Fig. 1: ASF1 forms a complex with RIF1 and is recruited to DSB sites by 53BP1-RIF1.

a A silver-stained SDS-PAGE gel showing the polypeptides that were immunopurified from extracts of HEK293 cells expressing FLAG-tagged RIF1, ASF1a, and ASF1b using the anti-FLAG antibody. HEK293 cells without expressing exogenous protein were included as a control (mock). The major polypeptides on the gel were identified by mass spectrometry. The mean numbers of peptides discovered for each protein from two replicates are listed at the bottom. A full list of mass spectrometry data is available in Supplementary Data 1. b, c Immunoblot showing IP of FLAG-tagged RIF1 (b), ASF1a, and ASF1b (c). HEK293 cells expressing FLAG-RIF1 were treated with/without bleomycin (20 μg/mL) for 3 h before harvest. d Schematic representation of RIF1 and sequence alignment of the B domains of RIF1, CAF-1 P60, HIRA, and CDAN1. For proteins with two motifs, both are aligned and designated (1) and (2). hu Homo sapiens, mu Mus musculus, ch Gallus gallus, xp Xenopus laevis, fish Danio rerio. e Immunoblot showing FLAG-IP from extracts of HEK293 cells expressing FLAG-tagged ASF1a and GFP-RIF1 wild-type (WT) or the AAD mutant. f–i Recruitment of GFP-ASF1a (f) and GFP-ASF1b (h) to laser-induced DNA damage sites in wild-type, 53BP1^∆ or RIF1^∆ HEK293T cells and quantification (g, i). Mean ± s.e.m. is shown for every time point. The numbers of cells pooled from two independent experiments are indicated. **P < 0.01; ****P < 0.0001; statistical analysis was performed using two-way ANOVA. j, k Recruitment of GFP-tagged wild-type or mutated ASF1a to laser-induced DNA damage sites (j) and quantification (k). Mean ± s.e.m. is shown for every time point. The numbers of cells pooled from two independent experiments are indicated. Representative images were taken 10 min after laser irradiation. Scale bar, 5 μm. Uncropped images of the gels and statistical source data including the precise P values are provided in Source data.