Mph1 (60 fmol)1 2 3 4 5 6 7 9 10 11 128 13 14 15 16B
17 18- B - B - + + + + + + + + + + + +
- - - - - - 1 4 10 25 1 4 10 25 1 4 10 25
1 nt 5 nt 15 nt
Lane
Time (min)5’ flap length
5’*
11
12
1, 5,15 nt ( )
25 nt25 nt
50 nt
15 16 17
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)1 2 3 4 5 6 7 9 10 11 128 13 14 15B - + + +- - 2 8 25
0 nt 27 nt 48 nt
Lane
Time (min)
5’ tail length
- - 2 8 25B - + + +
- - 2 8 25B - + + +
5’
*
0, 27, 48 nt ( )
25 nt
50 nt
14 10 18
11
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 nt25 nt
50 nt
10
11
12
19 20 21
Mph1 (60 fmol)1 2 3 4 5B - + + +- - 2 8 25
random
Lane
Time (min)
5’ flap sequence
6 7 8 9 10B - + + +- - 2 8 25
11 12 13 14 15B - + + +- - 2 8 25
16 17 18 19 20B - + + +- - 2 8 25
dT dA dC
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 nt25 nt
50 nt 11
12
1 3 4 5 6 7 8 92 Lane
Mph1 (fmol)
- - -60 120 60 120 60 120
random dT dA
- 60 120 Mph1 (fmol)S
ubst
rate
bou
nd
(%)
10 19 20
Fig. S2B Gel mobility shift assay with Mph1 on DNA substrates used in unwindingassays 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 nt25 nt
50 nt 11
12
10 19 20 21
27 nt (random, dT, dA, dC)
5’*
25 nt25 nt
50 nt 11
10 19 20 21
Mph1 (60 fmol)1 2 3 4 5B - + + +- - 2 8 25
random
Lane
Time (min)
5’ tail sequence
6 7 8 9 10B - + + +- - 2 8 25
11 12 13 14 15B - + + +- - 2 8 25
16 17 18 19 20B - + + +- - 2 8 25
dT dA dC
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 nt25 nt
50 nt
20 nt (dT)
10
11
29
5’
25 nt25 nt
50 nt
20 nt (dA) 5’
25 nt25 nt
50 nt
20 nt (dC)* *
1 2 3 4 5 6 7 8 9 10 11 12- - 100 100
Bo - 3010- - 100 100
Bo - 3010- - 100 100
Bo - 3010Mph1 (fmol)Time (min)
Lanes
(fmol)
Substrateunwound
*
*
6.3
11.9
5.5
10.5 8.
212
.4
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.
42 43
