Fig 2: Loss of ASF1 phenocopies RIF1 deficiency in the regulation of telomere fusion in TRF2-deficient cells and PARPi sensitivity in BRCA1-deficient cells.A, Western blot was conducted to determine ASF1A and ASF1B knockdown efficiency as well as TRF2 KO efficiency in inducible TRF2 KO HeLa cells. B, representative images of metaphase DNA fluorescence in situ hybridization with telomere probe in siControl and siASF1A + siASF1B-transfected inducible TRF2 KO HeLa cells. C, statistical quantification of percentage of telomere fusions in inducible TRF2 KO HeLa cells after knockdown of ASF1A and ASF1B (with chromosome counts >1000) from (A). Data are represented as mean ± SD (n = 2). ∗∗∗p < 0.001 (Student's t test). D, Western blotting for confirmation of sgASF1A, sgASF1B, and sgRIF1 knockdown efficiency is indicated in sgRNA-transfected stable RPE1 FLAG-Cas9 P53−/− BRCA1−/− cells. E, results of long-term clonogenic assays performed using cells from (D). About 2 × 103 cells were seeded in triplicate in each well of 6-well plate and incubated with the indicated concentration of olaparib for 14 days. Colonies were fixed and stained with 0.5% crystal violet. F, quantification of relative cell survival in the clonogenic assays in (E). Data are presented as means (±SD) (n = 3). ∗∗p < 0.01; ∗∗∗p < 0.001; ns (Student's t test). Dox, doxycycline; ns, not significant; PARPi, poly (ADP-ribose) polymerase inhibitor; RIF1, replication timing regulatory factor 1; sgRNA, single-guide RNA; TRF2, telomeric repeat–binding protein 2.

Fig 3: Identification of RIF1-associated proteins.A, strategy for tandem affinity purification (TAP) of 293T RIF1 SFB N-terminal knock-in cells. B, selected lists of RIF1-associated proteins in soluble and chromatin fractions analyzed by mass spectrometry. C, endogenous RIF1 interacts with ASF1A and MRN complexes. Cell lysates from 293T RIF1 SFB N-terminal knock-in cells were prepared and pulled down with S protein–conjugated beads. Pull-down samples were blotted using antibodies as indicated. MRN, MRE11–RAD50–NBS1; RIF1, replication timing regulatory factor 1; SFB, S protein, FLAG, and streptavidin-binding peptide; 293T, human embryonic kidney 293T cell line.

Fig 4: FOXK1 Depletion Impairs DNA Damage Response(A) Proximity ligation assay (PLA) showed the interaction of endogenous 53BP1 and FOXK1 (red spots) in non-treated (NT) and IR (10 Gy, released for 1 h)-treated cells. RIF1–53BP1 interaction was included as a positive control.(B) Quantification of PLA from (A). More than 80 cells of each group were quantified. Data are represented as mean ± SD (n = 2); ***p < 0.001, Student’s t test.(C) Generation of FOXK1 KO, FOXK2 KO, and FOXK1/2 DKO in HeLa cells. Immunoblot was performed with indicated antibodies.(D) Clonogenic survival of indicated cells after exposure to indicated doses of IR. Results are the means (± SD) of three independent experiments.(E) Cell cycle profile in WT, FOXK1 KO, FOXK2 KO, and FOXK1/2 DKO HeLa cells in NT and IR (2 Gy)-treated cells for different time points after IR.(F) Statistical quantification of the percentages of cells in different cell cycles from (E). Data are represented as mean ± SD (n = 3); **p < 0.01, ***p < 0.001, Student’s t test.

Fig 5: Mapping the binding regions on RIF1 and ASF1A.A, schematic presentation of full-length and truncation mutants of RIF1 used in this study. B and C, 293T cells were transfected with plasmids encoding various truncation mutants of SFB-tagged RIF1. Pull downs were performed with the use of streptavidin-conjugated beads, and Western blotting was conducted with FLAG and ASF1A antibodies. D, schematic presentation of full-length and truncation mutants of ASF1A used in this study. E, 293T cells were transfected with plasmids encoding WT or various mutants of SFB-tagged ASF1A. Pull downs were performed with the use of streptavidin-conjugated beads, and Western blotting was conducted with FLAG and RIF1 antibodies. F, 293T cells were transfected with plasmids encoding SFB-tagged WT or V94R mutant ASF1A. Pull downs were performed with the use of streptavidin-conjugated beads, and Western blotting was conducted with FLAG and RIF1 antibodies. RIF1, replication timing regulatory factor 1; SFB, S protein, FLAG, and streptavidin-binding peptide; 293T, human embryonic kidney 293T cell line.