ReviewAIDing antibody diversity by error-prone mismatch repair
Highlights
► The DNA mutator AID catalyzes U:G mismatches recognized by mismatch repair (MMR). ► MMR generally fixes DNA mismatches faithfully, except at the immunoglobulin genes. ► MMR differentially promotes A:T mutagenesis at V regions and DSB at S regions. ► The MMR sensor complex MutSα (MSH2/MSH6) promotes both SHM and CSR. ► The MMR adaptor complex MutLα (MLH1/PMS2) has no role in SHM but promotes CSR.
Introduction
Organisms at every level of evolutionary development are constantly exposed to genotoxic stress that can damage and alter the structure of their genetic material. To counteract this threat, a large variety of highly conserved repair mechanisms have been generated throughout evolution [1]. It is estimated that 2–4% of the genes in mammalian cells are devoted to the repair of DNA damage and these are tightly integrated through various signal transduction pathways with the regulation of the cell cycle and cell death [2]. Paradoxically, the generation and propagation of “useful” mutations could potentially confer an evolutionary advantage [3]. Furthermore, in complex organisms it is sometimes important to somatically produce increased levels of genetic diversity [4], [5].
Perhaps the most extreme example of engineering and tightly regulating genomic instability for a selective advantage is the adaptive immune response in higher organisms, where it is essential to spawn an enormous repertoire of antigen binding sites in B and T cell antigen receptors. This is achieved by the combinatorial rearrangement of a small number of variable (V), diversity (D) and joining (J) genes so that the organism can mount a protective immune response against most foreign antigens that it encounters [6]. B cells need to generate antibodies of high affinity to neutralize and inactivate pathogenic agents in the blood stream, in tissues and even in mucosal spaces. To achieve this V(D)J regions that encode low affinity antibodies are somatically hypermutated (SHM) so that they achieve the high affinities required to neutralize toxic foreign agents [7], [8]. In order to distribute these protective antibodies throughout the body and enable them to carry out different effector functions, it is also necessary to mutate the switch (S) regions that are upstream from each of the constant region genes and to convert those mutations into double stranded DNA breaks (DSBs). This allows the heavy chain V(D)J regions encoding the antigen-binding site to be rearranged from the μ constant region to the downstream γ, ɛ, and α constant regions through a process, termed class switch recombination (CSR) [9], [10].
In B cells, a potent mutator known as activation-induced deaminase (AID), initiates SHM of the Ig V(D)J and CSR of the Ig S regions by deaminating Cs and generating U:G DNA mismatches at a very high frequency. This occurs primarily in the activated B cells in the germinal centers of secondary lymphoid organs, such as the lymph nodes, spleen and tonsils where AID is expressed at very high levels. In those germinal center B cells, AID induced mutations recruit base excision (BER) and mismatch repair (MMR) processes that in other cell types and at non-Ig genes repair DNA lesions with high fidelity [11], [12]. However, in B cells, the BER and MMR that are recruited by AID-induced U:G mismatches facilitate an error-prone repair of these mismatches, and MMR is responsible for as many as half of the mutations that arise during SHM and for most of the mutations that occur at A:T bases [13]. While a great deal is known from biochemical and yeast studies about the role of MMR proteins in the normal process of high fidelity MMR, less is known about how these factors are targeted to particular mismatches in vivo and to the regulation of the repair process in different lineages of mammalian cells.
In this review we will describe the orchestration of MMR-mediated error-prone repair in both antibody V(D)J and S regions following the enzymatic production of DNA mismatches by AID. We will also point out the many unresolved features of this atypical and potentially dangerous repair process. Through comparison of the complete loss and separation-of-function mutants in the MMR proteins in vivo and the detailed examination of the outcome of this process as it is reflected in the sequences of individual antibody V(D)J and S regions, we are learning new aspects of how error-prone MMR generates antibody diversity in B cells. We are also gaining new insights into how high fidelity MMR is regulated in general and how its misregulation can lead to tumorigenesis.
Section snippets
AID-mediated cytosine deamination instigates a highly mutagenic cascade
Because it is extremely mutagenic, highly expressed in centroblast B cells, and generates mutations characteristic of antibody V and S regions, it was originally thought that AID was a B cell specific deaminase primarily devoted to the generation of antibody diversity [8]. However, subsequent studies revealed that many other genes were mutated in activated B cells some of which were repaired with high fidelity while others were also subjected to error-prone repair [14], [15] (see Saribasak and
MMR mediates the resection of ssDNA patches and the introduction of A:T mutations
The current model (Fig. 1) suggests, the U:G mismatch that is generated when AID mutates an Ig V(D)J region is either replicated over to produce a U → T mutation, recognized by UNG to initiate a sometimes error-prone BER (see also Saribasak and Gearhart, in this issue), or recognized by the MutSα MMR heterodimer composed of the MSH2 and MSH6 proteins (Fig. 1). This MMR-mediated sensing of the U:G lesion initiates a series of processes that are responsible for the mutations at surrounding A:T
MMR promotes DSB formation to facilitate class switching
The mechanisms by which the signaling cascade downstream of AID diverges so as to independently mediate SHM or CSR remains puzzling. One clear distinction is that DSBs are required to initiate CSR [10], [71] (Fig. 2), yet exactly how the DSB is initiated continues to be a matter of debate. Much like SHM, ssDNAs are often created as an intermediary step following AID-mediated U:G mismatches [72]. But instead of creating single point mutations - directly or through the recruitment of MMR, PCNA
Summary and future perspectives
While an enormous amount has been learned in the last decade about the biochemical, molecular and genetic mechanisms responsible for the generation of antibody diversity, there is still much to be uncovered. One issue is that all of the molecular mechanisms of affinity maturation, have in fact been hijacked and in various ways perverted from normal mechanisms that repair or modify DNA. AID may have evolved as a cytosine deaminase whose role was to modify the epigenetic landscape by triggering
Acknowledgments
This work was supported by the NIH Grants CA72649 and CA102705 (to M.D.S.) and CA76329 and CA93484 (to W.E.). M.D.S. is supported by the Harry Eagle Chair, provided by the National Women's Division of the Albert Einstein College of Medicine. S.R. is supported by the PTQ-11-04774 Grant from the Spanish Ministerio de Economía y Competitividad.
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