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...

Activation-induced deaminase (AID) mediates the somatic hypermutation (SHM) of Ig variable (V) regions that is required for the affinity maturation of the antibody response. An intensive analysis of a published database of somatic hypermutations that arose in the IGHV3-23*01 human V region expressed in vivo by human memory B cells revealed that the focus of mutations in complementary determining region (CDR)1 and CDR2 coincided with a combination of overlapping AGCT hotspots, the absence of AID cold spots, and an abundance of polymerase eta hotspots. If the overlapping hotspots in the CDR1 or CDR2 did not undergo mutation, the frequency of mutations throughout the V region was reduced. To model this result, we examined the mutation of the human IGHV3-23*01 biochemically and in the endogenous heavy chain locus of Ramos B cells. Deep sequencing revealed that IGHV3-23*01 in Ramos cells accumulates AID-induced mutations primarily in the AGCT in CDR2, which was also the most frequent site of mutation in vivo. Replacing the overlapping hotspots in CDR1 and CDR2 with neutral or cold motifs resulted in a reduction in mutations within the modified motifs and, to some degree, throughout the V region. In addition, some of the overlapping hotspots in the CDRs were at sites in which replacement mutations could change the structure of the CDR loops. Our analysis suggests that the local sequence environment of the V region, and especially of the CDR1 and CDR2, is highly evolved to recruit mutations to key residues in the CDRs of the IgV region.A fter an encounter with antigen and subsequent migration into the germinal centers of the secondary lymphoid organs, B cells undergo a regulated cascade of mutational events that occur at a very high frequency and are largely restricted to the variable (V) and switch (S) regions of the Ig heavy chain locus and the V region of the light chain locus. These mutagenic events are responsible for the somatic hypermutation (SHM) of the V regions and the class switch recombination of the constant (C) regions that are required for protective antibodies (1, 2). 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 (3-7). 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 (8-10), 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).The specificity of AID targeting to the Ig gene has been under intense investigation. Studies have shown that AID deamination and mutagenesis targets single-stranded DNA substrates generated during ...

IntroductionChronic lymphocytic leukemia (CLL) follows either an indolent or an aggressive course 1 and clinical decompensation is often accompanied by the appearance of new or increasing numbers of genetic aberrations associated with shorter survival, "clonal evolution." 2 The mechanism(s) responsible for the generation of these genetic abnormalities are not defined in CLL, which is not the case in certain other human cancers, especially lymphoid malignancies of germinal center (GC) origin, in which activation-induced deaminase (AID) appears to be pathogenic. [3][4][5] AID is required for the beneficial generation of Ab diversity in normal B lymphocytes by inducing IGV somatic hypermutation (SHM) and helps in the development of protective effector mechanisms by mediating IGH class-switch recombination (CSR). 6,7 These beneficial on-target AID activities occur primarily during a GC reaction and involve conversion of cytidine to uridine on single-stranded DNA at the IG locus. Such on-target actions in CLL B cells have been a matter of interest for several years, primarily because the presence or absence of IGHV mutations (which require AID) in CLL cells is closely linked to clinical outcome. Patients with leukemic clones with minimal (Ͻ 2% difference from germline) or no mutation in the IGHV (unmutated CLL [U-CLL]) have a far worse prognosis than patients with IGHV-mutated CLL (M-CLL). 8,9 Despite this SHM-based subcategorization of CLL cases, some clones exhibit ongoing IGHV diversification in vivo and in vitro, [10][11][12] with an antigen-driven pattern present in some cases, 13 and up to 50% of patients exhibit molecular evidence for intraclonal isotype CSR. [14][15][16][17][18] AID activity focused elsewhere 19 ) can lead to mutations, deletions, or translocations outside of the IG locus, as in GC-derived lymphomas. [3][4][5] However, such a role for AID in CLL has been questioned for several reasons: (1) although circulating CLL cells can express AID mRNA, [20][21][22] the number of such cells is exceedingly low (0.01%-0.2%) 22 ; (2) AID protein synthesis by these same cells has not been demonstrated 18,[20][21][22][23] ; (3) demonstration of the full range of AID functions is lacking in CLL, for example, by failure of cells to demonstrate SHM, especially for U-CLL clones, even on stimulation and induction of AID mRNA, 21 thereby creating the apparent paradox that U-CLL patients express more AID mRNA than M-CLL patients yet exhibit no or minimal SHM; and (4) despite association with several prognostic markers, 20,21,[24][25][26] no prospective analysis linking AID expression and disease severity has been performed.In the present study, we aimed to address these issues as a means of determining whether AID could be involved in the evolution of CLL to a more aggressive disease. We report that CLL cells are able to produce AID protein, but synthesis is The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and...