Unwinding of partial duplex DNA substrates by Fancm. Helicase reactions were performed as described in Materials and Methods. The indicated concentrations of Fancm were incubated with 0.1 nM of the indicated substrate for 15 min. Colored strand on each substrate represents radiolabeled 5′ strand. Quantitative data from at least three experiments were plotted as the average for each protein concentration. Error bars represent the SEM. Oligonucleotides used to make these substrates can be found in Table S1. (A) Comparison of the fraction of substrate unwound with partial duplex substrates of different duplex lengths. Pink ●, 15-bp duplex region with a 25-nt overhang. Blue ●, 20-bp duplex region with a 20-nt overhang. Blue ○, 20-bp duplex region with a 25-nt overhang; Blue ♦, 25-bp duplex region with 25-nt single-stranded arms. (B) Unwinding of D-loop intermediate substrates by Fancm. Pink ●, front; blue ▪, middle; pink ♦, end. Bubble structures were made using a two 90-nt oligonucleotides with 25 bp of complementary ends with a 40-nt noncomplementary middle (A1/A2). Substrate oligonucleotides are in Table S1.