M01654

Yeast application in biomedical research :

a study of Mcb1 in fission yeast

Yeast

Bakery and food products

Beverages

Yeast applications

Science

Bioethanol

Characteristics of Schizosaccharomyces pombe

Phyllum Ascomycota

archaeascomycetes

hemiascomycetes (budding yeast)

Schizosaccharomyces pombe

Saccharomyces cerevisiae Candida albicans

Pischia pastoris

euascomycetes (filamentous yeast)

Neurospora Aspergillus Penicillium

Kingdom : Fungi

Penicillium

S. pombe S. cerevisiae

Cell morphology rod round to ovoid

Cell size 3-4 µm (diameter), 7-14 µm (length) 5-10 µm in diameter

Number of chromosomes 16 3

Cell state haploid diploid

Number of introns ± 5000 250

S. pombe

as model organism

•Small genome

•Haploid

•Fully sequenced

•Short generation time

•Short generation time

•Easy handling and manipulation

•Similarities to mammalian genome

Basic principles of cells

Mitosis

(dormant)

Cell cycle phases

Gap 2

5 5

5

3 53 ORC

Cdt1 4 6 2 Cdc18 GINS Mcm10 Dpb11 Cdc45 Pol polymerase

G1

ORC: Origin Recognition Complex CDC: Cell Division Cycle

starting point of DNA replication origin 4 6 2 ORC Cdt1 Cdc18 2

4 24

7 7

DNA replication initiation

MCM complex 5 3 5 3 6 GINS Cdc45 Pol

Subcomplexes

dimer _{core complex}

5

2

Open ring for loading

7

4

6

3

5

2

6

Mcm = Mini Chromosome Maintenance

3

5

7

7

4

6

•Conserved in eukaryotes

• Essential

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

•Human

•Fission yeast

•Budding yeast

Novel binding component of MCMs

MCM-BP

function relation

7

4

6

3

5

2

• A. thaliana(ETG1 / At2g40550)

• Os01g0166800 (rice)

• C10orf119 (human)

• 1110007A13Rik (mouse)

• CAJ81286 (frog)

• CG3430 (fruitfly)

Research questions

Research questions

What

What is

is the

the biological significance of

biological significance of MCM

MCM--BP?

BP?

Fission yeast MCM-BP

•Human

•Arabidopsis •Xenopus •Drosophila

mcb1

+

is an essential gene

•Drosophila •S. japonicus •Mus musculus •Selaginella •Physcomitrella •Sorghum

•Oryza •Vitis •Ricinus •etc.

Terminal phenotype

PCR

Approach :

Isolation of conditional (

ts

) mutants of

MCM-BP in fission yeast

mcb1+ _{5Flag kanMX6} Approach:

transformation into wild type strain

G418+

ts check

mcb1

L363P

= mcb1

ts1

mcb1

ts2

mutants elongated at restrictive

temperature

25C _{34C 6h}

38_C 38C

cut phenotype abnormal genetic material distribution

25C

mcb1

ts2

mutants show abnormal nuclear

phenotype at restrictive temperature

34_C 34_C

cut phenotype abnormal septation cut : cell uniform tone

derived from disturbed DNA replication

mcb1

ts2

viability decreased at

restrictive temperature

0 2 4 6 8

mcb1

mcb1ts2

h at 36_C

mcb1

0 2 4

100

6 8

hours at 36C 80

60 40 20 110 90 70 50 30 10 0

%

v

ia

bi

lit

y mcb1ts2

mcb1

ts

mutants had decreased

association with MCMs

Mcm7-3HA Mcb1-5F Mcb1ts1_-5F

+ + + + + + -- -- + + + + + + -- -- + ₊ + + + + -- -- + + + + + + --

-Input Flag-IP Input Flag-IP

25C 34C 6h

Flag Co-IP

Mcb1ts1_-5F

1.4 1.6

mcb1ts2_-5Flag

mcb1-5Flag

Mcm7-3HA ChIP

Result 4 :

mcb1

ts

showed reduced Mcm7 binding onto origin

28C

20C 4h

-240’ 20’…………..120’Time 0 (M-phase arrest)

Early origins: used every cell cycle

ars2004

Late origins: used rarely

non-ars1

%

IP

/I

np

ut

20 40 60 80 100120

0 0 20 40 60 80 100120

1.0

0.2 0.4 0.8 0.6 1.2

0 1.4

mcb1ts2_-5Flag

(min)

ars2004 non-ars1

m

cb

1

+

0 hMcm2-GFP6 h 0 hMcm5-RFP6 h _{0 h}Mcm7-GFP_{6 h}

MCM2, 4-7 mislocalized in

mcb1

ts

at

high temperature

34_C

25C

34C

m

cb

1

ts

2

m

cb

1

ts

1

34C

100 130

180 25 34 25 34 25 34 25 34 25 34 C

2

Mcms-GFP

mcb1ts2 3 4 6 7

25 34°C

Mcm5-RFP mcb1ts2

(kDa) _anti-GFP

anti-RFP anti-tubulin anti-tubulin

mcb1

influenced Mcm localization in

Crm1-dependent fashion

25_mcm2-GFP C 34C 25_mcm6-GFP C 34C 25_C 34C

mcm2-GFP crm1-11R mcm6-GFP crm1-11R

25_C 34C

5 µm

25_C 34C 25_C 34C

18

m

cb

1

ts

2

Crm1: exportin

crm1-11R

inactivation of protein export

Summary

Summary

mcb1

+

•_••_•Essential gene_{Association with Mcms}Essential gene_{Association with Mcms}

•Lost association to Mcms

•Lost association to Mcms

mcb1

ts

•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

•Lost association to Mcms

•Impair the DNA damage repair

•Delay in S-phase progression

•Reduction of MCM chromatin binding

cytoplasm

Export OFF Mcb1 Mcb1

ts

BP _{2 2} 2 _Crm1

Export ON