Fig. 4
From: The Rad51 paralogs facilitate a novel DNA strand specific damage tolerance pathway
Csm2-Psy3 binds an abasic site analog in double-flap DNA. a Schematic of double-flap substrates containing abasic site analogs (tetrahydrofuran; indicated by an arrow and highlighted text) on the 3′ (AP1–4) or 5′ (AP5–8) DNA strand. Note that the complementing oligonucleotide, which is unmutated, is FITC labeled on the ssDNA end of the double-flap substrate. b Csm2-Psy3 binds to double-flap substrates with abasic site analogs along the 3′ DNA with equal affinity. DNA binding by Csm2-Psy3 was measured by fluorescence anisotropy, with increasing concentrations of the Csm2-Psy3 heterodimer added to 25 nM fluorescein-labeled double-flap substrate (AP1–4). Error bars indicate standard deviations measured in triplicate experiments. Data were fit to a one-site binding model and average dissociation constants (Kd) were calculated. Note the improved binding compared to Fig. 1 due to increased sample purity (described in detail in Supplementary Methods). c Csm2-Psy3 binds more tightly to a double-flap substrate containing an abasic site analog on the 5′ oligonucleotide at the junction (AP6, highlighted in red). In vitro-binding assays as described in b except with double-flap substrates containing 5′ abasic site analogs (AP5–8). d DNA binding by the Csm2-Psy3-Shu1-Shu2 was measured by fluorescence anisotropy, with increasing concentrations of the indicated proteins added to 5 nM AP6 (highlighted in red) or 2.5 nM double-flap DNA substrate. The data were fit to a quadratic equation
