Hoogsteen base-pairing allows four G nucleotides to associate and form a planar G-tetrad, and the stacking of multiple G-tetrads results in a stable G4 structure. The G-quadruplex (G4) is a four-stranded nucleic acid structure formed from G-rich DNA. Conversely, the SY C (S=C/G, Y=C/T) “coldspot” is targeted significantly less by AID ( 6).ĭNA secondary structures play an active role in regulating the transcriptional process. In addition, the A GCT motif, a palindromic variation of WR C, is enriched in the S region ( 4) and is targeted at particularly high rates in the V region as well ( 5). The canonical AID hotspot is defined as WR C (W=A/T, R=A/G and the underlined base indicates the site targeted). Class switching is achieved when the IgM heavy chain constant (C) region of the antibody is swapped for another one of the downstream isotypes, thus changing the effector function of the antibody while maintaining antigen specificity.Ĭertain DNA sequence contexts are mutated preferentially by AID, and are collectively referred to as “hotspots” ( 3). During CSR, AID mutations lead to DNA double-stranded breaks (DSBs) in the Ig switch (S) region. In the GC light zone, positive selection of B cells that acquire mutations, leading to higher binding affinity of the B cell receptor, takes place. Ig variable (V) region SHM occurs in the dark zone of the germinal center (GC) at a rate of around 10 -3 per bp per cell division. During SHM, co-opted DNA repair pathways, including base-excision repair and mismatch repair, result in unfaithfully repaired AID-induced lesions. AID binds to single-stranded DNA (ssDNA) and deaminates cytosine (C) to uracil (U). The IGHV genes are further categorized into families, defined by sequence similarity ( 2). There are ~55 Ig heavy chain variable (IGHV) genes in human, each of which may have one or more alleles. Activation-induced deaminase (AID) is a critical enzyme responsible for initiating somatic hypermutation (SHM) and class-switch recombination (CSR) at the Immunoglobulin (Ig) loci.

Our overall assessment reveals plausible evidence of functional G4s forming in the Ig V region.Īntibody diversification in antigen-stimulated B cells is instrumental in driving effective humoral immunity against a wide range of pathogens. In addition, CCC coldspots opposite a predicted G4 were shown to be targeted significantly more for mutation. G4 potential is also associated with higher mutability of certain WRC hotspots on the same strand. In silico mutagenesis experiments showed that G4 potentials were generally robust to mutation, although large deviations from germline states were found, mostly in framework regions. However, AGCT motifs in both V and C regions were less abundant than in S regions. The density of AGCT motifs, where two AGC hotspots overlap on both strands, was roughly 2.6-fold greater in the V region than the Constant (C) region, which does not mutate despite having predicted G4s at similar levels. G4-seq also revealed variation between S and V regions in the distribution of potential G4s, with the V region having overall reduced G4 abundance compared to the S region. Using a high-resolution (G4-seq) dataset of biochemically confirmed potential G4s in IGHV genes, we validated our computational predictions. Different IGHV alleles also showed variability in G4 potential.

This revealed that many genes from the IGHV3 and IGHV4 families are predicted to have high G4 potential in the top and bottom strand, respectively.

To assess the possibility of G4 formation in human V regions, we analyzed germline human Ig heavy chain V (IGHV) sequences, using a pre-trained deep learning model that predicts G4 potential. However, it is still unclear whether G4s form in the variable (V) region, or how they may affect SHM. Alterations in the gene encoding AID can further disrupt AID-G4 binding and reduce CSR in vivo. In vitro studies have shown G4s to form and bind AID in Ig switch (S) regions. G-quadruplex (G4) structures are four-stranded DNA secondary structures with key functions in transcription, translation and replication. AID preferentially targets WRC (W=A/T, R=A/G) hotspot motifs and avoids SYC (S=C/G, Y=C/T) coldspots. 2Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY, United StatesĪctivation-induced deaminase (AID) is a key enzyme involved in antibody diversification by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) of the Immunoglobulin (Ig) loci.1Department of Applied Mathematics and Statistics, Stony Brook University, Stony Brook, NY, United States.Catherine Tang 1 and Thomas MacCarthy 1,2*

Lightzone syc