Fig 2: siRNA-based Cdc42 knockdown modulated the CTD phosphorylation code and affected the level of CTD phosphatases and kinases in HeLa cells. (A) HeLa cells 48 hr after Cdc42 siRNA transfection. (B) Western blot showing the levels of Cdc42, Ser2P, Ser5P, Ser7P and CTD phosphatases (FCP1 and RPAP2) and kinases (CDK7 and CDK13) in Cdc42 siRNA-transfected HeLa cells. Tubulin was used as the protein loading control. Quantitative analysis of the Western blot results from two independent biological replicates was shown. Protein level in the control was set as 1.0. Significance level is indicated by * (p < 0.05) or ** (p < 0.01) vs. control. The letter a above the column indicates a marginal difference (p = 0.058) compared to control.

Fig 3: CTDP1 promotes homologous recombination DNA repair.a Schematic of the DR-GFP reporter homologous recombination assay. b Expression of transfected CTDP1-DDK in HeLa-DR cells. c HR efficiency of transfected cells validated in panel b was measured by flow cytometry quantitation of GFP-positive cells. n = 3. Data represented as mean ± standard error of the mean (SEM); paired two-tailed Student’s t-test. d Knockdown of CTDP1 (shCTDP1) and non-targeting scrambled shRNA control (shScr) in HeLa-DR cells evaluated by western blot. e HR efficiency in response to CTDP1 knockdown from cells validated in panel d was measured by flow cytometry quantitation of GFP-positive cells. n = 3; mean ± SEM; paired two-tailed Student’s t-test. f Confirmation of protein expression knock down in shScr, shCTDP1, shFANCA, and shBRCA2 targeted cells by western blot for the indicated proteins. g HR efficiency was measured by flow cytometry quantitation of GFP-positive cells in the cells validated in panel f. n = 3; mean ± SEM; paired two-tailed Student’s t-test

Fig 4: CTDP1 knockdown promotes ICL sensitivity and ICL-induced apoptosis.a Western blot confirmation of shRNA targeting CTDP1 with two independent shCTDP1 constructs (#1-TRCN0000002996 and #2-TRCN0000436164). b Representative clonogenic survival assay in MCF-10A cells expressing non-targeting shScr and shCTDP1 constructs treated with control or 5 μM melphalan. c Quantification of the clonogenic survival assays using MCF-10A cells expressing shScr or shCTDP1 #1 treated with 5 μM melphalan (Mel). n = 3 independent experiments. Data represented as mean ± SEM; paired two-tailed Student’s t-test. d Clonogenic survival assay using MCF-10A cells expressing shScr or shCTDP1 #1 treated with 0, 2, 4, 6, or 8 Gy ionizing radiation. n = 3; mean ± SEM; Asterisks indicate p-value ≤ 0.05; paired two-tailed Student’s t-test. e Clonogenic survival assay using MCF-10A cells expressing shScr or shCTDP1 #1 treated with 0, 0.1, 0.2, 0.3, 0.4, or 0.5 μM MMC. n = 3; mean ± SEM; Asterisks indicate p-value ≤ 0.05; paired two-tailed Student’s t-test. f Clonogenic survival assay using MCF-10A cells expressing shScr or shCTDP1 #1 treated with 0, 10, 20, 30, 40, or 50 J/m2 ultraviolet radiation. n = 3; mean ± SEM. g Clonogenic survival assay using MCF-10A cells expressing shScr or shCTDP1 #1 treated with 5 μM cisplatin or 12 μM 5-Fluorouracil (5-FU). n = 3; mean ± SEM; paired two-tailed Student’s t-test. h Caspase-3 activity assay using MCF-10A cells expressing shScr or shCTDP1 treated with control or melphalan for 24 h. Y-axis units are represented as the change in fluorescent units per microgram protein extract per hour of reaction incubation (ΔFU/μg protein/h). n = 3; mean ± SEM; paired two-tailed Student’s t-test. i Caspase-3 activity assay using MDA-MB-231 cells shScr or shCTDP1 treated with control or Melphalan for 0, 6, and 24 h. n = 3; mean ± SEM; paired two-tailed Student’s t-test. j Western blot confirmation of CTDP1 knockdown and FANCD2 expression in PD20 (FANCD2 deficient) and PD20 + FANCD2 fibroblasts. k Clonogenic survival assay using PD20 and PD20 + FANCD2 cells expressing shScr or shCTDP1 #1 treated with 0, 25, 50, 75, 100, or 200 nM MMC. n = 3; mean ± SEM. l Table of p-values determined using the paired two-tailed Student’s t-test for each listed comparison of treatment groups from results in panel k

Fig 5: CTDP1 BRCT domain TAP-MS identifies interactions with Fanconi anemia proteins.a Representation of CTDP1 and its modular domain organization. The BRCT region cloned for TAP–MS experiments is indicated between amino acids 619–738. b Workflow diagram of the TAP–MS experiment. c SAINT output for Bayesian false discovery rate (SAINT-BFDR) was used to create a high confidence interaction list. Graph represents the fold change in protein representation between CTDP1 BRCT purification and control and the number of proteins at binned intervals of 0.5 SAINT-BFDR scores. d TAP–MS experiments generate a comprehensive interaction network of 103 proteins interacting with the CTDP1 BRCT domain. Red outlined nodes indicate proteins with GO annotations for DNA damage (GO:0006281) and DNA replication (GO:0006260). Edge color represents source of protein interactions (gray: TAP-MS, blue: BisoGenet). e KEGG pathway enrichment determined by ClueGO of the 103 CTDP1 interacting proteins identified by TAP–MS, excluding CTDP1 itself. The most significant term for each cluster is presented in bold font and term p-value corrected with Bonferroni step down is presented in parentheses. Threshold for visualization was p-value ≤ 0.05 and represented inversely proportional to node size. Exact p-values are displayed under most significant group node determined by two-sided hypergeometric test corrected using Bonferroni step down method. f Specificity of protein interactions with the CTDP1 BRCT domain in comparison to 27 other BRCT domain interaction datasets. FANCI and FANCA passing SAINT-BFDR cutoff of ≤0.05 are represented as red circles. g Table detailing central FA pathway proteins’ (FANCA, FANCI, and FANCD2) average spectrum abundance (Ave. Spec.), peptide coverage of protein sequence (Percent Coverage), and SAINT BFDR from TAP-MS. h Analysis of CTDP1 and FANCI expression in the input and immunoprecipation (IP) of control (IgG) or endogenous CTDP1 in complex with FANCI from untreated 293FT cells. i Top: Whole cell lysate (WCL) blot of GFP-FANCI and CTDP1-DDK constructs co-overexpressed in 293FT cells with and without 0.1 μM MMC treated for 24 h. Bottom: IP of DDK-tagged CTDP1 co-immunoprecipitates GFP-tagged FANCI independent of MMC treatment