Abstract
The 53BP1-dependent end-joining pathway plays a critical role in double strand break repair and is uniquely responsible for cellular sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPi) in BRCA1-deficient cancers. We and others have investigated the downstream effectors of 53BP1, including replication timing regulatory factor 1 (RIF1) and Pax transactivation domain-interacting protein (PTIP), in the past few years to elucidate how loss of the 53BP1-dependent repair pathway results in PARPi resistance in BRCA1 patients. However, questions regarding the upstream regulation of the 53BP1 pathway remain unanswered. In this study, we identified the Tudor-interacting repair regulator (TIRR) that specifically associates with the ionizing radiation-induced foci formation region of 53BP1. 53BP1 and TIRR form a stable complex, which is required for their expression. Moreover, the 53BP1-TIRR complex dissociates after DNA damage, and this dissociation may be ataxia telangiectasia mutated-dependent. Similar to 53BP1, loss of TIRR restores PARPi resistance in BRCA1-deficient cells. Collectively, our data identified a novel 53BP1-TIRR complex in DNA damage response. TIRR may play both positive and negative roles in 53BP1 regulation. On the one hand, it stabilizes 53BP1 and thus positively regulates 53BP1. On the other hand, its association with 53BP1 prevents 53BP1 localization to sites of DNA damage, and thus TIRR is also an inhibitor of 53BP1.
Highlights
The 53BP1 anti-HA (53BP1)-dependent end-joining pathway plays a critical role in double strand break repair and is uniquely responsible for cellular sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPi) in BRCA1-deficient cancers
We identified the Tudor-interacting repair regulator (TIRR) that associates with the ionizing radiation-induced foci formation region of 53BP1. 53BP1 and TIRR form a stable complex, which is required for their expression
We and others have shown that a central region containing the Tudor domain and ubiquitination-dependent recruitment (UDR) motif of 53BP1 is required for the accumulation of 53BP1 at sites of DNA breaks [7, 8], which we named the ionizing radiation-induced foci (IRIF) region (Fig. 1A)
Summary
We and others have shown that a central region containing the Tudor domain and ubiquitination-dependent recruitment (UDR) motif of 53BP1 is required for the accumulation of 53BP1 at sites of DNA breaks [7, 8], which we named the IRIF region (Fig. 1A). To verify that TIRR associates with 53BP1, we repeated tandem affinity purification using 293T cell lines stably expressing SFB-tagged TIRR and identified 53BP1 as a TIRR-associated protein (Fig. 1D). To further confirm this interaction, we generated anti-TIRR antibodies and showed that endogenous TIRR strongly associates with 53BP1 (Fig. 1E). Using a bacterially expressed and purified GST-fused IRIF region of 53BP1 protein, we showed that the 53BP1 IRIF region binds to TIRR in vitro (Fig. 1F), indicating that the IRIF region of 53BP1 is sufficient for its binding to TIRR. We observed that 53BP1 D1521R mutant dramatically reduced 53BP1-TIRR interaction, whereas 53BP1 L1619A mutant did not notably affect its binding to TIRR
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