Ubiquitylated PCNA plays a role in somatic hypermutation and class-switch recombination and is required for meiotic progression

Abstract

Somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes are dependent upon activation-induced cytidine deaminase (AID)-induced mutations. The scaffolding properties of proliferating cell nuclear antigen (PCNA) and ubiquitylation of its residue K164 have been suggested to play an important role organizing the error-prone repair events that contribute to the AID-induced diversification of the Ig locus. We generated knockout mice for PCNA (Pcna ؊/؊ ), which were embryonic lethal. Expression of PCNA with the K164R mutation rescued the lethal phenotype, but the mice (Pcna ؊/؊ tg K164R ) displayed a meiotic defect in early pachynema and were sterile. B cells proliferated normally in Pcna ؊/؊ tg K164R mice, but a PCNA-K164R mutation resulted in impaired ex vivo CSR to IgG1 and IgG3, which was associated with reduced mutation frequency at the switch regions and a bias toward blunt junctions. Analysis of the heavy chain V186.2 region after NP-immunization showed in Pcna ؊/؊ tg K164R mice a significant reduction in the mutation frequency of A:T residues in WA motifs preferred by polymerase-(Pol ), and a strand-biased increase in the mutation frequency of G residues, preferentially in the context of AID-targeted GYW motifs. The phenotype of Pcna ؊/؊ tg K164R mice supports the idea that ubiquitylation of PCNA participates directly in the meiotic process and the diversification of the Ig locus through class-switch recombination (CSR) and somatic hypermutation (SHM).T o mount an effective antibody response, mice and humans create a highly diverse repertoire of antigen binding sites through the rearrangement of the germ line variable (V), diversity (D), and joining (J) Ig locus. Following interaction with antigen, B cells in the germinal centers (GCs) of secondary lymphoid organs express activation-induced cytidine deaminase (AID). AID, together with other enzymes, causes a very high rate (10 Ϫ5 -10 Ϫ3 /base pair/generation) of point mutations in Ig V regions resulting in the affinity maturation and the changes in fine specificity required to produce protective antibodies (1, 2). AID also initiates class-switch recombination (CSR) by mutating the switch regions (SRs) that are located just 5Ј of the constant region genes (3, 4). CSR allows antibodies to be distributed throughout the body and to carry out a wide variety of effector functions. AID deaminates deoxycytidines (dC) in single-stranded DNA in the V and SRs to generate deoxyuridine (dU) (1, 2). However, more than half of the mutations in the V and SRs of mice and humans are in A:T bases and are not the result of the direct biochemical action of AID. Rather, these mutations arise during a second phase of SHM and result from the error-prone base excision repair (BER) and mismatch repair (MMR), both of which are recruited to the dU:dG mismatch generated by AID (1, 2, 4).When critical MMR genes are deleted from mice, most of the mutations in A:T in the V region no longer occur, suggesting that MMR is responsible for the majority of the mutations that ar...

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

“…Unlike APE1, APE2 has a functional PCNA-interacting domain (48). PCNA monoubiquitinated at lysine 164 is known to recruit the translesional polymerases Pol η and Rev1 (23), and SHM analyses in mice expressing a knock-in PCNA K164R mutation show a reduction in A:T mutations (49,50). The 3′ to 5′ exonuclease processivity of APE2 is also stimulated by PCNA (25) and could contribute to mutations by excising a few nucleotides at a SSB, exposing a short singlestrand patch 5′ to the AP site that could be filled in by Pol η, also resulting in mutations at A:T bp.…”

PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance

“…TRIzol extraction (Invitrogen) of RNA was prepared 7 d after the boost from splenic B cells. Then high-fidelity RT-PCR and nested PCR were performed to amplify V H 186.2 joined to the IgG 1 constant region, as described previously (39). Analysis of unique mutations was done with SHMTool (38).…”

“…After 4 d in culture, surface IgM and IgG were stained and analyzed by FACS. PCR amplification from genomic DNA, sequencing, and analysis of unique junctional Sμ-Sγ3 regions were performed as described previously (39).…”

Antitumor Effects of a Novel Small Molecule Targeting PCNA Chromatin Association in Prostate Cancer

“…Each PCNA monomer is composed of 261 amino acids and contains two globular domains, providing the trimeric ring 6-fold symmetry (15). The toroid structure of PCNA is evolutionally well conserved; implicating the essential role for PCNA in basic cellular metabolism, which is underscored by the fact that homozygous deletion of PCNA results in embryonic lethality in mice (15)(16)(17). Trimerization of PCNA is crucial for carrying out its physiologic functions, as demonstrated in studies in which formation of the PCNA trimer was disrupted via a single mutation of tyrosine 114 (Y114A; ref.…”