GENESDEV/2008/105015, Supplementary Figure 1, Xu et al. RMI2 RPA14 BLM RMI2 Topo 3α RMI1 RPA70 RPA32 RMI1 IP Complex1 Complex2 Complex2 -BLM NE Topo 3α RMI1 B 1 2 3 4 BLAP250 BLM Topo 3α RMI1 RMI2 RPA70 RPA32 Complex1 Complex2 Complex2 -BLM NE Complex1 Complex2 Complex2 -BLM NE Input RMI1 IP C BLM Topo 3α RMI1 RMI2 Load 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 670 kDa 440 kDa Void Complex1 Complex2 A 66 kDa 1 2 3 4 5 6 7 8 Legend. (A) Immunoblotting shows that RMI2 in HeLa nuclear extract fractionates in 2 major peaks by Superose 6 gel-filtration chromatography, which correspond to two complexes designated as Complex 1 and 2 (marked by arrows on top). Notably, the elution profiles of RMI2, RMI1 and Topo 3α are coincident, suggesting that they always associate in complexes. In addition, BLM overlaps with the peak of complex 1 but not complex 2, suggesting that BLM is absent in complex 2. (B)(C) Silver-stained SDS gels (B), and immunoblotting (C), show that while Complex 1 contains all BLM complex components, complex 2 consists of only Topo 3α, RMI1 and RMI2. Immunoprecipitation was done by using RMI1 antibody from crude nuclear extract (NE), fractions containing either Complex 1 or Complex 2, or Complex 2-containing fractions after immuno-depletion of BLM (Complex 2-BLM), as indicated on top of the figure. Notably, RMI1 co-immunoprecipitated with Topo 3α and RMI2 but not BLM (lane 4 in B; lane 8 in C), indicating that the two RMI proteins and Topo 3α form a stable subcomplex. RMI2 associates with RMI1 and Topoisomerase 3α in two different complexes: one contains BLM and the other one does not
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GENESDEV/2008/105015, Supplementary Figure 1, Xu
et al.
RMI2RPA14
BLM
RMI2
Topo 3αRMI1
RPA70
RPA32
RMI1 IP
Com
plex
1Co
mpl
ex2
Com
plex
2-B
LM
NE
Topo 3αRMI1
B
1 2 3 4
BLAP250 BLM
Topo
3α
RMI1
RMI2
RPA70
RPA32
Com
plex
1Co
mpl
ex2
Com
plex
2-B
LM
NE Com
plex
1Co
mpl
ex2
Com
plex
2-B
LM
NE
Input RMI1 IPC
BLM
Topo 3α
RMI1
RMI2
Load 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
31 32 33 34
670 kDa 440 kDaVoid
Complex1 Complex2A
66 kDa
1 2 3 4 5 6 7 8
Legend. (A) Immunoblotting shows that RMI2 in HeLa nuclear extract fractionates in 2 major peaks by Superose 6 gel-filtration chromatography, which correspond to two complexes designated as Complex 1 and 2 (marked by arrows on top). Notably, the elution profiles of RMI2, RMI1 and Topo 3α
are coincident, suggesting that they always associate in complexes. In addition, BLM overlaps with the peak of complex 1 but not complex 2, suggesting that BLM is absent in complex 2. (B)(C) Silver-stained SDS gels (B), and immunoblotting (C), show that while Complex 1 contains all BLM complex components, complex 2 consists of only Topo 3α, RMI1 and RMI2. Immunoprecipitation was done by using RMI1 antibody from crude nuclear extract (NE), fractions containing either Complex 1 or Complex 2, or Complex 2-containing fractions after immuno-depletion of BLM (Complex 2-BLM), as indicated on top of the figure. Notably, RMI1 co-immunoprecipitated with Topo 3α
and RMI2 but not BLM (lane 4 in B; lane 8 in C), indicating that the two RMI proteins and Topo 3α
form a stable subcomplex.
RMI2 associates with RMI1 and Topoisomerase 3α in two different complexes: one contains BLM and the other one does not
The RMI1-OB1 domain has sequence similarityto the “Wedge” domain of bacterial RecG helicase
GENESDEV/2008/105015, Supplementary Figure 2, Xu et al.
Legend. Sequence alignment of RMI1-OB1 domain with the wedge domain of RecG helicase. The 5 predicted β-strands of OB-folds are underlined with arrows. The identical and conserved residues are highlighted with red and yellow, respectively. Green highlights residues only conserved in some OB1 and wedge domains. Solid arrow indicates a highly conserved aromatic residue which is important for DNA binding activity of the Wedge domain and also conserved inRMI1-OB1. An empty arrow indicates another aromatic residue critical for DNA binding activity of RecG but notconserved in RMI1. The abbreviations for RMI1 proteins are shown in Fig. 2 Legend.
Moc
k71
2.5
356.
2517
8.13
89.0
644
.53
22.2
711
.13
5.57
2.78
1.39
0.7
0.35
0.17
0.09
0.04
0.02
0.00
22.5 nM
hTOPO3α
+ 10 nM
BLM +
nM
hRMI1-hRMI2(WT)
*
RsaI
HhaI
*
Moc
k50
025
012
562
.531
.25
16.6
37.
813.
911.
950.
980.
490.
240.
120.
060.
030.
020.
00
22.5 nM
hTOPO3α
+ 10 nM
BLM +
nM
hRMI1-hRMI2(K121A)
*
RsaI
HhaI
*
Moc
k
hRM
I2 o
nly
2500
hTO
PO3α
only
1250
625
312.
515
6.3
78.1
39.1
19.5
9.8
0.00
22.5 nM
hTOPO3α
+ 66 nM
BLM +
nM
hRMI2BLM
only
*
RsaI
HhaI
*
Moc
k57
028
514
2.5
71 35.6
17.8
8.9
4.5
2.23
1.11
0.56
0.28
0.14
0.07
0.03
0.02
0.00
22.5 nM
hTOPO3α
+ 10 nM
BLM +
nM
hRMI1 (yeast)
*
RsaI
HhaI
*
RMI1-RMI2 wildtype and RMI1-RMI2(K121A) mutant complexes have similar activity in stimulating double-Holliday Junction dissolution compared to RMI1 alone; RMI2 displays no detectable activity
GENESDEV/2008/105015, Supplementary Figure 3, Xu et al.
A B
D C
Legend. Audioradiographs showing stimulation of double-Holliday Junction (dHJ) dissolution by the recombinant RMI1-RMI2 wildtype complex (A), RMI1 purified from yeast (B), and RMI2 (C), and the RMI1-RMI2 (K121A) mutant complex (D). The
presence of BLM and Topo 3α
is indicated on top. The dHJ substrate and the resolved product are shown on the right.
Purification of human RMI1 expressed in Yeast
GENESDEV/2008/105015, Supplementary Figure 4, Xu et al.
Legend. (A) 10% SDS-PAGE of fractions from the hRMI1 purification. The crude extract, flow-through (FT), and eluate (EL) from the IgG-agarose column, and the flow-through and fractions 1 through 7 from the calmodulin-sepharose column are shown. Proteins were detected by silver staining. The sizes of the molecular weight markers are indicated, as is hRMI1. (B) 10% SDS-PAGE of 1 µg of concentrated, purified hRMI1, stained with Coomassie blue. The asterisk indicates a proteolytic breakdown product of hRMI1 identified by mass spectrometry.
Purification of human RMI1 expressed in Yeast
GENESDEV/2008/105015, Supplementary Figure 4, Xu et al.
Legend. (A) 10% SDS-PAGE of fractions from the hRMI1 purification. The crude extract, flow-through (FT), and eluate (EL) from the IgG-agarose column, and the flow-through and fractions 1 through 7 from the calmodulin-sepharose column are shown. Proteins were detected by silver staining. The sizes of the molecular weight markers are indicated, as is hRMI1. (B) 10% SDS-PAGE of 1 µg of concentrated, purified hRMI1, stained with Coomassie blue. The asterisk indicates a proteolytic breakdown product of hRMI1 identified by mass spectrometry.
Purified recombinant RMI2 and the RMI complex containing either wildtype or K121A mutant of RMI2 from bacteria
K121A
RMI1
RMI2
WTMW
200
11697
66
45
31
2114
RMI1-RMI2complex
GENESDEV/2008/105015, Supplementary Figure 5, Xu et al.
Legend. (A) A Coomassie gel showing recombinant RMI2 protein purified from E.coli. (B) A Coomassie gel showing recombinant RMI complex containing either wildtype (WT) or K121A mutant of RMI2 purified from E.coli. The molecular weight markers (MW, in KDa) were included.
BA
20011697
66
45
31
21
14
MW RMI2
RMI2
Background
Well
10 30 100
300
1 3
RPA (30 nM)RMI (1 µM)
10 30 100
300
1 310 30 100
300
1 3ssDNA (nM)
None
*
RPA-ssDNAcomplexes
The RMI complex displays no detectable ssDNA binding activity, unlike RPA
GENESDEV/2008/105015, Supplementary Figure 6, Xu et al.
Legend. Autoradiographs showing that the RMI complex displays no detectable binding activity to ssDNA (middle panel). RPA was included as a positive control, and the RPA-ssDNA complexes are indicated by arrows. Reactions containing no added proteins (None) were included as a negative control, which reveals the presence of background signals possibly due to DNA mixtures yielded by non-specific annealing of ssDNA.
GENESDEV/2008/105015, Supplementary Figure 7, Xu et al.
The RMI2-deficient DT40 cells differ from the BLM-deficient cells in that they lack hypersensitivity to DNA damage drugs
Legend. (A) and (B) Histograms showing sensitivity of DT40 cells with various genotypes to cisplatin and methyl methanesulfonate (MMS). Mean and standard deviation from three independent experiments are shown. (C) A histogram showing growth curves of cells of various genotypes as indicated with different color codes. Cells were counted by flow cytometry, and at least three independent experiments are performed.