Based on a correlation between strand-specific hotspots for somatic mutations at WA sequence motifs and the error specificity of mammalian DNA pol η, we previously suggested that errors by pol η primarily during synthesis of the nontranscribed strand of Ig genes may contribute to somatic hypermutation (SHM) at A-T base pairs1. Dörner and Lipsky now conclude that any possible role for pol η in SHM is likely to be minor. They base this conclusion on an analysis implying that most mutations at WA motifs (mutable position underlined) also encompass the well known RGYW motif for SHM2. They suggest that the major target for SHM activity is actually the RGYW motif and that the DNA strand bias for mutations at WA reflects the asymmetry of the RGYW/WRCY motif.
The mutational analysis by Dörner and Lipsky differs in several significant ways from our study, which focused primarily on mutational hotspots representing the context specificity of hypermutation. This makes a direct comparison of the two studies difficult. Nonetheless, that the WA motif may also be a part of RGYW motifs is clearly correct. Indeed, such "fused" motifs (such as WAGYW, TACY or RGTW, potential hotspot positions underlined) are frequent in Ig genes. This fact and Dörner and Lipsky's interesting analysis and interpretations prompted us to subcategorize the WA hotspots reported in our study of 15 SHM spectra1. Twelve of 13 mutational hotspots at A-T pairs were found in WA motifs. Importantly, no simultaneous WA and RGYW hotspots were observed in fused motifs and only four WA hotspots were fused with an RGYW motif that was not a hotspot. Also, when we then analyzed 916 unselected somatic mutations in the VκOx1Jκ5 transgene (the largest spectrum available3), only five of 13 WA hotspots were fused to an RGYW sequence. Moreover, no rare-strand variant TW sites were found among eight nonfused WA hotspots, indicating that the strand bias for WA sites does not depend on fused motifs. Taken together, the data clearly show that WA hotspots do not require the simultaneous presence of RGYW and, further, that the strand specificity for WA hotspots that we reported earlier is not a specific feature of fused motifs. These interpretations are supported further by analysis of all WA sites (both hotspot and nonhotspot sites) in the VκOx1Jκ5 transgene, showing that the proportion of hotspot sites was similar for fused (0.44) and nonfused WA sites (0.42). In addition, the frequencies of the different types of substitutions were very similar in fused and nonfused WA motifs, suggesting that the mechanism of hypermutation is similar in both types of motifs. Finally, results in XP-V patients lacking pol η4 clearly show a significant decrease in somatic mutations at A-T base pairs for both fused (for example, codon 56, sequence GGTATAA, seven mutations in control clones and no mutations in XP-V clones) and independent WA motifs (for example, codon 31, sequence GCAACAG, five mutations in control clones and no mutations in XP-V clones). The total increase of mutation frequency for normal individuals compared to XP-V patients in fused WA sites was 4.3-fold, while in nonfused WA sites it was 2.9-fold. Thus, the absence of pol η in XP-V patients had a significant influence (P<0.01, Fisher's exact test) on both types of sites. All of these results suggest that pol η may contribute to SHM at strand-specific WA motifs irrespective of RGYW context. It is important to mention that the RGYW hotspots also do not depend on WA motifs, as both fused and nonfused RGYW hotspots can be observed (19 and 13 hotspots, respectively).
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