Supplemental Materials
Molecular Biology of the Cell
Kumagai and Dunphy
Figure S1.
Recombinant protein complexes for add
-
back e
xperiments
. Recombinant versions
of full
-
length human Treslin alone, full
-
length Treslin in a complex with human MTBP, the
Treslin
-
N fragment in a complex with MTBP, and MTBP alone were purified as
described in the
Materials and Methods. Preparations were subjected to SDS gel electrophoresis and stained with
Coomassie blue.
Figure S2.
Properties of the CTM
d
omain
.
(
A) A structural model for the CTM domain from
human MTBP (residues 813
-
904) was g
enerated by the Robetta server (left panel). The right
panel depicts the published structure of the C
-
terminal region from budding yeast Sld7 (residues
178
-
257)
(
Itou
et al.
, 2015
)
. (B) Sequence alignment of CTM domain from human MTBP and
residues 178
-
257 from yeast Sld7. Predicted and actual helical regions for MTBP and Sld7,
respectively, are indicated. (C) A preparation of GST
-
CTM was c
leaved with AcTEV protease
and subjected to gel filtration in a Superdex
-
200 column. Fractions from the column were
subjected to SDS gel electrophoresis and stained with Coomassie blue.
Data are representative
of two independent experiments.
Figure S3.
EMSA e
xperiments.
(A) The EMSA data from Figure 4A was quantitated and
plotted as indicated. Bars depict mean ± SEM (n=3). (B) The EMSA data from Figure 4D was
quantitated and plotted as indicated. Bars depict mean ± SEM (n=3).
Figure S4.
Characterization of mutant MTBP protein c
omplexes.
Recombinant complexes of
the Treslin
-
N fragment bound to wild
-
type (WT),
∆
C, or 7A versions of MTBP were purified as
described in the Materials and Methods. Preparations were subjected to SDS gel electro
phoresis
and stained with Coomassie blue.
Figure S5.
FACS p
rofiles of
c
ell
s expressing wild
-
type and mutant MTBP p
roteins.
Different
lines of human U2OS Flp
-
In T
-
REx cells (control and harboring inducible versions of the
indicated forms of MTBP) were i
ncubated with doxycycline and treated with control siRNA
(
left
panels
) or MTBP siRNA (
right panels) as described in Materials and Methods. Cells were
labeled with EdU for 30 min and processed for FACS analysis as described in Materials and
Methods.
Data
are representative of three independent experiments.
hMTBP
BSA
Treslin-MTBP
Treslin-N-MTBP
Treslin
MTBP
hTreslin
hTreslin-N
Figure S1
GST
MTBP-CTM
43 kD
12.4 kD
Sld7 C-term
MTBP-CTM
----------HHHHHHHHHHHHHHHHHHHHHH-------HHHHHHHHHHHHHHHHHHHHHH-----------HHHHHHHHHHHHHHHHHHHHHHHH---
MTBP-CTM: MHESKTSRQIK
ESRSQKH
TRILKEVVTETLKKHSITETHECFTACSQRLFEISKFYLKDLKT-------SRGLFEEMKKTANNNAVQVIDWVLEKTSKK
::.* * : . *.::: *: :.*::: : : *: ::* :* . .. **.:::*.:. ::::
Sld7-C: ----------NDKRLQFN-ETLSKLILGGLRLRGISNSITDYQKLYKITFDAAEFTHRDELKRISMGSGEEVSFESLQETVETL-LKLFTKS-------
-----------HHHHHHHHHHHHHHHHHHHH-------HHHHHHHHHHHHHHHHHHHHHHHHHHH--------HHHHHHHHHHHHHHHHH--
A
B
C
Figure S2
0.00
0.25
0.50
0.75
1.00
10
−
7.5
10
−
7
10
−
6.5
10
−
6
Conc (M)
Bound
GST−CTM
GST−CTM−7A
GST
0.00
0.25
0.50
0.75
1.00
10
−
7
10
−
6.5
10
−
6
Conc (M)
Bound
30-mer dsDNA (GST−CTM)
G4 H45 (GST)
G4 H45 (GST−CTM)
G4 H45 (GST−CTM−7A)
A
B
Figure S3
Treslin-N
MTBP
Treslin-N-MTBP-WT
Treslin-N-MTBP-ΔC
Treslin-N-MTBP-7A
Figure S4
0
200
400
600
800
7-AAD
10
0
10
1
10
2
10
3
EdU
0
200
400
600
800
7-AAD
10
0
10
1
10
2
10
3
EdU
0
200
400
600
800
7-AAD
10
0
10
1
10
2
10
3
EdU
0
200
400
600
800
7-AAD
10
0
10
1
10
2
10
3
EdU
0
200
400
600
800
7-AAD
10
0
10
1
10
2
10
3
EdU
0
200
400
600
800
7-AAD
10
0
10
1
10
2
10
3
EdU
0
200
400
600
800
7-AAD
10
0
10
1
10
2
10
3
EdU
0
200
400
600
800
7-AAD
10
0
10
1
10
2
10
3
EdU
Control siRNA
MTBP siRNA
Control
MTBP-WT
MTBP-ΔC
MTBP-7A
Figure S5
Table S1
.
List of Oligonucleotides Used in This Study
siRNA
Control:
ON
-
TARGET plus non
-
coding siRNA #1 (Dharmacon)
ON
-
TARGET plus MTBP #1: UCACAUUGUUGGAUGCUAAUU (Dharmacon)
ON
-
TARGET plus MTBP #2:
GAGAGAAACAGUUAGCUAAUU (Dharmacon)
Cloning of
Xenopus
MTBP from
Xenopus
oocyte cDNA library
Forward: TTCTCCGCTTCCCCTGGTGT
Reverse: AGTCCTCTGTGCTTTCTTCAGC
Cloning of MTBP amino acids 436
-
860 into pH10UE for antibody production
Forward:
GGAATTCCATATGAGAGAGCAGATACTAGCC
Reverse: ATAAGAATGCGGCCGCCTCTGTGCTTTCTTCAGC
For Production of siRNA
-
resistant MTBP
MTBP #1
Forward: GGAAAAGCAACTGGCCAATGTTCAAGTTTTAGCTTTGGAAG
Reverse: GGCCAGTTGCTTTTCCCTCTGTACAATCTGCTCCC
MTBP
#2
Forward:
TACCCTCCTCGACGCCAAAGAATTGCTGAAGTACTTTACCTC
Reverse: GGCGTCGAGGAGGGTAATTGAGTCCCGAGGACC
Steps in Construction of pcDNA5/TO
-
MTBP
-
Twin
-
Strep and pcDNA5/FRT/TO
-
MTBP
-
Twin
-
Strep
i. Amplification of Human MTBP cDNA
Forward: GTTTAAACTTAAGCTTGGCCACCATGGATCGGT
ACCTGC
Reverse: CTTTCTTGCTTGTCTTTTCTAATACCC
ii. Amplification of pcDNA5/TO and pcDNA5/FRT/TO
1: CCAAGCTTAAGTTTAAACGC
2: TGAGCAGATATCCAGCACAGTGG
iii. Tagging C
-
terminal end of MTBP with Twin
-
Strep
1: Twin
-
Strep
-
5
AGACCCACCATGGCTAGCTGGAGCCACCCTCAGTTCGAGAAAGGCGGAGGCTCCGGAGGCG
GATCCGGAGGCAGCGCCTGGAGCCACCCTCAGTTCGAAAAAGGCGCCCTGGAGGTGCTGTT
CCAGGGCCCTGATATC
2: Twin
-
Strep
-
3
GATATCAGGGCCCTGGAACAGCACCTCCAGGGCGCCTTTTTCGAACTGAGGGTGGCTCCAGG
CGCTGCCTCCGGATCCGCCTCCGGAGCCTCCGCCTTTCTCGAACTGAGGGTGGCTCCAGCTA
GCCATGGTGGGTCT
3:
GGCCCCAAGAAGAAGAGGAAGGTGGAGGACAGCGCTTGGAGCCACCCTCAGTTC
4: CTGGATATCTGCTCATTTTTCGAACTGAGGGTGGC
Mutations of pcD
NA5/TO
-
MTBP
-
Twin
-
Strep
MTBP
-
N (1
-
532)
1: GGACAAAATTAAAACCTTCGGCGGCGAAAACCTG
2: GAAGGTTTTAATTTTGTCCATCTTTC
MTBP
-
C (526
-
904)
1: TTTAAACTTAAGCTTGGCCACCATGGACAAAATTAAAACCTTCAATATATTAAATG
2: CCAAGCTTAAGTTTAAACGC
MTBP
-
Δ
C
1: GAATCAAAAGGCGGCGAAAACCTG
2: G
CCGCCTTTTGATTCATGCATACTTTTTTGACCTG
Construction of pGEX4T
-
CTM
1: GAAGACCCCGAGGCAATGCATGAATCAAAAACATCAAGGC
2: TCAGTCAGTCACGATTCATTTCTTGCTTGTCTTTTCTAATACCCAG
MTBP
-
824
-
830 7A
1: CAGCGGCAGCCACACGGATACTGAAAGAAGTA
2:
GTGTGGCTGCCGCTGCTGCTGCTGCCTTAATTTGCCTTGATGTTTTTG
MTBP
-
874
-
876 3A
1: CTCTAAGTTCTATCTAAAGGATCTTAAAGCTGCAGCGGGTCTATTTGAAGAAATGAAG
2: CTTCATTTCTTCAAATAGACCCGCTGCAGCTTTAAGATCCTTTAGATAGAACTTAGAG
MTBP
-
881
-
884 4A
1:
CTTCAAGGGGTCTATTTGAAGCAGCGGCGGCAACAGCAAACAACAATGCTGTAC
2: GTACAGCATTGTTGTTTGCTGTTGCCGCCGCTGCTTCAAATAGACCCCTTGAAG
pGEX4T
-
TEV
-
CTM
1: GACTGAAAATACAGGTTTTCGGATCCACGCGGAAC
2: AAAACCTGTATTTTCAGTCTATGCATGAATCAAAAACATCAAGG
Oligonucleotides used for EMSA expe
riments
ds 30
-
mer
1:
ATAGAGTGTTGCCAGTCTCAAGTACCATGC
2:
GCATGGTACTTGAGACTGGCAACACTCTAT
AT 30
-
mer
1:
AAAAAATTTAAAAAAAAAAAAAAAAAAATT
2: AATTTTTTTTTTTTTTTTTTTAAATTTTTT
G4 17
-
mer (I100
-
15)
GTGGTGGGTGGGTGGGT
G4 17
-
mer AS (antisense of G4 17
-
mer, used
for 17
-
mer dsDNA)
ACCCACCCACCCACCAC
GC 30
-
mer
1:
CCGGGGGGGGGGGGGGGGGGGCCCGGGGGG
2: CCCCCCGGGCCCCCCCCCCCCCCCCCCCGG
G30
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
Pu27
TGGGGAGGGTGGGGAGGGTGGGGAAGG
Pu27 mutant
TGAGGAGAGTGAGGAGAGTGAGGAAGG
Py27
CCTTCCCCACCCTCCCCACCCTCCCCA
Tandem G4 H45
GGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG
Table S2
.
List of Antibodies Used in This Study
Antibodies
Company
Catalog Number
Anti
-
Xenopus
MTBP
This study
NA
Anti
-
Xenopus
Treslin
Kumagai et al. (2010)
NA
Anti
-
Xenopus
Orc2
Kumagai et al. (2010)
NA
Anti
-
Xenopus
Cdc45
Kumagai et al. (2010)
NA
Anti
-
Xenopus
TopBP1
Kumagai et al. (2010)
NA
Anti
-
Human Treslin
Kumagai et al. (2010)
NA
Anti
-
Human MTBP (B
-
5)
Santa Cruz Biotech
sc
-
137201
Anti
-
FLAG M2
Sigma
-
Aldrich
F1804
Anti
-
Histone H3
Abcam
ab1791
Anti
-
GAPDH (14C10)
Cell Signaling Technology
#2118
Anti
-
Human Mcm2 (BM28)
BD Biosciences
610700
Anti
-
BrdU (MoBU
-
1)
Thermo Fisher
B35128
Anti
-
BrdU (B44) (Mouse)
BD Biosciences
347580
Anti
-
BrdU (Bu1/75
ICR1) (Rat)
Novus
NB500
-
169
Anti
-
ssDNA (Rabbit)
IBL
18731
Anti
-
StrepMAB
-
Classic
IBA Life Sciences
2
-
1507
-
001
Anti
-
Myc
Abcam
ab9106