Fig 2: Phosphorylated peptides from RAD50, BRCA1, MLH1 and XPC interact with MAP7(A) Peptides from four DDR proteins containing the similar feature S/TE/DXE. Amino acids with a negative charge are highlighted in red.(B) Top 10 hits from the proteomics of peptide pulldowns from HeLa nuclear extract performed with pairs of phosphorylated and non-phosphorylated peptides from BRCA1, RAD50, MLH1 and XPC.(C) Western blot analysis of peptide pull-down from HEK293T whole cell extract overexpressing GFP-MAP7 with non-phosphorylated (nonP) and phosphorylated (P) RAD50 peptides.(D) Volcano plots compare proteins binding to phosphorylated and non-phosphorylated peptides. A Benjamini-Hochberg cut-off of 5% with an s0 of (0.1) was used to select significant binders.

Fig 3: The interactions between MAP7/MAP7D1 and DDR proteins are not altered by nocodazole or paclitaxel(A) Proximity ligation assay with endogenous RAD50 and Flag-MAP7 (top left) or empty Flag vector (top right) and with endogenous BRCA1 and Flag-MAP7 (bottom left) or empty Flag vector (bottom right) in U2OS cells.(B) Quantification of PLA staining showing the percentage of cells containing the PLA signal both in the cytoplasm and in the nucleus or only in the nucleus. The error bars represent SEM of values from three independent experiments in which at least 30 cells were quanified. Scale bar, 10 µm.(C) Top: immunoprecipitation of GFP-MAP7 with endogenous BRCA1 from HEK293T whole cell extracts from cells treated with 0.1 ng/mL nocodazole at different time points. Bottom: representative immunofluorescent images of microtubules in HEK293T treated with nocodazole at different time points.(D) Immunoprecipitation of GFP-MAP7 with endogenous RAD50 from HEK293T whole cell extracts from cells treated with 0.1 ng/mL nocodazole at different time points.(E) Immunoprecipitation of GFP-MAP7 with endogenous BRCA1 from HEK293T whole cell extracts from cells treated with increasing doses of paclitaxel (0–10 µM). Detyrosinated a-tubulin is used as indicator of paclitaxel treatment.(F) Immunoprecipitation of GFP-MAP7 with endogenous RAD50 from HEK293T whole cell extracts from cells treated with increasing doses of paclitaxel (0–10 µM).(G) Immunoprecipitation of mCherry-MAP7D1 with endogenous BRCA1 from HEK293T whole cell extracts from cells treated with 0.1 ng/mL nocodazole at different time points.(H) Immunoprecipitation of mCherry-MAP7 with endogenous BRCA1 from HEK293T whole cell extracts from cells treated with increasing doses of paclitaxel (0–10 µM).

Fig 4: Interactions between MAP7 and MAP7D1 with RAD50 and BRCA1(A) Immunoprecipitation of endogenous MAP7 and MAP7D1 with RAD50 from HEK293T whole cell extract (both MAP7 and MAP7D1 are recognized by MAP7 antibody).(B) Immunoprecipitation of endogenous MAP7 and MAP7D1 with BRCA1 from HEK293T whole cell extract.(C) Schematic representation of full-length MAP7 and MAP7 constructs used in immunoprecipitations. CC1 and CC2 are two coil-coiled domains.(D) Domain mapping by immunoprecipitation of full-length GFP-MAP7 (GFP-FL) and MAP7 truncation constructs by endogenous RAD50 from HEK293T extract overexpressing GFP-MAP7 constructs.(E) Domain mapping by immunoprecipitation of full-length GFP-MAP7 (GFP-FL) and GFP-MAP7 truncation constructs by endogenous BRCA1 from HEK293T extract overexpressing GFP-MAP7 constructs.(F) Immunoprecipitation of GFP-tagged MAP7 N-terminal fragment (aa1-152) wild type (GFP-N1) or with Arg144 and 145 mutated to Ala (GFP-N1RA) with endogenous BRCA1 from HEK293T extract overexpressing GFP-MAP7 constructs.(G) Immunoprecipitation of GFP-N1 with endogenous RAD50 from HEK293T whole cell extract overexpressing GFP-MAP7 construct, untreated or treated with ?-phosphatase.(H) Immunoprecipitation of full length MAP7 (GFP-FL) wild type and MAP7 mutated on arginine 144/145 to alanine (GFP-FL RA) with endogenous BRCA1 from HEK293T whole cell extract treated or untreated with ?-phosphatase.(I) Further domain mapping using MAP7 constructs mutated on Arg144/145 to Ala with deletion between aa151-270 (GFP- ? 1 RA) and 151–300 (GFP- ? 2 RA). See also Figure S1.

Fig 5: Direct interactions between MAP7, BRCA1 and RAD50 are phosphorylation-dependent and regulated by CK2(A) Pull down of Flag-RAD50 wt or TA with GFP-MAP7. Proteins were purified from HEK293T whole cell extracts by immunoprecipitation followed by high salt (4M NaCl) washes. Flag-tagged proteins were eluted from beads using Flag peptide.(B) Pull down of Flag-RAD50 wt untreated or treated with ?-phosphatase with GFP-MAP7. Proteins were purified from HEK293T whole cell extracts by immunoprecipitation followed by high salt (4M NaCl) washes. Flag-tagged proteins were eluted from beads using Flag peptide.(C) Pull down of Flag-tagged MAP7 with GFP-BRCA1 wt untreated or treated with ?-phosphatase or GFP-BRCA1 SA mutation. Proteins were purified from HEK293T whole cell extracts by immunoprecipitation followed by high salt washes (4M NaCl). Flag-tagged proteins were eluted from beads using Flag peptide.(D) Immunoprecipitation of endogenous BRCA1 from HEK293T whole cell extracts. Proteins on beads were untreated or treated with ?-phosphatase followed by re-phosphorylation by recombinant CK2.(E) Immunoprecipitation of GFP-BRCA1 wt and GFP-BRCA1 SA from HEK293T whole cell extract. Immunoprecipitation of endogenous BRCA1 from HEK293T whole cell extracts. Proteins on beads were untreated or treated with ?-phosphatase followed by re-phosphorylation by recombinant CK2.(F) Immunoprecipitation of endogenous BRCA1 from HEK293T whole cell extract untreated or treated with two different doses of 5 µM or 15µm CK2 inhibitor.