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