Differential expression of APE1 and APE2 in germinal centers promotes error-prone repair and A:T mutations during somatic hypermutation

Abstract

Somatic hypermutation (SHM) of antibody variable region genes is initiated in germinal center B cells during an immune response by activation-induced cytidine deaminase (AID), which converts cytosines to uracils. During accurate repair in nonmutating cells, uracil is excised by uracil DNA glycosylase (UNG), leaving abasic sites that are incised by AP endonuclease (APE) to create single-strand breaks, and the correct nucleotide is reinserted by DNA polymerase β. During SHM, for unknown reasons, repair is error prone. There are two APE homologs in mammals and, surprisingly, APE1, in contrast to its high expression in both resting and in vitro-activated splenic B cells, is expressed at very low levels in mouse germinal center B cells where SHM occurs, and APE1 haploinsufficiency has very little effect on SHM. In contrast, the less efficient homolog, APE2, is highly expressed and contributes not only to the frequency of mutations, but also to the generation of mutations at A:T base pair (bp), insertions, and deletions. In the absence of both UNG and APE2, mutations at A:T bp are dramatically reduced. Single-strand breaks generated by APE2 could provide entry points for exonuclease recruited by the mismatch repair proteins Msh2-Msh6, and the known association of APE2 with proliferating cell nuclear antigen could recruit translesion polymerases to create mutations at AIDinduced lesions and also at A:T bp. Our data provide new insight into error-prone repair of AID-induced lesions, which we propose is facilitated by down-regulation of APE1 and up-regulation of APE2 expression in germinal center B cells. D uring humoral immune responses, the recombined antibody variable [V(D)J] region genes undergo somatic hypermutation (SHM), which, after selection, greatly increases the affinity of antibodies for the activating antigen. This process occurs in germinal centers (GCs) in the spleen, lymph nodes, and Peyer's patches (PPs) and entirely depends on activation-induced cytidine deaminase (AID) (1, 2). AID initiates SHM by deamination of cytidine nucleotides in the variable region of antibody genes, converting the cytosine (dC) to uracil (dU) (1, 3, 4). Some AIDinduced dUs are excised by the ubiquitous enzyme uracil DNA glycosylase (UNG), resulting in abasic (AP) sites that can be recognized by apurinic/apyrimidinic endonuclease (APE) (4, 5). APE cleaves the DNA backbone at AP sites to form a singlestrand break (SSB) with a 3′ OH that can be extended by DNA polymerase (Pol) to replace the excised nucleotide (6). In most cells, DNA Pol β performs this extension with high fidelity, reinserting dC across from the template dG. In contrast, GC B cells undergoing SHM are rapidly proliferating, and some of the dUs are replicated over before they can be excised and are read as dT by replicative polymerases, resulting in dC to dT transition mutations. Unrepaired AP sites encountering replication lead to the nontemplated addition of any base opposite the site, causing transition and transversion mutations. However, it is not clear why dU...

APE2 Zf-GRF facilitates 3′-5′ resection of DNA damage following oxidative stress

“…In mammalian cells, APE2 is also necessary for normal B-cell development and recovery from chemotherapy drug-induced DNA damage (29). It was recently proposed that differential expression of APE2 in germinal centers promotes error-prone repair and mutations during somatic hypermutation (30). Importantly, APE2 is a key player in the PCNA-dependent repair of hydrogen peroxide-induced oxidative DNA damage (31)(32)(33).…”

Overlapping hotspots in CDRs are critical sites for V region diversification

“…Both SHM and class switch recombination are initiated by activationinduced deaminase (AID) that preferentially deaminates the dC residues in WRC (W = A/T, R = A/G) hotspot motifs at frequencies 2-10-fold higher than SYC (S = G/C; Y = C/T) cold spots (3)(4)(5)(6)(7). During V region SHM, the resulting dU:G mismatch can then be replicated during S-phase to produce transition mutations, be processed by uracil-DNA glycosylase 2 and apurinic/ apyrimidinic endonucleases through the base excision repair pathway to produce both transitions and transversions (8)(9)(10), or be recognized by MutS homolog (MSH)2/MSH6 of the mismatch repair (MMR) complex that recruits the low-fidelity polymerase eta (Polη) to generate additional mutations at neighboring A:T residues (11).…”

Deficiency of base excision repair enzyme NEIL3 drives increased predisposition to autoimmunity

“…WGS was performed through Complete Genomics Inc. and analyzed with Mol-BioLib (74 tent cells. TA-plasmids were extracted from 200 clones using the Gene Jet Plasmid Miniprep Kit (Thermo Scientific) and Sanger sequenced; then a 551-bp fragment was analyzed for the presence of mutations (59). RNA extraction and RT/PCR of LRBA.…”

“…To determine whether Neil3 deficiency alters SHM, gDNA was extracted from GC B cells isolated from the intestinal PP of Neil3 -/-mice and WT controls (n = 3 per group), the V H J558L framework 3 and J H 4 3′ flanking regions were amplified from the Igh locus, cloned in the TOPO-TA cloning vector, and 200 clones from each mouse were Sanger sequenced. A 551-bp fragment from each clone was analyzed for the presence of mutations and compared with the reference sequence of the C57BL/6J strain, as described previously (59). There was no significant difference in the frequency of total mutations in GC B cells between Neil3 -/-mice and WT controls (11.92 ± 0.70 per 1000 bp in WT mice versus 12.38 ± 3.45 in Neil3 -/-mice).…”

“…The VHJ558L framework3-JH4 3′ flanking region was PCR amplified as previously described (59). PCR product (600 bp) was purified (QIAquick Gel Extraction Kit; QIAGEN), dA tails were added with Taq polymerase (Thermo Scientific), cloned in TOPO-TA vectors (Invitrogen), and used to transform Escherichia coli DH5α compe- to amplify LRBA cDNA exons using the SuperScript III One-Step RT-PCR System with Platinum Taq (Life Technologies).…”