Analysis of 6912 unselected somatic hypermutations in human VDJ rearrangements reveals lack of strand specificity and correlation between phase II substitution rates and distance to the nearest 3' activation-induced cytidine deaminase target

The initial event of somatic hypermutation (SHM) is the deamination of cytidine residues by activation-induced cytidine deaminase (AID). Deamination is followed by the replication over uracil and/or different error-prone repair events. We sequenced 659 nonproductive human IgH rearrangements (IGHV3-23*01) from blood B lymphocytes enriched for CD27-positive memory cells. Analyses of 6,912 unique, unselected substitutions showed that in vivo hot and cold spots for the SHM of C and G residues corresponded closely to the target preferences reported for AID in vitro. A detailed analysis of all possible four-nucleotide motifs present on both strands of the V(H) gene showed significant correlations between the substitution frequencies in reverse complementary motifs, suggesting that the SHM machinery targets both strands equally well. An analysis of individual J(H) and D gene segments showed that the substitution frequencies in the individual motifs were comparable to the frequencies found in the V(H) gene. Interestingly, J(H)6-carrying sequences were less likely to undergo SHM (average 15.2 substitutions per V(H) region) than sequences using J(H)4 (18.1 substitutions, p = 0.03). We also found that the substitution rates in G and T residues correlated inversely with the distance to the nearest 3' WRC AID hot spot motif on both the nontranscribed and transcribed strands. This suggests that phase II SHM takes place 5' of the initial AID deamination target and primarily targets T and G residues or, alternatively, the corresponding A and C residues on the opposite strand.     

Somatic hypermutation and class switch recombination in Msh6(-/-)Ung(-/-) double-knockout mice

Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by activation-induced cytosine deaminase (AID). The uracil, and potentially neighboring bases, are processed by error-prone base excision repair and mismatch repair. Deficiencies in Ung, Msh2, or Msh6 affect SHM and CSR. To determine whether Msh2/Msh6 complexes which recognize single-base mismatches and loops were the only mismatch-recognition complexes required for SHM and CSR, we analyzed these processes in Msh6(-/-)Ung(-/-) mice. SHM and CSR were affected in the same degree and fashion as in Msh2(-/-)Ung(-/-) mice; mutations were mostly C,G transitions and CSR was greatly reduced, making Msh2/Msh3 contributions unlikely. Inactivating Ung alone reduced mutations from A and T, suggesting that, depending on the DNA sequence, varying proportions of A,T mutations arise by error-prone long-patch base excision repair. Further, in Msh6(-/-)Ung(-/-) mice the 5' end and the 3' region of Ig genes was spared from mutations as in wild-type mice, confirming that AID does not act in these regions. Finally, because in the absence of both Ung and Msh6, transition mutations from C and G likely are "footprints" of AID, the data show that the activity of AID is restricted drastically in vivo compared with AID in cell-free assays.     

The Fanconi anemia core complex is dispensable during somatic hypermutation and class switch recombination

To generate high affinity antibodies during an immune response, B cells undergo somatic hypermutation (SHM) of their immunoglobulin genes. Error-prone translesion synthesis (TLS) DNA polymerases have been reported to be responsible for all mutations at template A/T and at least a fraction of G/C transversions. In contrast to A/T mutations which depend on PCNA ubiquitination, it remains unclear how G/C transversions are regulated during SHM. Several lines of evidence indicate a mechanistic link between the Fanconi Anemia (FA) pathway and TLS. To investigate the contribution of the FA pathway in SHM we analyzed FancG-deficient B cells. B cells deficient for FancG, an essential member of the FA core complex, were hypersensitive to treatment with cross-linking agents. However, the frequencies and nucleotide exchange spectra of SHM remained comparable between wild-type and FancG-deficient B cells. These data indicate that the FA pathway is not involved in regulating the outcome of SHM in mammals. In addition, the FA pathway appears dispensable for class switch recombination.     

A regulatory role for NBS1 in strand-specific mutagenesis during somatic hypermutation

Activation-induced cytidine deaminase (AID) is believed to initiate somatic hypermutation (SHM) by deamination of deoxycytidines to deoxyuridines within the immunoglobulin variable regions genes. The deaminated bases can subsequently be replicated over, processed by base excision repair or mismatch repair, leading to introduction of different types of point mutations (G/C transitions, G/C transversions and A/T mutations). It is evident that the base excision repair pathway is largely dependent on uracil-DNA glycosylase (UNG) through its uracil excision activity. It is not known, however, which endonuclease acts in the step immediately downstream of UNG, i.e. that cleaves at the abasic sites generated by the latter. Two candidates have been proposed, an apurinic/apyrimidinic endonuclease (APE) and the Mre11-Rad50-NBS1 complex. The latter is intriguing as this might explain how the mutagenic pathway is primed during SHM. We have investigated the latter possibility by studying the in vivo SHM pattern in B cells from ataxia-telangiectasia-like disorder (Mre11 deficient) and Nijmegen breakage syndrome (NBS1 deficient) patients. Our results show that, although the pattern of mutations in the variable heavy chain (V(H)) genes was altered in NBS1 deficient patients, with a significantly increased number of G (but not C) transversions occurring in the SHM and/or AID targeting hotspots, the general pattern of mutations in the V(H) genes in Mre11 deficient patients was only slightly altered, with an increased frequency of A to C transversions. The Mre11-Rad50-NBS1 complex is thus unlikely to be the major nuclease involved in cleavage of the abasic sites during SHM, whereas NBS1 might have a specific role in regulating the strand-biased repair during phase Ib mutagenesis.     

Alkyladenine DNA glycosylase (Aag) in somatic hypermutation and class switch recombination

Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes require the cytosine deaminase AID, which deaminates cytosine to uracil in Ig gene DNA. Paradoxically, proteins involved normally in error-free base excision repair and mismatch repair, seem to be co-opted to facilitate SHM and CSR, by recruiting error-prone translesion polymerases to DNA sequences containing deoxy-uracils created by AID. Major evidence supports at least one mechanism whereby the uracil glycosylase Ung removes AID-generated uracils creating abasic sites which may be used either as uninformative templates for DNA synthesis, or processed to nicks and gaps that prime error-prone DNA synthesis. We investigated the possibility that deamination at adenines also initiates SHM. Adenosine deamination would generate hypoxanthine (Hx), a substrate for the alkyladenine DNA glycosylase (Aag). Aag would generate abasic sites which then are subject to error-prone repair as above for AID-deaminated cytosine processed by Ung. If the action of an adenosine deaminase followed by Aag were responsible for significant numbers of mutations at A, we would find a preponderance of A:T>G:C transition mutations during SHM in an Aag deleted background. However, this was not observed and we found that the frequencies of SHM and CSR were not significantly altered in Aag-/- mice. Paradoxically, we found that Aag is expressed in B lymphocytes undergoing SHM and CSR and that its activity is upregulated in activated B cells. Moreover, we did find a statistically significant, albeit low increase of T:A>C:G transition mutations in Aag-/- animals, suggesting that Aag may be involved in creating the SHM A>T bias seen in wild type mice.     

PCNA ubiquitination-independent activation of polymerase η during somatic hypermutation and DNA damage tolerance

The generation of high affinity antibodies in B cells critically depends on translesion synthesis (TLS) polymerases that introduce mutations into immunoglobulin genes during somatic hypermutation (SHM). The majority of mutations at A/T base pairs during SHM require ubiquitination of PCNA at lysine 164 (PCNA-Ub), which activates TLS polymerases. By comparing the mutation spectra in B cells of WT, TLS polymerase η (Polη)-deficient, PCNA(K164R)-mutant, and PCNA(K164R);Polη double-mutant mice, we now find that most PCNA-Ub-independent A/T mutagenesis during SHM is mediated by Polη. In addition, upon exposure to various DNA damaging agents, PCNA(K164R) mutant cells display strongly impaired recruitment of TLS polymerases, reduced daughter strand maturation and hypersensitivity. Interestingly, compared to the single mutants, PCNA(K164R);Polη double-mutant cells are dramatically delayed in S phase progression and far more prone to cell death following UV exposure. Taken together, these data support the existence of PCNA ubiquitination-dependent and -independent activation pathways of Polη during SHM and DNA damage tolerance.     

DNA polymerase eta contributes to strand bias of mutations of A versus T in immunoglobulin genes

DNA polymerase (pol) eta participates in hypermutation of A:T bases in Ig genes because humans deficient for the polymerase have fewer substitutions of these bases. To determine whether polymerase eta is also responsible for the well-known preference for mutations of A vs T on the nontranscribed strand, we sequenced variable regions from three patients with xeroderma pigmentosum variant (XP-V) disease, who lack polymerase eta. The frequency of mutations in the intronic region downstream of rearranged J(H)4 gene segments was similar between XP-V and control clones; however, there were fewer mutations of A:T bases and correspondingly more substitutions of C:G bases in the XP-V clones (p < 10(-7)). There was significantly less of a bias for mutations of A compared with T nucleotides in the XP-V clones compared with control clones, whereas the frequencies for mutations of C and G were identical in both groups. An analysis of mutations in the WA sequence motif suggests that polymerase eta generates more mutations of A than T on the nontranscribed strand. This in vivo data from polymerase eta-deficient B cells correlates well with the in vitro specificity of the enzyme. Because polymerase eta inserts more mutations opposite template T than template A, it would generate more substitutions of A on the newly synthesized strand.     

A role for the MutL mismatch repair Mlh3 protein in immunoglobulin class switch DNA recombination and somatic hypermutation

Class switch DNA recombination (CSR) and somatic hypermutation (SHM) are central to the maturation of the Ab response. Both processes involve DNA mismatch repair (MMR). MMR proteins are recruited to dU:dG mispairs generated by activation-induced cytidine deaminase-mediated deamination of dC residues, thereby promoting S-S region synapses and introduction of mismatches (mutations). The MutL homolog Mlh3 is the last complement of the mammalian set of MMR proteins. It is highly conserved in evolution and is essential to meiosis and microsatellite stability. We used the recently generated knockout mlh3(-/-) mice to address the role of Mlh3 in CSR and SHM. We found that Mlh3 deficiency alters both CSR and SHM. mlh3(-/-) B cells switched in vitro to IgG and IgA but displayed preferential targeting of the RGYW/WRCY (R = A or G, Y = C or T, W = A or T) motif by Sgamma1 and Sgamma3 breakpoints and introduced more insertions and fewer donor/acceptor microhomologies in Smu-Sgamma1 and Smu-Sgamma3 DNA junctions, as compared with mlh3(+/+) B cells. mlh3(-/-) mice showed only a slight decrease in the frequency of mutations in the intronic DNA downstream of the rearranged J(H)4 gene. However, the residual mutations were altered in spectrum. They comprised a decreased proportion of mutations at dA/dT and showed preferential RGYW/WRCY targeting by mutations at dC/dG. Thus, the MMR Mlh3 protein plays a role in both CSR and SHM.     

Expression of the cancer-associated DNA polymerase ε P286R in fission yeast leads to translesion synthesis polymerase dependent hypermutation and defective DNA replication

Somatic and germline mutations in the proofreading domain of the replicative DNA polymerase ε (POLE-exonuclease domain mutations, POLE-EDMs) are frequently found in colorectal and endometrial cancers and, occasionally, in other tumours. POLE-associated cancers typically display hypermutation, and a unique mutational signature, with a predominance of C > A transversions in the context TCT and C > T transitions in the context TCG. To understand better the contribution of hypermutagenesis to tumour development, we have modelled the most recurrent POLE-EDM (POLE-P286R) in Schizosaccharomyces pombe. Whole-genome sequencing analysis revealed that the corresponding pol2-P287R allele also has a strong mutator effect in vivo, with a high frequency of base substitutions and relatively few indel mutations. The mutations are equally distributed across different genomic regions, but in the immediate vicinity there is an asymmetry in AT frequency. The most abundant base-pair changes are TCT > TAT transversions and, in contrast to human mutations, TCG > TTG transitions are not elevated, likely due to the absence of cytosine methylation in fission yeast. The pol2-P287R variant has an increased sensitivity to elevated dNTP levels and DNA damaging agents, and shows reduced viability on depletion of the Pfh1 helicase. In addition, S phase is aberrant and RPA foci are elevated, suggestive of ssDNA or DNA damage, and the pol2-P287R mutation is synthetically lethal with rad3 inactivation, indicative of checkpoint activation. Significantly, deletion of genes encoding some translesion synthesis polymerases, most notably Pol κ, partially suppresses pol2-P287R hypermutation, indicating that polymerase switching contributes to this phenotype.     

Hypermutation at A/T sites during G.U mismatch repair in vitro by human B-cell lysates

Somatic hypermutation in the variable regions of immunoglobulin genes is required to produce high affinity antibody molecules. Somatic hypermutation results by processing G.U mismatches generated when activation-induced cytidine deaminase (AID) deaminates C to U. Mutations at C/G sites are targeted mainly at deamination sites, whereas mutations at A/T sites entail error-prone DNA gap repair. We used B-cell lysates to analyze salient features of somatic hypermutation with in vitro mutational assays. Tonsil and hypermutating Ramos B-cells convert C-->U in accord with AID motif specificities, whereas HeLa cells do not. Using tonsil cell lysates to repair a G.U mismatch, A/T and G/C targeted mutations occur about equally, whereas Ramos cell lysates make fewer mutations at A/T sites (approximately 24%) compared with G/C sites (approximately 76%). In contrast, mutations in HeLa cell lysates occur almost exclusively at G/C sites (> 95%). By recapitulating two basic features of B-cell-specific somatic hypermutation, G/C mutations targeted to AID hot spot motifs and elevated A/T mutations dependent on error-prone processing of G.U mispairs, these cell free assays provide a practical method to reconstitute error-prone mismatch repair using purified B-cell proteins.     

Hypermutation at A-T base pairs: the A nucleotide replacement spectrum is affected by adjacent nucleotides and there is no reverse complementarity of sequences flanking mutated A and T nucleotides

Hypermutation is thought to be a two-phase process. The first phase is via the action of activation-induced cytidine deaminase (AID), which deaminates C nucleotides in WRC motifs. This results in the RGYW/WRCY hot spot motifs for mutation from G and C observed in vivo. The resemblance between the hot spot for C mutations and the reverse complement of that for G mutations implies a process acting equally on both strands of DNA. The second phase of hypermutation generates mutations from A and T and exhibits strand bias, with more mutations from A than T. Although this does not concur with the idea of one mechanism acting equally on both strands, it has been suggested that the AT mutator also has a reversible motif; WA/TW. We show here that the motifs surrounding the different substitutions from A vary significantly; there is no single targeting motif for all A mutations. Sequence preferences associated with mutations from A more likely reflect an influence of adjacent nucleotides over what the A mutates "to." This influence tends toward "like" replacements: Purines (A or G) in the 5' position bias toward replacement by another purine (G), whereas replacement with pyrimidines (C or T) is more likely if the preceding base is also a pyrimidine. There is no reverse complementarity in these observations, in that similar influences of nucleotides adjacent to T are not seen. Hence, WA and TW should not be considered as reverse complement hot spot motifs for A and T mutations.     

Ectopic expression of AID in a non-B cell line triggers A:T and G:C point mutations in non-replicating episomal vectors

Somatic hypermutation (SHM) of immunoglobulin genes is currently viewed as a two step process initiated by the deamination of deoxycytidine (C) to deoxyuridine (U), catalysed by the activation induced deaminase (AID). Phase 1 mutations arise from DNA replication across the uracil residue or the abasic site, generated by the uracil-DNA glycosylase, yielding transitions or transversions at G:C pairs. Phase 2 mutations result from the recognition of the U:G mismatch by the Msh2/Msh6 complex (MutS Homologue), followed by the excision of the mismatched nucleotide and the repair, by the low fidelity DNA polymerase eta, of the gap generated by the exonuclease I. These mutations are mainly focused at A:T pairs. Whereas in activated B cells both G:C and A:T pairs are equally targeted, ectopic expression of AID was shown to trigger only G:C mutations on a stably integrated reporter gene. Here we show that when using non-replicative episomal vectors containing a GFP gene, inactivated by the introduction of stop codons at various positions, a high level of EGFP positive cells was obtained after transient expression in Jurkat cells constitutively expressing AID. We show that mutations at G:C and A:T pairs are produced. EGFP positive cells are obtained in the absence of vector replication demonstrating that the mutations are dependent only on the mismatch repair (MMR) pathway. This implies that the generation of phase 1 mutations is not a prerequisite for the expression of phase 2 mutations.