Mechanisms of transversion mutation are dependent on sequence context and nucleotide paucity during antibody somatic hypermutation

Somatic hypermutation of antibodies during humoral immune responses depends on expression of Activation Induced Deaminase (AID) in antibody-producing B cells. AID initiates somatic hypermutation by converting cytosine (C) residues in antibody genes into uracil (U) residues, by deamination. Alone, conversion of cytosine into uracil can only produce C:G to T:A transition mutations, by replication across U (phase 1A mutation). Processing of C deaminations by base excision repair (BER) or mismatch repair (MMR) diversifies mutation, predominantly at C:G (phase 1B mutation) and A:T (phase 2 mutation), respectively. Mutations at C along the Ig variable region are not equally distributed. AID de-aminates C more often if they occur as part of WRCY motif (A/T,A/G,C,C/T). WRCY sequences are concentrated in hypervariable regions of Ig genes, where nucleotide substitutions are likely to be effective at generating useful amino acid substitutions to optimize affinity maturation. Of all WRCY motifs, AGCT and AACT are the most mutated hotspots. AGCT is also enriched in switch regions and facilitates CSR. In Chapter three, using large datasets of a transgenic mouse model, I compared Igh hypermutation between SWHEL B cells, SWHEL B cells deficient for UNG2 via retroviral expression of the uracil glycosylase inhibitor (ugi), SWHEL B cells deficient for MutSα by crossing Msh2ko alleles into SWHEL mice and SWHEL B cells deficient for both UNG2 and MutSα. I found that phase 1B mutations occur by distinct MMR-independent or MMR dependent pathways. At or in proximity to AGCW motifs, phase 1B mutations were driven by UNG2 without requirement for mismatch repair. Deaminations in AGCW were refractive both to processing by UNG2 and to high-fidelity base excision repair (BER) downstream of UNG2, regardless of mismatch repair activity. Outside AGCW motifs, transversions at C:G are co-dependent on UNG2 and MMR. Classically, MMR mediates high fidelity repair of mismatches introduced during replication. The reasons for the profound differences in repair accuracy between classical and AID-induced MMR have not been elicited. During S-phase of the cell replication cycle, when classical post-replication MMR occurs, nucleotide triphosphate (dNTP) levels are optimal for DNA replication, while in G1-phase dNTP levels are lower. Since there is evidence that AID is active in G1-phase, we hypothesized that low dNTP levels may be the cause of low fidelity MMR. Two enzymes are the major determinant of dNTP pools: ribonucleotide reductase (RNR), which converts ribonucleotides into deoxyribonucleotides predominantly during S-phase, and SAMHD1, which degrades dNTPs predominantly outside of S-phase. In Chapters four and five, I quantified antibody hypermutation in B cells lacking SAMHD1 and/or over-expressing RNR. I observed a 2-fold decrease in mutations at A:T bases in cells lacking SAMHD1. This decrease was comparable to the decrease induced by RNR over-expression and was consistent with our hypothesis. Unexpectedly, loss of SAMHD1 also decreased transversion mutations at C:G by about 70%, and almost doubled transition mutations at C:G bases. RNR over-expression had no obvious impact on transversion mutations at C:G, but increased transition mutations at C:G bases similarly to loss of SAMHD1. Furthermore, loss of SAMHD1 decreased AID/BER-dependent antibody class switch recombination, while RNR over-expression did not. These findings indicate that dNTPs play a role in MMR-mediated antibody mutation, as predicted by our hypothesis, but they also indicate a major role for SAMHD1 in AID-induced BER that was not predicted by our hypothesis or by current models of antibody hypermutation. This important finding warrants further investigation to identify the mechanism

Proximity to AGCT sequences dictates MMR-independent versus MMR-dependent mechanisms for AID-induced mutation via UNG2

AID deaminates C to U in either strand of Ig genes, exclusively producing C:G/G:C to T:A/A:T transition mutations if U is left unrepaired. Error-prone processing by UNG2 or mismatch repair diversifies mutation, predominantly at C:G or A:T base pairs, respectively. Here, we show that transversions at C:G base pairs occur by two distinct processing pathways that are dictated by sequence context. Within and near AGCT mutation hotspots, transversion mutation at C:G was driven by UNG2 without requirement for mismatch repair. Deaminations in AGCT were refractive both to processing by UNG2 and to high-fidelity base excision repair (BER) downstream of UNG2, regardless of mismatch repair activity. We propose that AGCT sequences resist faithful BER because they bind BER-inhibitory protein(s) and/or because hemi-deaminated AGCT motifs innately form a BERresistant DNA structure. Distal to AGCT sequences, transversions at G were largely co-dependent on UNG2 and mismatch repair. We propose that AGCTdistal transversions are produced when apyrimidinic sites are exposed in mismatch excision patches, because completion of mismatch repair would require bypass of these sites