Nbs1 ChIP-Seq Identifies Off-Target DNA Double-Strand Breaks Induced by AID in Activated Splenic B Cells

Activation-induced cytidine deaminase (AID) is required for initiation of Ig class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs) involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP) for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq). We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID.     

Activation-Induced Cytidine Deaminase-Initiated Off-Target DNA Breaks Are Detected and Resolved during S Phase

Activation-induced cytidine deaminase (AID) initiates DNA double-strand breaks (DSBs) in the IgH gene (Igh) to stimulate isotype class switch recombination (CSR), and widespread breaks in non-Igh (off-target) loci throughout the genome. Because the DSBs that initiate class switching occur during the G(1) phase of the cell cycle, and are repaired via end joining, CSR is considered a predominantly G(1) reaction. By contrast, AID-induced non-Igh DSBs are repaired by homologous recombination. Although little is known about the connection between the cell cycle and either induction or resolution of AID-mediated non-Igh DSBs, their repair by homologous recombination implicates post-G(1) phases. Coordination of DNA breakage and repair during the cell cycle is critical to promote normal class switching and prevent genomic instability. To understand how AID-mediated events are regulated through the cell cycle, we have investigated G(1)-to-S control in AID-dependent genome-wide DSBs. We find that AID-mediated off-target DSBs, like those induced in the Igh locus, are generated during G(1). These data suggest that AID-mediated DSBs can evade G(1)/S checkpoint activation and persist beyond G(1), becoming resolved during S phase. Interestingly, DSB resolution during S phase can promote not only non-Igh break repair, but also Ig CSR. Our results reveal novel cell cycle dynamics in response to AID-initiated DSBs, and suggest that the regulation of the repair of these DSBs through the cell cycle may ensure proper class switching while preventing AID-induced genomic instability.     

Recombinogenic phenotype of human activation-induced cytosine deaminase

Class switch recombination, gene conversion, and somatic hypermutation that diversify rearranged Ig genes to produce various classes of high affinity Abs are dependent on the enzyme activation-induced cytosine deaminase (AID). Evidence suggests that somatic hypermutation is due to error-prone DNA repair that is initiated by AID-mediated deamination of cytosine in DNA, whereas the mechanism by which AID controls recombination remains to be elucidated. In this study, using a yeast model system, we have observed AID-dependent recombination. Expression of human AID in wild-type yeast is mutagenic for G-C to A-T transitions, and as expected, this mutagenesis is increased upon inactivation of uracil-DNA glycosylase. AID expression also strongly induces intragenic mitotic recombination, but only in a strain possessing uracil-DNA glycosylase. Thus, the initial step of base excision repair is required for AID-dependent recombination and is a branch point for either hypermutagenesis or recombination.     

Interplays between ATM/Tel1 and ATR/Mec1 in sensing and signaling DNA double-strand breaks

DNA double-strand breaks (DSBs) are highly hazardous for genome integrity because they have the potential to cause mutations, chromosomal rearrangements and genomic instability. The cellular response to DSBs is orchestrated by signal transduction pathways, known as DNA damage checkpoints, which are conserved from yeasts to humans. These pathways can sense DNA damage and transduce this information to specific cellular targets, which in turn regulate cell cycle transitions and DNA repair. The mammalian protein kinases ATM and ATR, as well as their budding yeast corresponding orthologs Tel1 and Mec1, act as master regulators of the checkpoint response to DSBs. Here, we review the early steps of DSB processing and the role of DNA-end structures in activating ATM/Tel1 and ATR/Mec1 in an orderly and reciprocal manner.