Fission Yeast MCM-binding Protein
Mcb1 is Related to MCM Complex and
Important for Efficient Pre-RC formation
Fission Yeast MCM-binding Protein
Mcb1 is Related to MCM Complex and
Important for Efficient Pre-RC formation
3W5II 12.12.13
Venny Santosa, Sabrina Martha, Noriaki Hirose and Katsunori Tanaka
Kwansei Gakuin University
Department of Bioscience
3
5 35
ORC Cdt1 7 4 6 3 5 2 ORC Cdt1 Cdc6 Cdc6 Mcm10 Dpb11 MCM hexamer complex
MCM complex as a helicase in DNA replication
polymerase 2 7 4 6 7 4 6 2
•
AAA+ ATPase family
•
Walker A, B, RING Finger
•
Essential
•
Conserved in eukaryotes
Background 1:
G1
Pre-RC
GINS
Mcm10
Cdc45 Cdc6Cdt1
SPAC1687.04
=
mcb1
+
(MCM Binding Protein 1)
Background 2:
Fission Yeast MCM
Fission Yeast MCM--BP
BP
highly conserved among eukaryotes
except for budding yeast and
Caenorhabditis elegans
MCM-BP: Novel binding component of MCMs
Terminal phenotype
essential
3 5 3 5 ORC ORC Cdt1 Cdc6 Cdc6 GINS Mcm10 Dpb11 Sld2 Cdt1 Cdc6
Pol DNA helicase
MCM-BP
Sakweet al., 2007; Ding and Forsburg,2011
Nishiyamaet al., 2011
2
BP
Background 3:
Cross-organism studies of MCM-BP
7 4 6 3 5 2 7 4 6 2 7 4 6 2 Cdt1 GINS Cdt1 ORC Pol Mcm10 GINSPol ORC Mcm10 GINSPol DNA helicase Replication fork Termination of replication Mitosis
Fission Yeast
? ?Takahashi et al., 2008; Takahashi et al., 2010
•Mcm2 alternate within MCM complex
•Aided survival
•Required for sister chromatids cohesion
•MCM complex unloader
•Re-replication prevention
•MCM helicase antagonist
Co-IP
Background 4:
Fission yeast Mcb1 associates with all of Mcm subunits
Y2H assay
GA
L4
-AD
vector Mcm2 Mcm3 Mcm4 Mcm5 Mcm6 Mcm7
vector Mcb1 GAL4-DBD
+His+Ade –His-Ade +His+Ade –His-Ade
in vitro
binding assay
T7-His6-Mcb1
GST-Mcm2-7
GST
T7-His6-Mcb1 + + + + + + +
GST pull-down
GST-Mcm2-7 - 2 3 4 5 6 7
Result 1 :
Isolation of temperature sensitive mutants of
mcb1
mcb1
ts1=mcb1
L363Pmcb1
ts2=mcb1
L254PYES
0.005% MMS
10 µM CPT
WT
mcb1-5Flag mcb1ts2-5Flag
mcb1ts1-5Flag
28C 30C 32C
25C
25
C
36
C 10h
cdc phenotype
34
C 4h
cut phenotype 5µm
10 µM CPT
5 mM HU
Mcm7-3HA Mcb1-5F Mcb1ts1-5F
Mcb1ts2-5F
+
+
+
+ + + +
--
--
+
+
+ + + +
--
-- +
+
+
+
+ + + +
--
--
+
+
+ + + +
--
-- +
Input
Flag-IP
Input
Flag-IP
25
C
36
C 6h
Mcm2 Result 2 :
Mcb1-Mcms interactions are affected in
mcb1
tsmutants
Mcb1-Flag Co-IP
Mcm2 Mcm4 Mcm5 Mcm6
Mcm7-3HA Mcb1-5FLAG
in vitro
binding assay
Poster: 3P-0195
mcb1-5Flag ce lls 100 90 100 120 (min) 100 120 (min)
28C
20C 4h -240’
Time 0 (M-phase arrest) 20’…………..120
’ Result 3 :
mcb1
tsshowed delayed S-phase progression
FACS
Septation index
nda3-KM311
block and release
28C
mcb1-5Flag % se pt at ed ce lls (min) 0 20 40 60 80 100 120
0 20 40 60 80 10 50 30 70 90
mcb1ts2-5Flag
mcb1
ts2-5Flag
2C 4C 2C 4C
%
IP
/In
pu
t 1.0
0.8 1.2 1.4 1.6
mcb1ts2-5Flag
mcb1-5Flag
Mcm7-3HA ChIP
Result 4 :
mcb1
tsshowed reduced Mcm7 binding onto origin
%
IP
/In
pu
t
20 40 60 80 100120
0 0 20 40 60 80 100120
0.2 0.4 0.8 0.6
0
(min)
ars2004 non-ars1
9
Synthetic effects with mutants involved in S phase
Isolation of multicopy suppressors
Genetic analysis using
mcb1
ts
mutant
10
Isolation of multicopy suppressors
Result 5 :
Mutant Synthetic effect
Pre-RC
nda1-376 (Mcm2) synthetic lethal cdc19-PI (Mcm2) + + +
cdc21-M68 (Mcm4) + + + mis5-28 (Mcm6) + + +
orp5-H19 + +
orp1-H5 + +
cdc18-K46
-hsk1-1312 + + + nda4-108 (Mcm5) + + +
mcm10-5 + + +
mcb1
ts2showed synthetic defects with
mutants of S-phase genes
+++ strong ++ moderate
+ slight
Pre-IC
mcm10-5 + + +
cut5-T401 + + +
hsk1-89
-goa1-U53 (Cdc45) + sna41-928 (Cdc45)
-sld3-10
-cdc2-33
-psf3-1
-Polymerase
cdc27-P11 (Polδ) + + + cdc6-121 (Polδ) + +
swi7-H4 (Polα) +
pola-ts13 +
cdc20-M10 (Polε) - 11 + slight
Screening of multicopy suppressors of mcb1
ts2Isolation of
mcm5
+/nda4
+plasmid
vector
p
mcb1
+pmcm5
+25
°
C 34
°
C 36
°
C
m
cb
1
ts
2
Result 6 :
Multicopy plasmid of
mcm5
+specifically suppresses
mcb1
ts2vector pmcm2+
pmcm3+
pmcm4+
pmcm5+ pmcm6+
pmcm7+
25°C 30°C 32°C 34°C
m
cb
1
ts
2
pmcm5
+m
cb
1
Result 7 :
mcb1
ts2
is suppressed by
cig2
D
or
rum1
+
ded1
csmutant
cig2
repression
(Grallert, et al., JCS, 2000)
Screening of spontaneous intergenic suppressors of
mcb1
ts23 copies of
mcm5+
32C
28C 34C
20C
wt
mcb1ts2
mcb1ts2+X
mcb1ts2+Y
mcb1ts2+Z
mcb1ts2+X
mcb1ts2+Y
vector
pcig2+
vector
pcig2+
32C
28C 34C
25C
prum1+ vector
25°C 30°C 32°C
mcb1ts2
ded1 mutant
cig2
repression
(Grallert, et al., JCS, 2000)CDK modulator
MCM
G1 phase S phase
Sld3
DDK
ORC
pre-RC
cdc19-P1/mcm2 nda1-376/mcm2 cdc18-K46
cdc21-M68/mcm4 mis5-268/mcm6 cdc2-33
cdc2-56 cdc10-129
suppressed by
cig2
D
or rum1
+not suppressed by
cig2D or rum1+
CDK modulation suppresses mutations in pre-RC components
Screening of spontaneous intergenic suppressors of
mcb1
ts2Cdc45
CDK
Cut5
GINS Drc1
RPA RPA
mis5-268/mcm6 orp1-4/orc1
mcb1
ts1,2nda4-108/mcm5 cdc20-M10
cdc22-M45 cdc24-M38 pol1-1
polδts
cig2
D
or rum1
+Jallepalliet al., 1997; Lopez-Girona et al., 1998; Grallertet al., 2001; Yamada et al., 2004
not suppressed by
cig2D or rum1+
Result 9 :
Crm1-dependent mislocalization of MCM proteins
in
mcb1
ts2mutants
Mcm2-GFP
Mcm6-GFP
25 34
C
25 34
Tubulin
Mcm
mcm2-GFP mcm6-GFP
mcm2-GFP
mcb1
ts2mcb1
+mcm6-GFP
25
C 34
C
25
C 34
C
25C 34C
mcm2-GFP
mcm2-GFP crm1-11R
mcm6-GFP
mcm6-GFP crm1-11R
25C 34C
5 µm 25C 34C 25C 34C
mcb1
s1Involvement of Mcb1 in pre-RC assembly
Hypothesis
cytoplasm
Export OFF
3 5 4 6Mcb1 Mcb1
L254P
Dr. Masukata, Dr. Nakagawa, Dr. Ishii (Osaka Univ., Japan)
Dr. Masai (Tokyo Metropolitan Institute, Japan)
Dr. Niki, Dr. Aoki, Dr. Kanemaki (NIG, Japan)
Dr. Furuya (Kyoto Univ., Japan)
Acknowledgements
Acknowledgements
Dr. Furuya (Kyoto Univ., Japan)
Eishi Noguchi (Drexel Univ., USA)
Susan Forsburg (Univ. of Southern California, USA)
Beáta Grallert & Erik Boye (ICR, Norway)
NBRP
