Generation and repair of AID-initiated DNA lesions in B lymphocytes

Eder

Elos

et al. 2016

Self Cite

Activation-induced deaminase (AID) functions by deaminating cytosines and causing U:G mismatches, a rate-limiting step of antibody gene diversification. However, precise mechanisms regulating AID-deamination frequency remain incompletely understood. Moreover, it is unknown whether different sequence contexts influence the preferential access of mismatch repair (MMR) or uracil glycosylase (UNG) to AID-initiated U:G mismatches. Here, we employed two knock-in models to directly compare the mutability of core Sµ and VDJ exon sequences and their ability to regulate AID-deamination and subsequent repair process. We find that S region is a much more efficient AID deamination target than V region. Igh locus AID-initiated lesions are processed by error-free and error-prone repair. S region U:G mismatches are preferentially accessed by UNG, leading to more UNG-dependent deletions, enhanced by MMR deficiency. V region mutation hotspots are largely determined by AID deamination. Recurrent and conserved S region motifs potentially function as spacers between AID-deamination hotspots. We conclude that the pattern of mutation hotspots and DNA break generation is influenced by sequence-intrinsic properties, which regulate AID deamination and affect the preferential access of downstream repair. Our studies reveal an evolutionarily conserved role for substrate sequences in regulating antibody gene diversity and AID targeting specificity.

Section: Introductionmentioning

confidence: 99%

Interplay between Target Sequences and Repair Pathways Determines Distinct Outcomes of AID-Initiated Lesions

Eder

Elos

et al. 2016

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“…Cγ, Cε, and Cα), respectively (1). The essential molecular components of CSR include: (1) active germline transcription of C H genes that renders a given C region accessible for recombination (1, 3, 4); (2) switch (S) regions that are highly repetitive and specific DNA sequences, located 5’ of each set of C H exons except Cδ (5); (3) activation induced deaminase (AID) that deaminates cytosine (C) and converts it into uracil (U), thereby resulting in U:G mismatch; (4) subsequent recognition and processing of the AID-initiated U:G mismatch by mismatch repair (MMR) and base excision repair (BER) pathways that generate DNA double strand breaks (DSBs) in the upstream donor Sμ and a downstream acceptor S region (6, 7); (5) repair of the AID-initiated DSBs via non-homologous end-joining (NHEJ) that eventually completes CSR via re-joining the two broken S regions (8, 9). Both classical and alternative NHEJ contribute to the repair of S region DSBs (8, 9).…”

Section: Introductionmentioning

confidence: 99%

Imbalanced PTEN and PI3K Signaling Impairs Class Switch Recombination

Getahun

et al. 2015

Class switch recombination (CSR) generates isotype-switched antibodies with distinct effector functions. B cells express phosphatase and tensin homolog (PTEN) and multiple isoforms of class IA phosphoinositide 3-kinase (PI3K) catalytic subunits, including p110α and p110δ, whose roles in CSR remain unknown or controversial. Here, we demonstrate a direct effect of PTEN on CSR signaling by acute deletion of Pten specifically in mature B cells, thereby excluding the developmental impact of Pten deletion. We show that mature B cell-specific PTEN overexpression enhances CSR. More importantly, we establish a critical role of p110α in CSR. Furthermore, we identify a cooperative role of p110α and p110δ in suppressing CSR. Mechanistically, dysregulation of p110α or PTEN reversely affects activation-induced deaminase expression via modulating AKT activity. Thus, our study reveals that a signaling balance between PTEN and PI3K isoforms is essential to maintain normal CSR.

“…In the previous model, we found that the inserted Sγ1 region mutated at a 10 fold higher frequency than the intronic sequence of Bcl6 (39). While S region sequences are indeed optimal targets of AID in the context of the Igh locus (18, 30), we propose that the capacity of such sequences to increase the frequency of mutations or DSBs at a given locus is dependent on the “genomic context” of the particular locus. However, genomic context represents a broad concept; to define its specific influence on AID-mediated mutagenesis, we propose that locus-specific cis regulatory elements probably account for the disparity between c-myc and Bcl6 loci in terms of AID targeting.…”

Section: Discussionmentioning

confidence: 91%

AID-Initiated DNA Lesions Are Differentially Processed in Distinct B Cell Populations

Ranganath

Viboolsittiseri

et al. 2014

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Activation-induced deaminase (AID) initiates U:G mismatches, causing point mutations or DNA double-stranded breaks at immunoglobulin (Ig) loci. How AID-initiated lesions are prevented from inducing genome-wide damage remains elusive. Differential DNA repair mechanism might protect certain non-Ig loci such as c-myc from AID attack. However, determinants regulating such protective mechanisms are largely unknown. To test whether target DNA sequences modulate protective mechanisms via altering the processing manner of AID-initiated lesions, we established a knock-in model by inserting an Sγ2b region, a bona fide AID target, into the first intron of c-myc. Unexpectedly, we found that the inserted S region did not mutate or enhance c-myc genomic instability, due to error-free repair of AID-initiated lesions, in antigen-stimulated germinal center (GC) B cells. In contrast, in vitro cytokine-activated B cells display a much higher level of c-myc genomic instability in an AID- and S region-dependent manner. Furthermore, we observe a comparable frequency of AID deamination events between the c-myc intronic sequence and inserted S region in different B cell populations, demonstrating a similar frequency of AID targeting. Thus, our study reveals a clear difference between GC and cytokine-activated B cells in their ability to develop genomic instability, attributable to a differential processing of AID-initiated lesions in distinct B cell populations. We propose that locus-specific regulatory mechanisms (e.g. transcription) appear to not only override the effects of S region sequence on AID targeting frequency but also influence the repair manner of AID-initiated lesions.