Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
A DNA nick at Ku-blocked double-strand break ends serves as an entry site for exonuclease 1 (Exo1) or Sgs1-Dna2 in long-range DNA end resection
Weibin Wang, James M. Daley, Youngho Kwon, Xiaoyu Xue, Danielle S. Krasner,Adam S. Miller, Kevin A. Nguyen, Elizabeth A. Williamson, Eun Yong Shim, SangEun Lee, Robert Hromas, and Patrick Sung
Supporting Information
Supplemental Figure S1: related to Figure 1Supplemental Figure S2: related to Figure 2Supplemental Figure S3: related to Figure 5
S‐1
−− + + + + + +− + + + + + +
− Dna2− + + + + + + Sgs1− + + + + + + RPA
0
20
40
60
80
100
0 5 10 15 20 25 30 35
dsDNA
nicked dsDNA
DN
A r
esec
ted
(%)
Dna2 (nM)
Supplemental Figure S1
A
DN
A r
esec
ted
(%)
Exo1 (nM)
0
20
40
60
80
100
120
0 2 4 6 8 10 12 14 16 18
dsDNA
nicked dsDNA
B
Nucleaseproduct
Substrate
Nucleaseproduct
Substrate
0 0.12
5
0.25
0.5
1 2 4 8 16 Exo1 (nM)0 0.12
5
0.25
0.5
1 2 4 8 16
dsDNA nicked dsDNA5’3’
3’5’*
*5’3’
3’5’*
*
dsDNA nicked dsDNA5’3’
3’5’*
*5’3’
3’5’*
*
Supplemental Figure S1. Nuclease activity of Exo1 or Dna2 on linear dsDNA substrate with or without an incision site
A, The indicated concentration of Exo1 was incubated with linear dsDNA substrate (1 nM) with or without a nick. The resultsfrom three independent experiments were graphed, with the error bars representing SD. B, DNA substrates used in A wereincubated with Dna2 (1, 2, 4, 8, 16 and 32 nM) in the presence of Sgs1 (16 nM) and RPA (800 nM). The results were graphed asin A. The asterisk denotes the 32P label in the substrate in all the figure parts. C, DNA substrate (1 nM) with a nick 41-nt, 59-ntor 82-nt away from the DNA end was pre-incubated with Ku (48 nM), followed by the addition of Exo1 (1.5, 3 and 6 nM). Theresults were quantified and presented as in A. D, Ku-blocked DNA substrates used in C were incubated with Dna2 (2, 8 and 32nM) in the presence of Sgs1 (16 nM) and RPA (800 nM). The quantification of the results was shown as in A.
Nucleaseproduct
Substrate
+ + + + Ku− Exo1
nick 82-nt from DNA end
+ + + +−
nick 59-nt from DNA end
+ + + +−
nick 41-nt from DNA end
0
20
40
60
80
100
0 2 4 6 8
41-nt
59-nt
82-nt
DN
A r
esec
ted
(%)
Exo1 (nM)
0
20
40
60
80
100
0 1 0 2 0 3 0 4 0
41-nt
59-nt
82-nt
Nucleaseproduct
Substrate
+ + + + Ku− + + + Sgs1− + + + RPA− Dna2
nick 82-nt from DNA end
+ + + +− + + +− + + +−
nick 59-nt from DNA end
+ + + +− + + +− + + +−
nick 41-nt from DNA end
DN
A r
esec
ted
(%)
Dna2 (nM)
C D
S‐2
Exo1 (nM)A
0
20
40
60
80
100
120
0 0.5 1 1.5 2
T7 Exo (U/μl)
DN
A r
esec
ted
(%)
T7 Exo (U/μl)
Supplemental Figure S2
B
0
20
40
60
80
100
120
0 1 2 3 4 5
DN
A r
esec
ted
(%)
Exo1 (nM)
0 0.06
25
0.12
5
0.25
0.5
1 2 4 0 0.21
0.42
0.84
1.68
*
*
*
*
*
*
Supplemental Figure S2. Nick processing activities of Exo1 and T7 Exo on circularnicked dsDNA
A, Nuclease activity of the indicated concentrations of Exo1 was tested on 1 nM circularnicked dsDNA substrate. The asterisk denotes the 32P label in the substrate. The resultsfrom three independent experiments were quantified and plotted, with the error barsrepresenting SD. B, Nuclease assay was carried out as in A, except that the indicatedconcentrations of T7 Exo were used. The results were quantified and plotted as in A.
S‐3
SOSS1 SOSS2
INTS3
hSSB1
hSSBIP1
A C
− − − TR− + + + + − − Sgs1− + + + + − − RPA
E
1 2 3 4 5 6 7 Lane
Supplemental Figure S3
B
− − − MRX− + + + + − − Sgs1− + + + + − − RPA
− − − Mph1− + + + + + + + − − RPA
HD
dsDNAssDNA
37 -50 -
75 -
kDa
15 -
20 -25 -
10 -
150 -100 -
250 -
37 -50 -
75 -
kDa
15 -
20 -25 -
10 -
150 -100 -
250 -
INTS3
hSSB2
hSSBIP1
1 2 3 4 5 6 7 Lane
dsDNAssDNA
dsDNAssDNA
HD HD
RPA70
Sgs1 (His)
RPASgs1RPA
S W E S W E
hRPASgs1hRPA
S W E S W E
hRPA70
Sgs1 (His)
0 5 15 45 0 0 5 15 45 0 0 5 15 45 0 Time (min)HD HD HD
nicked DNAwith 3’ label
nicked DNAwith 5’ labelgapped DNA
G 3’*
5’ 3’*
5’ 3’*5’
dsDNA
ssDNA
0
10
20
30
40
50
60
70
0 10 20 30 40 50
nicked DNAwith 3' label
gappedDNA
nicked DNAwith 5' labelD
NA
unw
oun
d (%
)
Time (min)
F
Supplemental Figure S3. Helicase activity of Sgs1 on circular dsDNA substrate with a nick or a gap
A, Mph1 helicase (1, 2, 4, 8, 16 and 32 nM) failed to unwind the nick-containing circular DNA substrate with RPA (800 nM) beingpresent. The assay was conducted as described in Figure 5B. HD, heat denaturation. B, SOSS1 and SOSS2 were analyzed by SDS-PAGE and Coomassie blue staining. C, Species-specific interaction between Sgs1 and yeast RPA. 6xHis-tagged Sgs1 was incubatedwith yeast or human RPA, followed by the addition of Ni-NTA resin to capture protein complexes. The supernatant (S) containingunbound proteins, the wash (W), and the eluate (E) fractions were analyzed by SDS-PAGE and immunoblotting using antibodiesagainst the largest subunit of yeast or human RPA or against the poly-histidine tag. D, The indicated concentrations of hRPA, SOSS1and SOSS2 were tested for their binding to 90-nt ssDNA (20 nM). The results were quantified and graphed, with the error barsrepresenting SD. E, Stimulation of unwinding of the nick-containing DNA catalyzed by Sgs1 (8 nM) in conjunction with RPA (800nM) by the TR complex (2, 4 and 8 nM). The results from three independent experiments were graphed, with the error barsrepresenting SD. F, DNA unwinding by Sgs1-RPA was assessed with MRX (4, 8 and 16 nM) as in E. The results were graphed as in E.G, Time-dependent unwinding of the nick-containing and gapped circular DNA substrates (0.5 nM each) was tested with Sgs1 (8 nM)and RPA (400 nM). The results from three independent experiments were graphed, with the error bars representing SD. The asteriskdenotes the 32P label in the substrate.
75 -
50 -
250 -150 -
kDa
75 -
50 -
250 -150 -
kDa
1 2 3 4 5 60
20
40
60
80
1 2 3 4 5 60
10
20
30
40
DN
A u
nwou
nd
(%)
Lane
DN
A u
nwou
nd
(%)
Lane
- 10 20 40 80 160
320
10 20 40 80 160
320
nM- 10 20 40 80 160
320
hRPA SOSS1 SOSS2
Protein-DNAcomplex
DNA
0
20
40
60
80
100
120
0 1 0 0 2 0 0 3 0 0 4 0 0
hRPASOSS1SOSS2
DN
A b
ound
(%
)
Concentration (nM)
D
S‐4