Fig. 2: BLM interacts with condensin II through a short linear N-terminal motif.

a BLM truncations tested in mammalian-two-hybrid (M2H) assay against full-length SMC2. Interaction is indicated by secreted alkaline phosphatase (SEAP) activity as ++ (strong), + (mild), and - (absent). See Supplementary Fig. S1c for measurements of SEAP activity from three experiments. Red box indicates BLM region required for interaction with SMC2. b Alignment of BLM residues 150-184 with BLM from other vertebrates. Residues W154, M157, and F160 were mutated to lysine, referred to as BLM-WMF^mut. c SEAP activity detected in M2H assay between two independently generated pVP16-BLM-WMF^mut clones (#1, #2) and pM-SMC2. pM3-VP16 is a positive control from the manufacturer expressing a fusion of the GAL4 DNA-binding domain to the VP16 activation domain. Significance of differences between means ± SD was determined by a t-test and is reported as ****p ≤ 0.0001. d Plasmid expressing BLM-WMF^mut was stably transfected into BLM-KO cells. Western blot of nuclear extracts from two independently generated BLM-WMF^mut clones (#1, #2) and of WT, BLM-KO, and BLM-COMP cells was probed with BLM antibody. PCNA was used as a loading control. Expression levels were analyzed 3 h after release from G1/S arrest. e Co-immunoprecipitations of BLM and BLM-WMF^mut #1 were probed with antibodies against SMC2, condensin II-specific subunit NCAPH2, and known BLM-interacting proteins. Whole-cell extracts were prepared from cells 3 h after release from G1/S arrest. f PLA using antibodies against endogenous BLM and NCAPH2 in BLM-COMP, BLM-WMF^mut #1, and BLM-KO cells 3 h after release from G1/S arrest. Scale bars, 10 μm. BLM-NCAPH2 PLA foci/nucleus were quantified (BLM-COMP, n = 102 nuclei; BLM-WMF^mut #1, n = 130 nuclei; BLM-KO, n = 100 nuclei). Significance of differences was determined by a Mann–Whitney test and is reported as ****p ≤ 0.0001. For additional PLA controls, see Supplementary Fig. S1b.