Here we have shown that AID initiates non-Igh (off-target) DSBs during the G1 phase of the cell cycle and these breaks can persist until S-phase for repair. We further demonstrate that AID initiated DSBs arise independently of DNA replication. We provide evidence that AID-mediated DNA breaks are generated during G1, but initially avoid activating the G1/S checkpoint, and then become sensed and repaired during S-phase. Interestingly, we find that CSR-initiating DSBs within Igh may, at least under some circumstances, undergo bona fide class switch recombination during S-phase. We propose that these cell cycle dynamics allow for coordination of repair at both Igh and non-Igh locations in cells harboring widespread damage from AID (). This would promote immunoglobulin class switching while simultaneously reducing risk of deleterious chromosomal rearrangements.
Model for cell cycle modulation and coordinated repair of AID initiated DSBs. AID initiates DSBs in G1 phase of the cell cycle. DSBs that fail to resolve can bypass the G1/S checkpoint, and proceed into S-phase for resolution.
There is a growing body of evidence that shows AID induces both point mutations and DSBs at numerous non-Igh
(off-target) locations throughout the genome (23
). While the Igh
locus is recognized as the preferred, physiological target for AID, recent studies have suggested that other, non-Igh
genes may also be targeted by AID for point mutations, albeit at significantly lower frequencies (23
). AID has also been implicated in DSBs at non-Igh
genes, although the precise mechanisms, frequency, and relationship to off-target point mutations has been unresolved (24
). We now show that, in addition to the Igh
locus, AID initiates recurrent DSBs at a multiple other sites throughout the genome. We further demonstrate that homologous recombination defective cells accumulate unrepaired DNA breaks in Igh
and in various non-Igh
sites, demonstrating a role for HR in the resolution of both on- and off-target damage. Because productive class switch recombination is known to require DNA end-joining, we suggest that HR mediates high fidelity repair of AID-induced Igh
DSBs leading to restoration of the original template without class switching. Although XRCC2
mediated HR is known to promote avian immunoglobulin pseudogene conversions, this is the first study to document a role for HR within the Igh
locus in mammalian cells (46
Two possible models for AID-initiated off-target DSBs can be envisaged. First, AID may generate base-pair mismatches that are directly converted into DSBs via processing of the mutated bases. Excision of two closely apposed bases, leading to juxtaposed single strand nicks, would generate a DSB. Alternatively, induction of single strand nicks could produce fragile sites that are converted to frank DSBs by mechanical stress. In either case, AID-mediated DSBs could arise independent of cell cycle stage. The second model, by contrast, suggests that off-target DSBs are replication dependent, arising either by passage of a replication fork over an AID-initiated single strand break (SSB), or by direct attack on the fork itself by AID (22
). By this model, AID-initiated off-target DSBs should largely coincide with sites of active nucleotide incorporation during S-phase. We now demonstrate that AID-dependent off-target DSBs, like those occurring in Igh
, are generated during the G1 phase of the cell cycle and occur largely independently of DNA replication sites. Our findings indicate that off-target DSBs arise by mechanisms analogous to those that induce CSR within the Igh
locus. In this context, non-Igh
DSBs could be considered the products of an ectopic CSR-like process.
This poses an interesting dichotomy. CSR-initiating DSBs within Igh
resolve predominantly in G1 via classical non-homologous end joining (NHEJ) or micro-homology mediated end joining (MMEJ). By contrast, off-target DSBs are preferentially repaired by HR, which is repressed during G1 but active in post-G1 cells (25
). We now find that both off-target DSBs and Igh
breaks are enhanced by HR deficiency (), suggesting a multi-phasic response to AID-initiated DSBs. We speculate that, under normal circumstances, the majority of Igh
S regions DSBs are resolved via end-joining during G1 (15
). However, we show that S region DSBs can
be resolved during S-phase. Together, these findings suggest a cellular fail-safe mechanism — S region DSBs that fail to recombine in G1 can then be recombined later in S-phase. Moreover, we now demonstrate that HR can function in the Igh
locus. We propose that S region DSBs that fail to undergo a switch recombination (in either G1 or S), can be repaired by high-fidelity HR, ensuring cell survival and providing another chance to switch in the next cell cycle.
How some AID-mediated DSBs that arise during G1 evade canonical checkpoint activation and persist until S-phase for repair is not understood. One possibility is that there are too few AID-dependent DSBs to reach a threshold level for checkpoint activation. While we cannot definitively rule out this possibility, previous reports have documented p53 activation in B-cells stimulated by LPS plus anti-δ-dextran, demonstrating checkpoint competency in mature splenic B-cells (40
). Consistent with these, we show that splenic B-cells are acutely sensitive to checkpoint activation by IR-induced DSBs (). Thus, another possibility is that checkpoint initiation by AID-mediated DSBs is context dependent, such that some stimuli (e.g. TLR signaling after LPS exposure) initiate checkpoints, while others (e.g. CD40 signaling) do not. This may be physiologically important, as CD40 signaling is key in germinal center formation, while TLR signaling is not.
Given that we find neither ATM nor p53 to be initially activated by AID-induced damage, Atm
mutant B-cells might be expected to class switch at least as efficiently as wild-type. This seems to be partly true in the case of p53, although the effect of p53 on class switching appears to be mediated by its antioxidant role, rather than its checkpoint function (3
). However, defects in Atm
generally impinge upon CSR, suggesting that, although ATM initially escapes activation by AID-mediated DSBs activation, there are subsequently direct roles for ATM in promoting class switching. The partial inhibition of CSR seen in AT patients further suggests a direct role for ATM in promoting class switching (47
). Taken together, these diverse observations support the conclusion that class switching B-cells mount a multifactorial response to AID-induced DNA damage, which includes checkpoint modulation and dynamic cell cycle regulation.
Why, and under what circumstances, should activated B-cells delay the repair of AID-initiated DSBs until S-phase? One possibility is that, in cells incurring numerous AID-initiated breaks, it is advantageous to delay repair until S-phase to ensure that both homologous and non-homologous modes of repair are available to properly repair appropriate targets. By this model, delayed repair would prevent resolution ofzs off-target, non-Igh DSBs by end-joining mechanisms, which could increase the likelihood of deleterious translocations in cells harboring numerous AID-induced DSBs (). Delaying repair until S-phase would allow homology-mediated repair of off-target DSBs, but preserve the opportunity for CSR by end-joining processes. Another intriguing possibility is that some CSR events may involve S-phase, even in the absence of supernumerary DSBs at non-Igh locations. Previous studies have shown that physiological class switching is cell-division linked, where increased efficiency of class switching correlates with increased number of cellular divisions. Our data now show that proliferation per se is not strictly necessary for B-cells to class switch. Because cells treated with 0.4μM aphidicolin, and therefore enriched in S-phase and prevented from undergoing multiple rounds of cell division, are able to switch as efficiently as untreated wild-type B-cells, we suggest that S-phase transit is sufficient to promote class switch recombination, but that proliferation enhances CSR and amplifies switched subclones within a B-cell population. In this context, both S-phase progression and overall proliferation combine to magnify physiological class switching.
Mechanistically, S-phase may promote long-range recombination, which occurs at an unexpectedly high rate in class switching B-cells (35
). Finally, it is clear that both end-joining and homologous recombination modes of DSB repair are used for different purposes in activated B-cells. A major question is how repair pathway choice is regulated and how specific modes of repair might be spatially directed to particular target sites. While this question is not yet resolved, it is likely that S-phase progression influences the balance between end-joining and homology mediated modes of repair. Repair choice may be particularly relevant in B-cells undergoing CSR. In support of this notion, 53BP1, a key regulator of DNA end resection, modulates both the type of end-joining repair within switch regions, and the likelihood of ATM activation by persistent switch region breaks (47
In summary, we have shown here that AID initiates recurrent DNA double strand breaks at numerous locations throughout the genome, that these DSBs can arise during G1 independently of DNA replication, and that HR preferentially influences off-target DSB repair. This highlights a dichotomous response to Igh versus non-Igh DSBs in activated B-cells. Overall, our findings indicate a tightly regulated and highly orchestrated response to AID-initiated genomic damage that involves both cell cycle regulation and DNA repair pathway choice. Elucidating the detailed mechanisms that link AID, cell cycle dynamics, and DSB repair pathways provides insight into normal adaptive immune development and the etiology of B-cell neoplasms associated with genomic instability.