Supplementary Figure 1. (A) Recruitment of GFP-APRIN in shCTL and shPALB2 Hela cells. (B) Representative images of laser-irradiated SKOV3 GFP-APRIN (green) cells and subjected to RAD51 immunofluorescence staining, 60 min post-DNA damage.
B
(kDa)
M2 Flag beads
APRIN
Flag-APRIN-His
MW
A
1
2
250 150 100 75
peptide 3x Flag
50 37 Flag-APRIN-His
Talon beads 25 20
Imidazole
15 Flag-APRIN-His
C 1 49
1 2 3 4 5 6 7 8 9 10
FLAG
611
469
HID
1083
1447
11 1213 14 15 16 17 18 19 20
NLS AT hooks
20 HEAT repeats
HIS
D KDa 150 37 37
15
CF
NF
ChF APRIN RAD51 GADPH H3
Supplementary Fig. 2: (A) Schematic representation of the double affinity purification of APRIN. Two-step affinity purification of Flag-APRIN-His from Sf9 soluble cell lysate: Flag binding, followed by elution of the protein with Flag peptides 3X, TALON-binding and elution with imidazole. (B) Silver-stained SDS-PAGE of APRIN. (C) Scheme of APRIN tagged Flag (red)/His (green). APRIN bears Heat repeats and AT hooks domains. (D) Fractionation from 293T cells. Endogenous APRIN was detected in the chromatin fraction (ChF). RAD51 is a control for the Nuclear soluble Fraction (NF), Histone H3 of the ChF fraction and GAPDH of the cytoplasmic Fraction (CF).
60nt
*
* APRIN (nM)
30nt *
60nt
0 1 2.5 5 7.5 10 15
30nt
0 1 2.5 5 7.5 10 15
0
1 2.5 5 7.5 10 15
DNA-protein complex
Probe 1 2
3
4
5
6 7
8 9 10 11 12 13 14 29nt 91nt
APRIN (nM)
0
60nt *
*
1 2.5 5 7.5 10 15
15 16 17 18 19 20 21
0
1 2.5 5 7.5 10 15 DNA-protein complex Probe
22 23 24 25 26 27 28
29 30 31 32 33 34 35
Supplementary Fig. 3: EMSA were performed with APRIN (0-15 nM) and ssDNA (lanes 1-7), or dsDNA (lanes 8-14), or Splayed Arms (lanes 15-21), or D-loop (lanes 22-28), or Holliday Junction substrates (lanes 29-35).
A
1 49
611
469
1 2 3 4 5 6 7 8 9 10
HID
1083
B
1447
(kDa)
MW GST
A1
A2
A3
A4
A5
APRIN
11 1213 14 15 16 17 18 19 20
NLS AT hooks 1 GST
245 246 GST
75
A1
1 2 3 4
569 6 7 8 9 10
HID
GST
A2
50
A3
37
863
570 11 1213 14 15
864 GST
1152 A4
17 18 19 20
1153
1447
GST
A5
25
NLS AT hooks
C 0
A2
0 50 10
50 10
A3
0
A4
0
50 10
A5
0
50 10
0 5
50 10
1 49
GFP
AP
A1
RIN
D
611
469
1 2 3 4 5 6 7 8 9 10
HID
1083
APRIN WT NLS AT hooks
(nM) DNA-protein complex DS
611
1 49
GFP
1 2 3 4
1083
APRINΔA2
1 49
611
469
1 2 3 4 5 6 7 8 9 10
HID
1447
APRINΔA4
11 1213 14 15
SS
NLS AT hooks 1 49
GFP
γ-H2AX
DAPI
611
469
1 2 3 4 5 6 7 8 9 10
HID
1 49
469
GFP NLS
E
1447
11 1213 14 15 16 17 18 19 20
NLS AT hooks
GFP
GFP-APRIN
1447
11 1213 14 15 16 17 18 19 20
1 2 3 4 5 6 7 8 9 10
GFP-APRIN
1083
APRINΔA5
11 1213 14 15 16 17 18 19 20
611
HID
γ-H2AX
1083
11 1213 14 15 16 17 18 19 20
NLS-APRINΔA5
DAPI GFP-APRIN
DelA2
DelA4
DelA5
DelA5+NLS
30 min
60 min
Supplementary Figure 4. (A) Scheme of APRIN deletion variants fused to GST, purified from bacteria. (B) Coomassie Blue stained SDS-PAGE gel of APRIN deletion variants before GST cleavage. (C) EMSAs were performed with both a ssDNA (ss) and dsDNA (ds) and APRIN fragments (50 or 100 nM): A1 (lanes 2-3), A2 (lanes 4-5), A3 (lanes 6-7), A4 (lanes 8-9), A5 (lanes 10-11), or full length APRIN (7.5 nM) (lane 12). (D) Scheme of APRIN deletion variants fused to GFP. (E) Representative images of laser-irradiated Hela cells expressing different deletions of APRIN (green – variants are mentioned on the right) and immunostained for γ-H2AX (red), 30 minutes or 60 minutes after DNA damage. DAPI (blue) represents the nucleus.
250
ds DNA
150
B
100 75
den. DNA
APRIN
PALB2
(kDa)
MW
A
7.5 10 15 20 0
0
0
0
0
0
0
0
7.5 10 15 20
APRIN (nM) PALB2 (nM) *
50
*
37 1
2
3
1
2
3
4
5
6
46
92
96 100 100
7
8
9
10
48
60
80
91
Annealing (%)
Supplementary Figure 5. (A) SDS-PAGE gel of purified His/Flag APRIN and PALB2 stained with Coomassie blue. Lane 1, Molecular weight markers; Lane 2, PALB2 purified protein (250 ng); Lane 3, APRIN purified protein (250 ng). (B) APRIN and PALB2 promote single-strand annealing. Lane 1, purified 60-bp duplex DNA (dsDNA). Reactions contained denatured 60-bp duplex DNA in buffer with no protein (lane 2; den. DNA: denatured DNA) or with purified APRIN (1–10 nM), lanes 3–6; or with purified PALB2 (1–10 nM), lanes 7–10. Percentage of annealing is depicted under the figure.
A WT
A7
C5
D1
PI
AnnexinV-APC
B Normalized AnnexinV (%)
150
100
50
0 WT
A7
C5
D1
Supplementary Figure 6. (A) AnnexinV staining of Mouse B-cell line CH12F3 depleted from APRIN. WT: wild-type CH12F3. A7 clone represents a deletion in exon 3. C5 and D1 clones represent deletion of exon 5 and a part of exon 6. (B) Quantification of the result.
Double-strand break
1.
IN
APR
4.
γH2AX
p-SMC1
RPA
Phosphorylation
RPA
? R51 R51
?
Replacement of RPA by HR mediators
BRCA2
R51 R51
PALB2
R51 R51
PALB2
IN
APR
R51
BRCA2
?
N
I APR
R51
PALB2
IN
APR
R51 R51
IN
APR
BRCA2 PALB2
BRCA2
2.
Strand invasion DNA strand exchange
R51 R51 R51 R51
IN R51 APR IN R AP
D-loop formation
3.
IN
Second-end capture? Annealing function?
APR
BRCA2
Resolution
DNA REPAIR Supplementary Figure 7. Model for the specific roles of APRIN in homologous recombination (HR). 1. APRIN is rapidly recruited after the DNA damage. 2. APRIN interacts with different proteins implicated in HR. 3. APRIN enhances different steps of HR. 4. APRIN activity could be modulated by phosphorylation.
