Journal: Open biology
Article Title: Opposing role of condensin hinge against replication protein A in mitosis and interphase through promoting DNA annealing
Figure Lengend Snippet: Interaction of isolated condensin and SMC dimer with different DNAs. ( a ) SDS-PAGE patterns of holocondensin (Cut3-Cut14-Cnd1-Cnd2-Cnd3), the SMC dimer (Cut3-Cut14) and the non-SMC trimer (Cnd1-Cnd2-Cnd3), together with single Cut3 and Cut14 as controls, stained with Coomasie brilliant blue. The procedures of isolation were previously described, and the degree of purity for these preparations was similar to those previously reported [ 20 , 21 ]. The Cut14 and Cnd1 overlap, and the Cnd2 band is diffuse and less intense than the other non-SMC subunits, probably owing to phosphorylation and/or degradation [ 9 ]. Limited proteolysis of Cut3 has been reported [ 20 ]. ( b ) Condensin and SMC dimer were incubated with a mixture of ssDNA and dsDNA, then analysed on a 10% non-denaturing acrylamide gel in the absence of SDS. DNA used was tagged with fluorescent FITC. ( c ) WT and mutant SMC dimer were incubated with M13 ssDNA with or without the pre-heat treatment at 42°C for 10 min, then analysed on a 0.7% native agarose gel in the absence of SDS. The mutant dimer was obtained by simultaneous overexpression of Cut3 and Cut14-Y1, and purified by affinity chromatography, stained with SYBR Gold. ( d ) WT and mutant SMC dimers were incubated with hdDNA with or without pre-heat treatment of the SMC dimers (see text), stained with ethidium bromide.
Article Snippet: Highly purified bacterial single-strand DNA binding protein (SSB; purchased from Promega)-coated hdDNA was incubated with the S. pombe SMC dimer.
Techniques: Isolation, SDS Page, Staining, Incubation, Acrylamide Gel Assay, Mutagenesis, Agarose Gel Electrophoresis, Over Expression, Purification, Affinity Chromatography