Correlation of somatic hypermutation specificity and A-T base pair substitution errors by DNA polymerase η during copying of a mouse immunoglobulin κ light chain transgene

Abstract

To test the hypothesis that inaccurate DNA synthesis by mammalian DNA polymerase (pol ) contributes to somatic hypermutation (SHM) of Ig genes, we measured the error specificity of mouse pol during synthesis of each strand of a mouse Ig light chain transgene. We then compared the results to the base substitution specificity of SHM of this same gene in the mouse. The in vitro and in vivo base substitution spectra shared a number of common features. A highly significant correlation was observed for overall substitutions at A-T pairs but not for substitutions at G-C pairs. Sixteen mutational hotspots at A-T pairs observed in vivo were also found in spectra generated by mouse pol in vitro. The correlation was strongest for errors made by pol during synthesis of the non-transcribed strand, but it was also observed for synthesis of the transcribed strand. These facts, and the distribution of substitutions generated in vivo, support the hypothesis that pol contributes to SHM of Ig genes at A-T pairs via short patches of low fidelity DNA synthesis of both strands, but with a preference for the non-transcribed strand.H igh affinity antibodies result from somatic hypermutation (SHM) of Ig genes followed by selection. The SHM process introduces base substitutions at a very high rate into DNA encoding the variable regions of immunoglobulins (1-4). Although the mechanism for introducing these sequence changes is currently unknown, several features of SHM specificity offer clues to the DNA transactions that might be involved. For example, SHM primarily occurs in two highly mutable DNA sequence motifs (5-9). One is the RGYW sequence (the underlined G is mutated, R ϭ A or G, Y ϭ T or C, and W ϭ A or T), which is found in SHM substitution spectra in equal proportions in both DNA strands. The other is the WA motif, where substitutions are more likely in one strand than the other (5, 7-10). A clue to the origins of the substitutions in the WA motif is the observation that their type and location correlates with the base substitution error specificity of human DNA polymerase when copying a bacterial gene sequence in a model system in vitro (8). Based on that correlation and additional considerations of the two mutable motifs (11), we suggested that errors at A-T base pairs by pol may contribute to as much as one third of somatic mutations in Ig genes, preferentially during synthesis of the non-transcribed strand. This hypothesis is supported by the observation that XP-V patients lacking active polymerase have a lower proportion of somatic substitutions at A-T base pairs in Ig genes (12). Because pol error specificity does not correlate with substitutions in the RGYW sequence motif, and because those substitutions are distributed equally on both strands, we further suggested that SHM may involve more than one DNA transaction and more than one DNA polymerase (8). This finding is consistent with the two-phase model of SHM proposed earlier (9, 13). Other DNA polymerases suggested to participate in SHM include pol (14-16), pol (17, 18) ...

Mechanism of somatic hypermutation at the WA motif by human DNA polymerase η

“…The base-substitution spectrum of SHM from XP-V patients is altered with severely decreased mutations at A:T pairs (11,17,18). Similar suppression of A:T to G:C mutations was observed in the POLH-deficient mice (19)(20)(21). Interestingly, the MSH2-MSH6 heterodimer interacts and stimulates Pol η activity in vitro (22), and mutations of A:T base pairs were abolished in SHM when both POLH and MSH2 genes were knocked out (23).…”

DNA polymerases in adaptive immunity

“…Whereas such a strand-specific repair would be compatible with the equalization of mutations between G and C bases, and the A over T bias observed in V gene mutations (Fig.4), the proposition deserves additional studies, as the mutations reported do not fit with the pattern expected from Polη-mediated repair 60 .…”

“…Polη is not a processive enzyme and therefore favours the synthesis of a few nucleotides (short-patch DNA synthesis). Mutagenicity of Polη was estimated using a Vκ template sequence when synthesizing either the transcribed or the non-transcribed strand in vitro 60 . Interestingly, despite the fact that this assay monitored the error rate of the polymerase during the synthesis of a few hundred bases that may not occur in vivo, a strong correlation with the in vivo pattern of mutations at A/T base pairs was observed, in particular during synthesis of the coding strand (the non-transcribed strand), although hotspots on both coding and non-coding strands were clearly observed.…”

Polymerase μ is up‐regulated during the T cell‐dependent immune response and its deficiency alters developmental dynamics of spleen centroblasts

“…Notably, we detected the opposite effect when we expressed Poll in a hypermutating cell line, with larger numbers of heavily mutated clones compared to control cells [15]. Polg deletion results in fewer mutations as well as targeting alteration [40], whereas Poll does not appear to affect targeting, nonetheless argues against this hypothesis.…”