Fig 2: RPA32 is phosphorylated and ubiquitylated in response to DNA damage that targets active replication forks. (A) Cells were transfected with a vector expressing His6-tagged ubiquitin and lysed under denaturing conditions. Ni-NTA pulldown was performed to isolate ubiquitylated proteins. The indicated proteins were detected with specific antibodies. (B) Cells were transfected with a vector expressing Strep-HA ubiquitin and treated with 1 or 5 μM CPT, 10 γ IR, 4 mM HU or 50 J/m2 UV for 4 h. Ubiquitylated proteins were isolated by Strep-Tactin pulldown under denaturing conditions. (C) Cells transfected as in (B) were treated with 1 μM CPT for the indicated times and total RPA32 or (D) phosphorylated RPA32 species were detected using specific antibodies after Strep-Tactin pulldown. (E) A stable HEK293T cell line expressing SFB-PRP19 was treated with CPT 1 μM for the indicated times. SFB-PRP19 and its interactors were isolated using streptavidin-associated beads.

Fig 3: EDC4 interacted with RPA and promotes RPA phosphorylation. a The interaction of EDC4 and RPA1 or RPA2 was analyzed by immunoprecipitation. b The phosphorylation of RPA was analyzed by Western blot

Fig 4: ZFP161 is enriched at replication forks. a Cells were depleted of RPA32 using shRNA. The co-localization of phospho-RPA32 (S4/S8) and ZFP161 were determined by immunofluorescence. b RPA32 deficient HCT116 cells were incubated with EdU and HU. Replication fork proteins were isolated by iPOND and immunoblotted with indicated antibodies. c Cells were depleted of ZFP161 using sgRNA. The co-localization of RPA32 and γH2AX were determined by immunofluorescence. d ZFP161 deficient HCT116 cells were incubated with EdU and HU. Replication fork proteins were isolated by iPOND and immunoblotted with indicated antibodies. e Purified GST-ZFP161 protein and RPA complex were incubated with biotin labeled single fork DNA. The proteins retained on DNA were determined by immunoblotting. Scale bars, 10 µm. Source data are provided as a Source Data file.

Fig 5: ZFP161 mediates the RPA-ATRIP interaction. a, b Chromatin fractionations from HCT116 cells in presence or absence of ZFP161 following 10 mM HU treatment for 2 h were used for immunoblotting with indicated antibodies. c Whole cell lysates from HCT116 cells were used for immunoprecipitation with ZFP161 antibody. d The schema of ZFP161 constructs used to identify the minimal region required for its interaction with RPA32. e HEK293T cells were transfected with S-tagged RPA32 and ZFP161 vectors. After 48 h, the interaction between ZFP161 and RPA32 was determined by immunoprecipitation and immunoblotting with indicated antibodies. f, g Whole cell lysates from HCT116 cells were used for immunoprecipitation with ZFP161 (f), or ATRIP (g) antibodies. h The schema of ZFP161 constructs used to identify the minimal region required for its interaction with ATRIP. i HEK293T cells were transfected with the indicated ZFP161 vectors. Aftter 48 h, ZFP161 and ATRIP interaction was determined by immunoprecipitation and immunoblotting with indicated antibodies. j, k HCT116 cells expressing or lacking ZFP161 were transfected with constructs encoding Myc-ATRIP, ZFP161 (WT) and D3 mutant (j) or T2 mutant (k). Whole cell lysates were immunoprecipitated with Myc antibody and immunoblotted with indicated antibodies. l ZFP161 deficient HCT116 cells were incubated with EdU and HU. Replication fork proteins were isolated by iPOND and immunoblotted with indicated antibodies. m Biotin labeled single fork DNA was incubated with HCT116 whole cell lysates and pulled down with streptavidin beads. The interaction of ATRIP and RPA on DNA was determined by immunoblotting. < : ZPF161 full length and mutants bands. Source data are provided as a Source Data file.