Interplay between Target Sequences and Repair Pathways Determines Distinct Outcomes of AID-Initiated Lesions

Abstract

Activation-induced deaminase (AID) functions by deaminating cytosines and causing U:G mismatches, a rate-limiting step of antibody gene diversification. However, precise mechanisms regulating AID-deamination frequency remain incompletely understood. Moreover, it is unknown whether different sequence contexts influence the preferential access of mismatch repair (MMR) or uracil glycosylase (UNG) to AID-initiated U:G mismatches. Here, we employed two knock-in models to directly compare the mutability of core Sµ and VDJ exon sequences and their ability to regulate AID-deamination and subsequent repair process. We find that S region is a much more efficient AID deamination target than V region. Igh locus AID-initiated lesions are processed by error-free and error-prone repair. S region U:G mismatches are preferentially accessed by UNG, leading to more UNG-dependent deletions, enhanced by MMR deficiency. V region mutation hotspots are largely determined by AID deamination. Recurrent and conserved S region motifs potentially function as spacers between AID-deamination hotspots. We conclude that the pattern of mutation hotspots and DNA break generation is influenced by sequence-intrinsic properties, which regulate AID deamination and affect the preferential access of downstream repair. Our studies reveal an evolutionarily conserved role for substrate sequences in regulating antibody gene diversity and AID targeting specificity.

SAMHD1 enhances immunoglobulin hypermutation by promoting transversion mutation

“…In remarkable convergence with this footprint size, Woodrick et al (46) showed that LP-BER produces excision patches centered on the excised AP site that are ≤20 nucleotides long; they further showed that LP-BER predominates over single-nucleotide BER in living cells. AGCW motifs are the only hotspots where AID can deaminate either DNA strand with comparable efficiency in vivo (8,9,47). This leads us to update the Ig hypermutation model and propose that MMR-independent C/G transversions occur via simultaneous G 1 -phase LP-BER of deaminations that have accumulated in Fig.…”

“…S1), presumably by the U:G mismatches deamination induces. Processing via BER or MMR predominantly restores C in place of U (6)(7)(8)(9) but is also error-prone. Error-prone BER converts deamination sites into transition or transversion mutations at C/G and occasionally produces mutations at bases flanking deamination sites, including A/T bases (1).…”

“…Only G 1 -phase activities of AID or UNG2 are mutagenic in mouse B cells (5,6,23). Strong evidence for mutagenic interaction between BER and MMR implies that mutagenic MMR also occurs in G 1 phase (2,3,8,9,12,21). dNTP levels were reported to be lowest in G 1 phase, especially in the nucleus (24).…”

Proximity to AGCT sequences dictates MMR-independent versus MMR-dependent mechanisms for AID-induced mutationviaUNG2

“…Loss of UNG2 activity can also increase the frequency of C:G to T:A transition mutations, presumably as a result of increased uracil replication. This suggests that high fidelity uracil BER does occur in hypermutating B cells ( 9 , 11 , 19 , 21 ).…”

The Number of Overlapping AID Hotspots in Germline IGHV Genes Is Inversely Correlated with Mutation Frequency in Chronic Lymphocytic Leukemia

“…While the mouse Sμ switch region contains a high density of AGCT hotspots, this density varies within the switch region, which led the authors to define sub-regions reflecting sparse, intermediate and dense AGCT densities. Interestingly, across the sub-regions mutation frequency did not correlate with AGCT density, suggesting that AID targeting is efficient once a threshold density is reached, and that higher densities do not necessarily increase the mutation targeting frequency [ 42 ].…”