M01654

Yeast application in biomedical research :
a study of Mcb1 in fission yeast

Venny Santosa
MB UKSW
Permi 06.06.15

Yeast applications
Bakery and food products
Beverages

Yeast

Supplements and probiotics

Science
Bioethanol

Characteristics of Schizosaccharomyces pombe
Kingdom : Fungi
Phyllum

Ascomycota

archaeascomycetes

Schizosaccharomyces pombe
Saccharomyces cerevisiae

hemiascomycetes
(budding yeast)
euascomycetes
(filamentous yeast)

Cell morphology
Cell size
Number of chromosomes
Cell state
Number of introns
Propagation method
Extended period of cell cycle

Candida albicans
Pischia pastoris
Neurospora
Aspergillus
Penicillium

S. pombe
rod
3-4 µm (diameter), 7-14 µm
(length)
16
haploid
± 5000
fission
G2

S. cerevisiae
round to ovoid
5-10 µm in diameter
3

diploid
250
budding
G1

S. pombe as model organism
•Small genome
•Haploid
•Fully sequenced
•Short generation time
•Easy handling and manipulation
•Similarities to mammalian genome

Basic principles of cells
Cell cycle and regulation

Cell cycle phases
Mitosis
(dormant)

Gap 2

Synthesis
5

DNA replication initiation
starting point of DNA replication

G1

origin
Cdc18

2 Cdt1 2
5
4
5 4
MCM
3 6
complex 3 7 6

7
Dpb11

polymerase
Pol

S

ORC

Mcm10

Cdc45 GINS

ORC: Origin Recognition Complex
CDC: Cell Division Cycle

2 Cdc18 Cdt1 2
5 4
5 4

ORC
3 6
3 6
7
7

2
5 4
ORC
3 6
7

Unwind the DNA
DNA helicase double helix
2
5 4
3 6
7

Replication fork

5
3

2
7

6
4
6

Mcm = Mini Chromosome Maintenance
Open ring for
loading

Subcomplexes
dimer

5
3

2
7

(Forsburg, 2004. Microbiol. Mol. Biol. Rev.)

•Conserved in eukaryotes
• Essential

core complex

6
4

2

5
6
3 7 4
•Human

•Fission yeast
•Budding yeast
•Xenopus
•Arabidopsis
•Drosophila
7

Novel binding component of MCMs

MCM-BP

•
•
•
•
•
•
•

function

relation

5
3

2
7

6
4

A. thaliana (ETG1 / At2g40550)
Os01g0166800 (rice)
C10orf119 (human)
1110007A13Rik (mouse)
CAJ81286 (frog)
CG3430 (fruitfly)
SPAC1687.04 (fission yeast)
8

Research questions

What is the biological significance of MCMMCM-BP?

Fission yeast

9

Fission yeast MCM-BP
mcb1+ is an essential gene
•Human
•Arabidopsis
•Xenopus
•Drosophila
•S. japonicus
•Mus musculus
•Selaginella
•Physcomitrella
•Sorghum
•Oryza
•Vitis
•Ricinus
•etc.

Terminal phenotype

Dissection of diploid of mcb1+/mcb1::hygr

10

Approach
:
Approach:

Isolation of conditional (ts) mutants of
MCM-BP in fission yeast
mcb1+

5Flag

kanMX6

PCR
transformation into wild type strain
G418+
ts check

mcb1L363P = mcb1ts1
mcb1L254P = mcb1ts2
11

mcb1ts2 mutants elongated at restrictive
temperature
25C

34C 6h

5 µm

12

mcb1ts2 mutants show abnormal nuclear
phenotype at restrictive temperature
25C

34-38C

cut : cell uniform tone
derived from disturbed
DNA replication

cut phenotype

abnormal genetic
material distribution

38C

38C

34C

34C

cut phenotype

abnormal septation
13

mcb1ts2 viability decreased at
restrictive temperature
0

h at 36C
4

2

6

8

mcb1

mcb1ts2

mcb1
mcb1ts2

% viability

110
100
90
80
70
60
50
40
30
20
10
0
0

2

6
4
hours at 36C

8
14

mcb1ts mutants had decreased
association with MCMs
Flag Co-IP
Mcm7-3HA
Mcb1-5F
Mcb1ts1-5F
Mcb1ts2-5F

25C

+
-

Input

+
+
-

+
+
-

+
+

+
-

Flag-IP

+
+
-

+
+
-

+
+

+
-

Input

+
+
-

+
+
-

34C 6h
Flag-IP

+
+

+
-

+
+
-

+
+
-

+
+

Mcm2
Mcm4
Mcm5
Mcm6
Mcm7-3HA
Mcb1-5FLAG
15

Result 4 :

mcb1ts showed reduced Mcm7 binding onto origin
Mcm7-3HA ChIP
28C
20C 4h 20’…………..120’
-240’
Time 0 (M-phase arrest)

Late origins: used rarely
non-ars1

mcb1-5Flag
mcb1ts2-5Flag

% IP/Input

1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0

Early origins: used every cell cycle
ars2004

n=3
0 20 40 60 80 100120
ars2004

0 20 40 60 80 100120 (min)
non-ars1

16

MCM2, 4-7 mislocalized in mcb1ts at
high temperature
34C
Mcm5-RFP
0h
6h

Mcm7-GFP
0h
6h

mcb1+

Mcm2-GFP
0h
6h

25C

mcb1ts1

mcb1ts2

34C

36C
5 µm

17

mcb1 influenced Mcm localization in
Crm1-dependent fashion
Mcms-GFP
mcb1ts2

(kDa) 180
130
100

2
3
4
6
7
25 34 25 34 25 34 25 34 25 34 C
anti-GFP

Mcm5-RFP mcb1ts2
25 34°C
anti-RFP

anti-tubulin

crm1-11R

inactivation of
protein export
to cytoplasm

mcb1ts2

Crm1: exportin

anti-tubulin

mcm2-GFP
34C
25C

mcm6-GFP
34C
25C

mcm2-GFP crm1-11R
34C
25C

mcm6-GFP crm1-11R
34C
25C

5 µm

18

Summary

mcb1+
mcb1ts

•Essential gene
•Association with Mcms

•Lost association to Mcms
•Impair the DNA damage repair
•Delay in S-phase progression
•Reduction of MCM chromatin binding
•Crm1-dependent mis-localization of Mcm proteins

19

Hypothesis
Involvement of Mcb1 in pre-RC assembly
Mcb1 Mcb1ts

cytoplasm

Export OFF

BP

2

5
3

BP

BP

7

6
4

5 6
3 4
7

2
2Cdt1
6 ORC 5 6
5 Cdc18
37 4
3 7 4 Cdt1

2

5 6
3 4
7

2

5 6
3 4
7

BP

5
3

2
5 6
3 4
7

BP

6
4
7

2

BP

Cdt1

2

2

BP

BP

2

Export ON

2
2
6
5 6Cdc18
5
ORC
3 4
3 4
7
Cdt1 7

ts

Crm1
7

6
4

2

2

5 6
3 4
7

5
3
ts

Cdt1

ts

2
6
5 Cdc18
3 4 ORC
7

2
5 6
3 4
7

2

2
5 6
3 4
7
ts
ts

5
3

ts
7

2
5 6
3 4
7

6
4

ts

Cdc18

ORC
20