(a) SMARCAD1 and TOPBP1 interact and their interaction depends on functional phospho-binding pockets in BRCT1 and BRCT2 of TOPBP1. lexA-BD TOPBP1 1–360 (harbouring BRCT0/1/2) or lexA-BD TOPBP1 1–766 (harbouring BRCT0-5) were tested as WT versions or as K155E, KK154,155AM (affecting BRCT1) or K250E (affecting BRCT2) mutant derivatives. Interaction was tested against the Gal4-AD SMARCAD1 55–274. 3AT was added to –His plates to suppress auto-activation and to increase the stringency of the two-hybrid. Two-hybrid interactions with the lexA-BD TOPBP1 1–360 construct were generally stronger compared to lexA-BD TOPBP1 1–766, leading to milder effects of the K155E and K250E single-mutants, particularly at low 3AT concentrations. (b) SMARCAD1 interacts with TOPBP1 after CDK phosphorylation. GFPSMARCAD1 (55-445) was bound to a GSTTOPBP1 BRCT0/1/2 construct after phosphorylation with CDK. This CDK-dependent interaction was seen with several N-terminal SMARCAD1 constructs, but not with FL, perhaps due to low expression. (c–d) Threonine 71 of SMARCAD1, a putative CDK phosphorylation site, is required for TOPBP1 binding. (c) Two-hybrid analysis of ADSMARCAD1 (1-220) and phospho-mutant derivatives to BDTOPBP1 BRCT0/1/2. (d) Co-IP as in (a), but additionally using a T71A variant of GFPSMARCAD1 (55-274). (e) Dpb11 can bind to human SMARCAD1, and T71 is important for the interaction. Two-hybrid analysis as in (b), but using a BDDpb11 BRCT1+2 construct. (f) A SMARCAD1-Fun30 chimera lacking the Dpb11-binding site of Fun30, but containing the TOPBP1-binding site of SMARCAD1 restores sensitivity to CPT. The SMARCAD1-Fun30 chimera is expressed from the pGAL1-10 promoter and induced by galactose. Spotting on CPT medium as in Figure 4A.