Figure 6: The Smc5/6-hinge preferentially binds to ssDNA.
From: Specialized interfaces of Smc5/6 control hinge stability and DNA association
(a) Fluorescence polarization assay. Wild-type (WT) Smc5/6-hinge binds with higher affinity to a 45 nt ssDNA (open circle) substrate than to a 45 bp dsDNA substrate (closed circle). (b) Representative EMSA gel, imaged by laser-scanning, for the interaction of wild-type Smc5/6-hinge with a 45 nt ssDNA substrate. The position of a fully resolved protein:DNA species is indicated by the ‘Complex’ label. At higher protein concentrations a second species, which does not enter the gel, is also observed (labelled with an asterisk). (c) Biotinylated-oligonucleotide pull-down assay. WT Smc5/6-hinge was incubated with magnetic beads coated with the indicated length of oligonucleotide. M=molecular mass marker. (d) Fluorescence polarization assays for binding of the indicated Smc5/6-hinge mutant to a 15mer oligonucleotide. (e) Sensorgrams for SwitchSENSE ‘Fdown’ experiments. Representative association (left) and dissociation curves (right), at a protein concentration of 5 μM, are shown for binding of WT and mutant forms of the Smc5/6-hinge (top) to immobilized ssDNA. The solid black lines indicate the fit of the indicated binding model to the experimental data. Fluorescence polarization data are the mean of either 4 (a) or 3 replicates (d,e) with error bars indicating 1 standard deviation. Dissociation constants (Kd) were calculated by least-squares fitting of a one-site binding model. ND, not determined.
