DNA repair and antibody diversification: the 53BP1 paradigm.

The DNA double-strand break (DSB) repair factor 53BP1 has long been implicated in V(D)J and class switch recombination (CSR) of mammalian lymphocyte receptors. However, the dissection of the underlying molecular activities is hampered by a paucity of studies [V(D)J] and plurality of phenotypes (CSR) associated with 53BP1 deficiency. Here, we revisit the currently accepted roles of 53BP1 in antibody diversification in view of the recent identification of its downstream effectors in DSB protection and latest advances in genome architecture. We propose that, in addition to end protection, 53BP1-mediated end-tethering stabilization is essential for CSR. Furthermore, we support a pre-DSB role during V(D)J recombination. Our perspective underscores the importance of evaluating repair of DSBs in relation to their dynamic architectural contexts.

Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region.

RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular feature that enables the genome-protective function of RIF1 during DNA replication stress.

Charting a DNA Repair Roadmap for Immunoglobulin Class Switch Recombination.

Immunoglobulin (Ig) class switch recombination (CSR) is the process occurring in mature B cells that diversifies the effector component of antibody responses. CSR is initiated by the activity of the B cell-specific enzyme activation-induced cytidine deaminase (AID), which leads to the formation of programmed DNA double-strand breaks (DSBs) at the Ig heavy chain (Igh) locus. Mature B cells use a multilayered and complex regulatory framework to ensure that AID-induced DNA breaks are channeled into productive repair reactions leading to CSR, and to avoid aberrant repair events causing lymphomagenic chromosomal translocations. Here, we review the DNA repair pathways acting on AID-induced DSBs and their functional interplay, with a particular focus on the latest developments in their molecular composition and mechanistic regulation.

53BP1 Supports Immunoglobulin Class Switch Recombination Independently of Its DNA Double-Strand Break End Protection Function.

Class switch recombination (CSR) is a DNA recombination reaction that diversifies the effector functions of antibodies. CSR occurs via the formation and non-homologous end joining (NHEJ) repair of programmed DNA double-strand breaks (DSBs) at the immunoglobulin heavy chain locus. The DNA repair factors 53BP1 and Rif1 promote NHEJ and CSR by protecting DSBs against resection. However, to what extent repression of DNA end resection contributes to CSR is unknown. Here, we show that B lymphocytes devoid of 53BP1-Rif1-dependent DSB end protection activity undergo robust CSR. Inactivation of specific sets of phospho-sites within 53BP1 N-terminal SQ/TQ motifs abrogates Rif1 recruitment and inhibition of resection but only mildly reduces CSR. Furthermore, mutations within 53BP1 oligomerization domain abolish CSR without substantially affecting DNA end processing. Thus, inhibition of DNA end resection does not correlate with CSR efficiency, indicating that regulation of DSB processing is not a key determinant step in CSR.

Prevention of radiation-induced bystander effects by agents that inactivate cell-free chromatin released from irradiated dying cells.

Radiation-induced bystander effect (RIBE) is a poorly understood phenomenon wherein non-targeted cells exhibit effects of radiation. We have reported that cell-free chromatin (cfCh) particles that are released from dying cells can integrate into genomes of surrounding healthy cells to induce DNA damage and inflammation. This raised the possibility that RIBE might be induced by cfCh released from irradiated dying cells. When conditioned media from BrdU-labeled irradiated cells were passed through filters of pore size 0.22 µm and incubated with unexposed cells, BrdU-labeled cfCh particles could be seen to readily enter their nuclei to activate H2AX, active Caspase-3, NFκB, and IL-6. A direct relationship was observed with respect to activation of RIBE biomarkers and radiation dose in the range of 0.1-0 Gy. We confirmed by FISH and cytogenetic analysis that cfCh had stably integrated into chromosomes of bystander cells and had led to extensive chromosomal instability. The above RIBE effects could be abrogated when conditioned media were pre-treated with agents that inactivate cfCh, namely, anti-histone antibody complexed nanoparticles (CNPs), DNase I and a novel DNA degrading agent Resveratrol-copper (R-Cu). Lower hemi-body irradiation with γ-rays (0.1-50 Gy) led to activation of H2AX, active Caspase-3, NFκB, and IL-6 in brain cells in a dose-dependent manner. Activation of these RIBE biomarkers could be abrogated by concurrent treatment with CNPs, DNase I and R-Cu indicating that activation of RIBE was not due to radiation scatter to the brain. RIBE activation was seen even when mini-beam radiation was delivered to the umbilical region of mice wherein radiation scatter to brain was negligible and could be abrogated by cfCh inactivating agents. These results indicate that cfCh released from radiation-induced dying cells are activators of RIBE and that it can be prevented by treatment with appropriate cfCh inactivating agents.