Cell cycle-dependent inhibition of 53BP1 signaling by BRCA1

Abstract

DNA damage response mediator protein 53BP1 is a key regulator of non-homologous end-joining (NHEJ) repair. 53BP1 protects DNA broken ends from resection by recruiting two downstream factors, RIF1 (RAP1-interacting factor 1) and PTIP (Pax transactivation domain-interacting protein), to double-stranded breaks (DSBs) via ATM (ataxia telangiectasia mutated)-mediated 53BP1 phosphorylation, and competes with BRCA1-mediated homologous recombination (HR) repair in G1 phase. In contrast, BRCA1 antagonizes 53BP1-direct NHEJ repair in S/G2 phases. We and others have found that BRCA1 prevents the translocation of RIF1 to DSBs in S/G2 phases; however, the underlying mechanism remains unclear. Here we show that efficient ATM-dependent 53BP1 phosphorylation is restricted to the G1 phase of the cell cycle, as a consequence RIF1 and PTIP accumulation at DSB sites only occur in G1 phase. Mechanistically, both BRCT and RING domains of BRCA1 are required for the inhibition of 53BP1 phosphorylation in S and G2 phases. Thus, our findings reveal how BRCA1 antagonizes 53BP1 signaling to ensure that HR repair is the dominant repair pathway in S/G2 phases.

Keywords: 53BP1; ATM; BRCA1; DNA repair choice; PTIP; RIF1; cell cycle; homologous recombination.

Figure 1

Cell cycle-dependent PTIP (Pax transactivation domain-interacting protein) foci formation. (a) Asynchronous HeLa cells were treated with γ-irradiation (ionizing radiation (IR), 10 Gy) and recovered for 4 h before fixation and stained with antibodies as indicated. Asterisks indicate PTIP foci-negative cells. (b) Asynchronous HeLa cells were treated as in a and co-stained with S phase marker, cyclin A. G1 phase cells were indicated. (c) Representative images showing PTIP, RIF1 (RAP1-interacting factor 1) and BRCA1 foci. HeLa cells were enriched in G1 phase by treating with nocodazole and then released for 6 h. S/G2 phase cells were enriched by double-thymidine block and released for 6 h. Two  hours before fixation, cells were irradiated with 10 Gy of IR. (d) A histogram showing the percentage of cells containing >10 nuclear foci. Experiments were performed in triplicate and at least 100 cells were counted in each experiment for each treatment.

Figure 2

Cell cycle-dependent 53BP1 phosphorylation by ATM (ataxia telangiectasia mutated). (a) Asynchronous HeLa cells were treated as described in Figure 1a, arrows indicate p-53BP1 foci-negative cells. Cells with >10 foci following ionizing radiation (IR) were counted as positive cells. (b) HeLa cells were processed as in a, and the asterisks indicate BRCA1 or p-53BP1 foci-negative cells. The percentage of asynchronous cells forming indicated foci were as follows: S25/S29 (58%), S1778 (60%), 53BP1 (98%), BRCA1 (65%), RPA2 (52%) and p-ATM (94%), at least 200 cells were counted. (c) Summary of cell cycle-dependent foci formation of BRCA1 and 53BP1 complexes. (d) HeLa cells were arrested in M phase with nocodazole (100 ng ml⁻¹) treatment for 16 h and then released for different time periods. The cells were irradiated with 10 Gy of IR 1 h or left untreated before harvesting.

Figure 3

BRCA1 inhibits 53BP1 phosphorylation. (a) BRCA1 deficiency restored PTIP (Pax transactivation domain-interacting protein) and p-53BP1 foci formation in S/G2 phase cells. HeLa cells infected with lentivirus carrying non-target or BRCA1 short hairpin RNAs were synchronized in S/G2 and irradiated as described in Figure 1c. (b) Quantification of 53BP1 complex foci formation, the data are represented as the mean±s.e. (n=3). More than 100 cells were counted to determine the percentages of foci forming cells in each sample. (c) Depletion of BRCA1 increases 53BP1 phosphorylation by ATM (ataxia telangiectasia mutated). Control and BRCA1 knockdown cells were synchronized in S/G2 phases as described in Figure 1c. Cells were mocked-treated or treated with ionizing radiation. One hour after ionizing radiation, cells were harvested, and the cell lysates were analyzed by immunoblotting using the indicated antibodies. (d) ATM kinase assays were performed with anti-ATM immunoprecipitates prepared from control (Con.) or BRCA1-depleted cells. ATM kinase was incubated with the purified GST-53BP1 fusion protein (1–910 aa) for the times as indicated. The phosphospecific antibody against phospho-Ser25 and Ser29 of 53BP1 was used for western blotting to detect phosphorylated 53BP1. IR, ionizing radiation; shRNA, short hairpin RNA.

Figure 4

The E3 ubiquitin ligase activity of BRCA1 is required for inhibition of 53BP1 signaling. (a) 293T cells were untreated or irradiated at 10 Gy and processed 1h later. Cell lysates were prepared and immunoprecipitated (IP) using rabbit immunoglobulin G (IgG) or anti-53BP1 antibody. (b) BRCA1 ubiquitinates 53BP1 in vivo. 293T cells were transfected with His-ubiquitin (Ub), SFB-BRCA1/BARD1 and HA-53BP1. The pull-down and immunoblotting analysis was conducted as indicated. (c) HeLa cells infected with lentivirus carrying control or BRCA1 short hairpin RNAs were irradiated or untreated. Total cell lysates were prepared. Immunoprecipitation using high-salt buffer was performed with normal rabbit IgG or anti-53BP1 antibody. Endogenous 53BP1 ubiquitination was detected with anti-Ub antibody (see Materials and Methods for details). (d) BRCA1-depleted HeLa cells were transfected with various BRCA1 constructs, synchronized in S/G2 phases and processed as described in Figure 1c. (e) Quantification of PTIP (Pax transactivation domain-interacting protein) foci formation by the indicated BRCA1 constructs, as described in c. More than 30 FLAG-positive cells and the cells with >20 PTIP foci were counted, results are presented as the means (±s.d.) of three independent experiments. Ni-NTA, nickel-nitrilotriacetic acid; WT, wild type.

Figure 5

A working model for an inhibitory role of BRCA1 in the regulation of 53BP1 signaling.