Fig. 5: The SiwR WYL domain binds ssDNA regions in the context of different nucleic acids molecules.
a Binding of Sumo-SiwR to FAM-labeled polyT20 ssDNA was measured using fluorescence anisotropy. While a KD of 182 nM could be determined for the wild-type form of Sumo-SiwR, the WYL domain variant R204A-R207A showed no binding. b Overview of further nucleic acid molecules tested for Sumo-SiwR binding. Nucleotides in dark green show the conjugated FAM position. We tested a 12-nucleotide ssDNA piece (black) or equivalent RNA piece (pink), the same ssDNA with an 8-oxoguanine mutation (red), a 5’ tailed DNA piece (green), a 3’ tailed DNA piece (blue) and a DNA fork mimic (orange). c Sumo-SiwR binds to ssDNA containing oxidized guanine with a similar affinity (227 nM) as to the same nucleotide piece without oxidized guanine (178 nM). d SiwR binds other forms of nucleic acids such as 5’ tailed, 3’ tailed and fork DNA mimic, but not ssRNA. The observed KD values for the tailed or fork ssDNA molecules are higher than for simple ssDNA fragments. Each anisotropy datapoint was measured at least five times. Every KD was calculated under the assumption that both WYL domains of dimeric SiwR can bind ligand. Error bars represent data points of at least four independent fluorescent anisotropy measurements.
