Comment
Somatic hypermutation (SHM) at immunoglobulin genes affects C·G (deoxycytidine·deoxyguanosine) and A·T (deoxyadenosine·deoxythymidine) pairs with approximately equal frequency in vivo. C and G residues are targeted with approximately equal frequency on both the transcribed strand (TS) and the non-transcribed strand (NTS). By contrast, A and T residues are targeted with different frequencies on the TS and the NTS. (On the NTS, A residues are targeted approximately twice as often as T residues.) That is, the targeting of A·T pairs is strand biased.
There is strong evidence to indicate that activation-induced deaminase (AID) deaminates C residues to U (deoxyuridine) residues in DNA, causing hypermutagenesis at C·G pairs (phase 1 of SHM) and the triggering of hypermutagenesis at A·T pairs (phase 2 of SHM)1,2,3,4,5,6. The proposition that error-prone, DNA-dependent short-patch repair of AID-generated U-residue lesions in DNA explains A·T-targeted hypermutagenesis7,8,9,10,11,12 seems to have mainstream support in the SHM research community at present. However, like us13,14, Neuberger and colleagues15 have recognized that this proposition is incompatible with quantitative and qualitative features that define SHM spectra (which have been established over many years using data sets that contain thousands of nucleotide substitutions). This realization stimulated Neuberger's research group to propose an alternative explanation of A·T-targeted hypermutagenesis — the dUTP (deoxyuridine triphosphate)-incorporation model — which states that dUTP is sometimes incorporated opposite A residues (instead of dTTP, deoxythymidine triphosphate) during DNA-dependent patch repair of AID-generated lesions in DNA15. Removal of a U residue from the resultant A·U pairs through the activity of a uracil-DNA glycosylase (UDG) other than UNG would generate an apyrimidinic site, subsequent synthesis opposite which would potentially lead to both transitions and transversions at A·T pairs. Critical analysis of this proposition uncovers the following three issues.
First, the dUTP-incorporation model implicitly assumes that, in phase 2 of SHM, mutation occurs only at A·T pairs. However, a comparison of the SHM spectra of mice that are deficient in UNG4 and mice that are deficient in both UNG and MSH2 (homologue 2 of Escherichia. coli MutS)6 shows that the infrequent C·G-targeted transversions that are detected in UNG-deficient mice are probably attributable to the mechanism of hypermutagenesis in phase 2 of SHM.
Second, the dUTP-incorporation model indicates that the strand bias in phase 2 of SHM might be a consequence of the strand preference of transcription-coupled repair. That is, although both DNA strands would be susceptible to dUTP incorporation opposite A residues (during patch repair of AID-generated U·G lesions) and UDG-mediated excision of U residues at resultant U·A pairs, apyrimidinic sites on the TS would presumably be preferentially repaired (leaving apyrimidinic sites mainly on the NTS). In this scheme, an apyrimidinic site on the NTS paired with an A residue on the TS would occur more frequently than an A residue on the NTS paired with an apyrimidinic site on the TS. However, bearing in mind that apyrimidinic sites opposite A residues originally contained a T residue, a dUTP-incorporation model seems to predict that, with respect to the NTS, T residues should be targeted more frequently than A residues (which is the inverse of what is observed in vivo). In fact, if phase 2 of SHM is mediated as is suggested in the dUTP-incorporation model, there would somehow need to be preferential error-free repair of the NTS, not the TS (even though AID seems to deaminate C residues to U residues in DNA on the NTS and TS with approximately equal frequency)6.
Third, to reiterate, the dUTP-incorporation model anticipates that transitions and transversions at A·T pairs are ultimately a consequence of synthesis opposite apyrimidinic sites15. Similarly, the DNA-deamination model of phase 1 of SHM predicts that C·G-targeted transversions (and a subset of C·G-targeted transitions) are the result of synthesis opposite apyrimidinic sites3. Assimilating the DNA-deamination and dUTP-incorporation models, transversions at C·G pairs should therefore accumulate in a substantially strand-biased manner during normal SHM. However, in vivo, this is not the case (as evaluated after correction for base composition of the target sequence)4.
It remains feasible that dUTP incorporation might account for a minor subset of mutations that accumulate at A·T pairs in vivo. However, for the reasons described above, we think that the dUTP-incorporation model15 (similar to the polymerase-error model of A·T-targeted hypermutagenesis through DNA-dependent short-patch repair)7,8,9,10,11,12 is incompatible with the quantitative and qualitative criteria that are demanded by the SHM spectra.
References
Milstein, C., Neuberger, M. S. & Staden, R. Both DNA strands of antibody genes are hypermutation targets. Proc. Natl Acad. Sci. USA 95, 8791–8794 (1998).
Rada, C., Ehrenstein, M. R., Neuberger, M. S. & Milstein, C. Hot spot focusing of somatic hypermutation in MSH2-deficient mice suggests two stages of mutational targeting. Immunity 9, 135–141 (1998).
Petersen-Mahrt, S. K., Harris, R. S. & Neuberger, M. S. AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature 418, 99–103 (2002).
Rada, C. et al. Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice. Curr. Biol. 12, 1748–1755 (2002).
Imai, K. et al. Human uracil-DNA glycosylase deficiency associated with profoundly impaired immunoglobulin class-switch recombination. Nature Immunol. 4, 1023–1028 (2003).
Rada, C., Di Noia, J. M. & Neuberger, M. S. Mismatch recognition and uracil excision provide complementary paths to both Ig switching and the A/T-focused phase of somatic mutation. Mol. Cell 16, 163–171 (2004).
Faili, A. et al. DNA Polymerase η is Involved in hypermutation occurring during immunoglobulin class switch recombination. J. Exp. Med. 199, 265–270 (2004).
Zeng, X. et al. DNA polymerase η is an A–T mutator in somatic hypermutation of immunoglobulin variable genes. Nature Immunol. 2, 537–541 (2001).
Pavlov, Y. I. et al. Correlation of somatic hypermutation specificity and A–T base pair substitution errors by DNA polymerase h during copying of a mouse immunoglobulin κ light chain transgene. Proc. Natl Acad. Sci. USA 99, 9954–9959 (2002).
Rogozin, I. B., Pavlov, Y. I., Bebenek, K., Matsuda, T. & Kunkel, T. A. Somatic mutation hotspots correlate with DNA polymerase η error spectrum. Nature Immunol. 2, 530–536 (2001).
Martomo, S. A. et al. Different mutation signatures in DNA polymerase η- and MSH6-deficient mice suggest separate roles in antibody diversification. Proc. Natl Acad. Sci. USA 102, 8656–8661 (2005).
Mayorov, V. I., Rogozin, I. B., Adkison, L. R. & Gearhart, P. J. DNA polymerase η contributes to strand bias of mutations of A versus T in immunoglobulin genes. J. Immunol. 174, 7781–7786 (2005).
Franklin, A. & Blanden, R. V. On the molecular mechanism of somatic hypermutation of rearranged immunoglobulin genes. Immunol. Cell Biol. 82, 557–567 (2004).
Steele, E. J., Franklin, A. & Blanden, R. V. Genesis of the strand biased signature in somatic hypermutation of rearranged immunoglobulin variable genes. Immunol. Cell Biol. 82, 209–218 (2004).
Neuberger, M. S. et al. Somatic hypermutation at AoT pairs: polymerase error versus dUTP incorporation. Nature Rev. Immunol. 5, 171–178 (2005).
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Franklin, A., Blanden, R. Somatic hypermutation at A·T pairs: critical analysis of the dUTP-incorporation model. Nat Rev Immunol 5, 180 (2005). https://doi.org/10.1038/nri1553-c1
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DOI: https://doi.org/10.1038/nri1553-c1
