Mph1 (60 fmol) B B-B nt 5 nt15 nt Lane Time (min) 5’ flap length 5’ * , 5,15 nt ( ) 25 nt 50 nt Fig. S1A Helicase activity of Mph1 on 5’ flap-structured substrates of various lengths (1, 5 and 15-nt) in the flap region. Gel of Fig. 2A

Mph1 (60 fmol) B nt 27 nt48 nt Lane Time (min) 5’ tail length B B ’ * 0, 27, 48 nt ( ) 25 nt 50 nt Fig. S1B Helicase assays were performed with fork-structured substrates of various lengths (0, 27 and 48-nt) in their 5’ extension region. Gel of Fig. 2B

27 nt (random, dT, dA, dC) 5’ * 25 nt 50 nt Mph1 (60 fmol) B random Lane Time (min) 5’ flap sequence B B B dTdAdC Fig. S2A Unwinding of 5’ flap-structured substrates of different nucleotides sequences in the 27-nt 5’ flap region (random, dT, dA, and dC). Gel of Fig. 2C

27 nt (random, dT, dA) 5’ * 25 nt 50 nt Lane Mph1 (fmol) random dTdA Mph1 (fmol) Substrate bound (%) Fig. S2B Gel mobility shift assay with Mph1 on DNA substrates used in unwinding assays in Fig. 2C and Fig. S2A. See ‘Materials and Methods’ for assay condition. Gel is shown in left panel and the amount of bound DNA was quantified and represented as a bar graph in right panel.

Fig. S2C ATPase activities of Mph1 in the presence of unlabeled DNA substrates used in unwinding assays in Fig. 2C, Fig. S2A and DNA binding assay in Fig. S2B (5’ flap-structured substrates of different nucleotides sequences in the 27-nt 5’ flap region; random, dT, dA, and dC). See ‘Materials and Methods’ for reaction condition. ATP hydrolyzed was quantified and plotted against incubation periods (5, 15, and 30 min). 27 nt (random, dT, dA, dC) 5’ 25 nt 50 nt

27 nt (random, dT, dA, dC) 5’ * 25 nt 50 nt Mph1 (60 fmol) B random Lane Time (min) 5’ tail sequence B B B dTdAdC Fig. S2D Unwinding of fork-structured substrates with sequence variations in the 27-nt 5’ tail region (random, dT, dA, and dC). Gel of Fig. 2D

27 nt 5’ * 25 nt 50 nt 20 nt (dT) ’ 25 nt 50 nt 20 nt (dA) 5’ 25 nt 50 nt 20 nt (dC) * * Bo Bo Bo Mph1 (fmol) Time (min) Lanes (fmol) Substrate unwound * * Fig. S3 Helicase assays were performed with double flap substrates of different nucleotide sequences (dT, dA and dC) in their 3’ flap region in standard reaction condition. 100 fmol of Mph1 was incubated with each substrate for 10 or 30 min. 4243