our ability to engineer sequences with desired mutational profiles, for example, to create broadly neutralizing antibodies for treatment or prevention of viral infections, as noted by the authors. 8 In addition, their results have the intriguing mechanistic implication that AID's in vivo DNA substrate is a long (> 20 nt) stretch of ssDNA, which is notable given that the ssDNA region in the elongating Pol2 complex has been reported in many studies to be only 10-20 nt long. 10,11 Future studies will be needed to validate and extend the model of Wang et al, particularly structural analyses of ssDNA-AID complexes and further studies of how mesoscale motifs affect AID targeting and SHM in the context of germinal center B cells and B cell lymphoma.…”

“…These findings led Wang et al to hypothesize that other local sequences or higherorder structures, operating on a mesoscale (5-50 bp), also play a role in influencing the targeting of AID and SHM. 8 In a recent paper published in Cell, Wang et al report a significant advance in this area by demonstrating that flexible pyrimidine-pyrimidine dinucleotides surrounding AID deamination hotspot motifs facilitate binding to and deamination by AID, helping to explain preferential SHM in CDRs. 8 Taking advantage of a large database of IgV sequences derived from out-of-frame Ig heavy chain gene rearrangements, Wang et al established that CDRs have a higher density of WRC motifs than FRs and, importantly, that cytidines in WRC motifs mutate at a higher frequency in CDRs than FRs (Fig.…”

“…Through systematic in vivo and in vitro sequence mapping, Wang et al provide evidence that the key feature is the intrinsic flexibility of the ~12 nucleotides upstream and ~6 nucleotides downstream of the WRC motif. 8 High flexibility, conferred by enrichment for pyrimidine-pyrimidine (Py-Py) dinucleotides (which have a low base stacking free energy), results in high mutability, whereas low flexibility, conferred by a high proportion of Purine-Purine (Pu-Pu) pairs (with high base stacking free energies), yields low mutability. The dinucleotide composition of the six nucleotides immediately 5′ of the WRC motif appears to be particularly important in this regard.…”