Proapoptotic Bid mediates the Atr-directed DNA damage response to replicative stress

Abstract

Pro-apoptotic Bid, a BH3-only Bcl-2 family member, is situated at the interface between the DNA damage response and apoptosis, with roles in death receptor-induced apoptosis as well as cell cycle checkpoints following DNA damage(Kamer et al., 2005; Yin et al., 1999; Zinkel et al., 2005). Here we demonstrate that Bid acts at the level of the sensor complex in the Atm and Rad3-related (Atr)-directed DNA damage response. Bid is found with Replication protein A (RPA) in nuclear foci and associates with the Atr/Atr-interacting protein (Atrip)/RPA complex following replicative stress. Furthermore, Bid-deficient cells demonstrate an impaired response to replicative stress manifest by reduced accumulation of Atr and Atrip on chromatin and at DNA damage foci, reduced recovery of DNA synthesis following replicative stress, and decreased Chk1 activation and RPA phosphorylation. These results establish a direct role for the BH3-only Bcl-2 family member, Bid, acting at the level of the damage sensor complex to amplify the Atr-directed cellular response to replicative DNA damage.

Bid protects the mouse hematopoietic system following hydroxyurea-induced replicative stress

“…Thus, the phenotype in IR-stressed Bid À / À bone marrow differs from that observed following long-term replicative stress, consistent with our previous finding of a prominent role for Bid in the Atr-directed DDR. 19 We have previously demonstrated that Bid À / À cells display limited Atr function and increased sensitivity to replicative stress. 19,26 Furthermore, bone marrow of Bid À / À mice displays increased sensitivity to intraperitoneal injection of HU but not to IR, 19 and Bid À / À but not Bid þ / þ MPCs and LSK cells demonstrate increased Annexin V þ cells following HU (Figures 2a and b).…”

“…6 To determine if the increased replating capacity of Bid À / À bone marrow was specific to treatment with HU, Bid þ / þ and Bid À / À mice were irradiated with low dose IR (2 Gy), chosen for a similar decrease in overall bone marrow cellularity in Bid þ / þ mice as observed with HU. 19 Bone marrow from 2 Gy IR-stressed Bid þ / þ and Bid À / À mice was harvested and cultured in methylcellulose. Both Bid þ / þ and Bid À / À bone marrow demonstrate modestly increased replating ability following 2 Gy relative to unstressed bone marrow.…”

“…19,20 Bid is a highly potent activator of intrinsic apoptosis following multiple cellular stresses; [21][22][23][24][25] however, a singular apoptotic function does not account for all of the current data regarding Bid's function. Bid-deficient myeloid progenitor cells (MPCs) display decreased viability in culture.…”

“…Indeed, Bid À / À MPCs and Bid-deficient U2OS cells demonstrate limited Atr function following HU treatment. 19 Furthermore, Bid associates with the Atr/Atrip/ RPA complex, 19 and the association between Atr/Atrip and RPA is significantly diminished in Bid-deficient cells following replicative stress, 19 suggesting that Bid has a role to maintain the DNA damage sensor complex. Bid is thus positioned to have a role in the DDR response to replicative stress in the hematopoietic system.…”

“…19,20 Cultured Bid À / À activated T cells and myeloid progenitor cells are hypersensitive to HU but not IR treatment in vitro. 26 Bid À / À bone marrow is hypersensitive to intraperitoneal injection of HU but not to IR in vivo, 19 consistent with an impaired DDR to replicative stress. Indeed, Bid À / À MPCs and Bid-deficient U2OS cells demonstrate limited Atr function following HU treatment.…”