Trends in Immunology
ReviewBalancing AID and DNA repair during somatic hypermutation
Introduction
Somatic hypermutation (SHM) occurs primarily in germinal center B cells and is the driving force for antibody affinity maturation (see Glossary). It introduces point mutations into the variable regions of immunoglobulin (Ig) genes at a rate of ∼1 mutation per 1,000 bp per cell generation, almost a million fold higher than the spontaneous mutation rate in somatic cells. C-G and A-T base pairs are mutated at roughly equal frequencies with certain ‘hotspot’ DNA motifs, RGYW and WA, in addition to their reverse complements WRCY and TW (where R=A or G, Y=C or T, W=A or T), being preferentially targeted. Although it has long been proposed that SHM involves two steps – the generation of a DNA lesion followed by its error-prone processing – it was not until the discovery of activation induced deaminase (AID) in late 1990s 1, 2, 3 that substantial progress has been made in elucidating the molecular mechanism of the reaction. It is now believed by most investigators that SHM and two other antibody diversification processes, namely class switch recombination (CSR) and Ig gene conversion (GCV), is initiated by cytosine deamination of Ig genes by AID. AID acts only on single-stranded DNA, which is probably generated during transcription in the form of transcription bubbles (Figure 1). Mutations are thought to arise from the resulting uracils by several different mechanisms (Figure 1). DNA replication across the uracil leads to C to T or G to A transition mutations. Alternatively, removal of the uracil by uracil-DNA glycosylase (UNG) generates an abasic site in the DNA, which when replicated across yields transition and transversion mutations at the C-G bp. The U-G mismatch can also be recognized by the mismatch repair heterodimer Msh2/Msh6, which is thought to trigger the excision and error-prone resynthesis of a short stretch of DNA, thus spreading the mutations to surrounding A-T bp. Error-prone DNA polymerase Polη has an important role in creating A-T mutations. In Msh2/Ung or Msh6/Ung double knockout (dKO) mice, mutations are exclusively C to T or G to A transition mutations, which presumably reveal the full extent of the action of AID (for excellent reviews, see Refs. 4, 5, 6).
Ung and Msh2/Msh6 are ubiquitously expressed factors whose primary function is as components of high fidelity base excision repair (BER) and mismatch repair (MMR) pathways that safeguard the genome from mutagenesis 7, 8. For example, uracils are generated in DNA during normal cell growth by deamination of cytosines and misincorporation of uracil in place of thymidine during DNA replication. Such events are quite frequent and are repaired with high accuracy by BER processes [9]. The mechanisms that allow Ung and Msh2/Msh6 to function in error-prone DNA repair during SHM in B cells while engaging in high-fidelity DNA repair in other cells are unknown. It is unlikely that error-prone repair during SHM is simply because of overloading of the high-fidelity repair systems, as discussed later.
How SHM is preferentially targeted to the Ig loci is poorly understood (Box 1 and Box 2) [10]. The reaction is tightly linked to gene transcription, although not all transcribed genes are mutated 11, 12. SHM represents a major risk to B-cell genomic stability with the potential to generate DNA lesions linked to large-scale chromosomal alterations and to cause deleterious point mutations 13, 14. An attractive mechanism to minimize this risk is careful targeting of AID. However, our recent study demonstrated that AID deaminates many more genes than previously thought (including numerous oncogenes implicated in B-cell tumorigenesis), with the majority of AID-generated uracils being corrected by gene-specific high-fidelity DNA repair (Figure 2) [11]. These findings reveal an additional layer of regulation of SHM, suggest new hypotheses for how the reaction is targeted in normal B cells and mistargeted in cancer and provide fresh interpretations of some previous findings in the field.
Section snippets
How widely do mutations occur as a consequence of SHM?
In the last decade, several non-Ig genes (BCL6, CD79A, CD79B and FAS) were demonstrated to undergo SHM in normal germinal center B cells [10], and four proto-oncogenes, RHOH, PIM1, MYC and PAX5, were shown to mutate in diffuse large B-cell lymphomas, but importantly, not in normal germinal center B cells [15]. Furthermore, when an AID-expressing B-cell line was infected with a retroviral construct bearing no Ig gene-derived sequences, the construct could mutate in many different locations in
How widely is AID targeted?
To distinguish between these two possibilities, we sequenced 83 transcribed non-Ig genes from Ung/Msh2 dKO germinal center B cells. As mentioned previously, the distinct pattern of C to T and G to A transition mutations in these cells is thought to reflect all of the deamination events catalyzed by AID. Remarkably, more than half of the transcribed genes analyzed were detectably deaminated by AID, as indicated by their mutation frequencies, mutation spectra and hotspot focusing, in addition to
An additional level of protection for the B-cell genome by high-fidelity repair
Interestingly, the genes most frequently hit by AID (as assessed in Ung/Msh2 dKO B cells) are not necessarily those with the highest number of mutations in WT B cells. For example, the proto-oncogene Myc, the mutation frequency of which in WT cells is only slightly above background, is hit by AID almost as frequently as Bcl6 (which is itself hit only an order of magnitude less than the Ig loci). The tumor suppressor gene H2afx, the mutation frequency of which in WT cells is not different from
How is differential repair achieved?
It is not known how error-prone repair is accomplished during SHM of Ig loci, although it probably involves the recruitment of error-prone polymerases (such as REV1 and Polη) and perhaps the inhibition or exclusion of higher fidelity polymerases (such as Polβ) (for a review see Ref. [17]). However, our results demonstrate that high-fidelity repair is not shut down throughout the B-cell nucleus, as Myc, H2afx, and numerous other genes are efficiently protected by the system. This is consistent
New ways of thinking about B-cell tumorigenesis
AID has been strongly implicated in the development of B-cell tumors [14]. DNA breaks in Ig V regions and switch regions associated with SHM and CSR, respectively, underlie large-scale chromosomal translocations, and the ability of AID to mutate oncogenes and tumor suppressors is also thought to contribute to B-cell tumorigenesis. The findings that several oncogenes are physiological targets of AID and that most deamination events in those genes are erased by high-fidelity repair have important
Is the high-fidelity repair system that protects Myc, H2afx, etc. B-cell specific?
During SHM in germinal center B cells, Ig genes employ an error-prone repair machinery to facilitate diversity rather than accuracy. In the same cells, however, some loci (Myc, H2afx, etc.) are protected by gene-specific high-fidelity repair. An interesting question arises: is high-fidelity repair of AID-generated uracils B-cell specific? Answering this question might help explain the interesting observation that mice containing a ubiquitously expressed AID transgene frequently develop T-cell
Could SHM of non-Ig genes be beneficial to the host?
The ability of AID to mutate non-Ig genes, including oncogenes, apparently has a negative outcome for the host because it leads to tumorigenesis. Interestingly, a recent study indicates that this ability might also be beneficial to the host as an anti-viral and anti-tumor mechanism [54]. In this study, infection of bone marrow-derived pre-B cells by Abelson-murine leukemia virus (AMuLV) induced AID expression and mutations in Ig and non-Ig genes. Interestingly, compared to AID−/− controls, WT
Conclusions
It was widely assumed that the targeting of SHM was achieved solely through the specific targeting of AID. New findings demonstrate that there is another level of regulation of SHM: gene-specific error-prone or high-fidelity DNA repair. Understanding how both levels of targeting are achieved will be crucial to understanding the regulation of SHM. Because some of the physiological targets of AID are oncogenes, it is extremely important to understand what regulates gene-specific DNA repair and
Acknowledgements
The authors would like to thank S. Unniraman for many helpful suggestions and insightful ideas over the years, and M. Nussenzweig and J. M. Buerstedde for sharing data before publication. D.G.S. is an investigator of the Howard Hughes Medical Institute.
Glossary
- Affinity maturation
- some SHM-generated mutations give rise to antibodies with increased affinity for antigen. B cells bearing such mutations preferentially survive, divide and undergo further rounds of mutation and selection. This process, which results in B cells capable of producing much higher affinity antibodies, is called affinity maturation.
- Base excision repair
- a DNA repair system that recognizes and repairs damaged or inappropriate DNA bases by a mechanism that involves base removal,
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2020, HeliyonCitation Excerpt :Low-fidelity cellular responses to the presence of dU, including the mismatch repair (MMR) and the base-excision repair (BER) pathways, then are thought to introduce mutations in SHM and CSR, as well as cause double-strand breaks (DSBs) repaired through non-homologous end-joining (NHEJ) and alternative end-joining (A-EJ) in CSR (Chaudhuri and Alt, 2004; Gearhart and Wood, 2001; Goodman et al., 2007). Though necessary for proper physiological function, these lymphocytic diversification processes are rife with danger for B cells; strong selective pressure exists to orchestrate and target them carefully so as not to threaten the genomic integrity of the cells through breaks or other mutations at non-Ig loci (Casellas et al., 2016; Liu et al., 2008; Liu and Schatz, 2009). Yet these deleterious events can nevertheless occur, as demonstrated by the involvement of AID in the mutagenic events at Ig and non-Ig loci that result in oncogenic translocations.
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