M267 Lecture 4, Cell Cycle
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M267 Lecture 4, Cell Cycle February 27, 2006. Cohesins. cyclin deg. cdc2/28. G 2. Prophase. Metaphase. Anaphase. Telophase. G 1. - p34 cdc2 cyB. +p34 cdc2 cyB. 0 15 30 50 0 15 30 50. min. min. interphase. mitotic. M extract. I extract. buffer.

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M267 Lecture 4, Cell Cycle February 27, 2006

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M267 lecture 4 cell cycle february 27 2006

M267 Lecture 4, Cell Cycle

February 27, 2006


M267 lecture 4 cell cycle february 27 2006

Cohesins

cyclin deg.

cdc2/28

G2

Prophase

Metaphase

Anaphase

Telophase

G1


M267 lecture 4 cell cycle february 27 2006

-p34cdc2cyB

+p34cdc2cyB

0 15 30 50 0 15 30 50

min

min

interphase

mitotic


M267 lecture 4 cell cycle february 27 2006

M extract

I extract

buffer

Time 0 6 12 20 25 30 35 40 60


M267 lecture 4 cell cycle february 27 2006

cyclin B

cyclin B

cdc2

p

cdc2

p

cdc25

p

p

p

p

cyclin B

cyclin B

+

cdc2

p

cdc2

cyclin B sythesis

p

p


M267 lecture 4 cell cycle february 27 2006

cyclin B

protein X

cyclin B

cdc2

p

cdc2

p

cdc25

p

p

p

p

protein X

p

p

cyclin B

cyclin B

protease

+

cdc2

p

cdc2

cyclin B sythesis

p

p


M267 lecture 4 cell cycle february 27 2006

Ub

Ub

Ub

Ub

p

p

APC

proteasome

Cyclin B

Cyclin B

Cyclin B

Ub

Ub

Ub

Ub


M267 lecture 4 cell cycle february 27 2006

Cln1-

Cln2-

Cln3-

G1

M

G2

S


M267 lecture 4 cell cycle february 27 2006

Presence of methionine

Cln1-

Cln2-

Cln3-

G1

M

G2

S

Met-cln2 (met repressible)

Gal-clb2 (gal inducible)


M267 lecture 4 cell cycle february 27 2006

Cyclin B RNA

Cyclin B protein

H1 kinase activitry

These cells do not express any G1 cyclins (clns). Consequently, they arrest in G1. They can accumulate cyclin B mRNA in response to galactose.

Although cyclin B mRNA can be made, cyclin B protein cannot be made – cyclin B protein continues to be degraded.

Since no cyclin is available to activate cdc28, no CDK activity (e.g. histone phosphorylation activity) is present.

+ methionine, + galactose


M267 lecture 4 cell cycle february 27 2006

Cyclin B RNA

Cyclin B protein

H1 kinase activitry

If one removes methionine, Cln2 (the G1 cyclin) is made. Now cyclin B protein can be made.

Now that cyclins are made, the CDK catalytic subunit can be activated, and CDK activity (histone phosphorylation) can occur


M267 lecture 4 cell cycle february 27 2006

APC mediated destruction machinery for cyclin B

Cyclin Oscillations in yeast

CLN1, CLN2 and CLN3 associated kinases are absent in early G1, reach peak levels at the G1-S transition, and decline as cells enter G2. These kinases not only promote budding and DNA synthesis, but also inactivate CLB cyclin proteolysis. Activation of the mitotic kinase during G2 causes repression of CLN1, CLN2 and CLN3 synthesis and onset of M phase and ultimately leads to the reactivation of CLB proteolysis. Cyclin B proteolysis then persists during G1 until the reactivation of CLN cyclins


M267 lecture 4 cell cycle february 27 2006

Cyclin B RNA

Cyclin B protein

H1 kinase activitry

What would happen to CLB2 synthesis if the cyclosome was “broken”?

Gal promoter

CLB2 protein

b-gal protein


M267 lecture 4 cell cycle february 27 2006

pGAL-CLB2

-GAL

+GAL

WT

Cdc16-123

Cdc23-304

Cse1-22


M267 lecture 4 cell cycle february 27 2006

Cse1

cdc16

cdc27

cdc23

The cyclosome, the “machine” that

ubiquitinates cyclin B, and leads to

cyclin B desruction


M267 lecture 4 cell cycle february 27 2006

Express a non-degradable Cyclin B

Cells arrest here

Cells do not arrest

at anaphase, even

though cyclin B is

present.


M267 lecture 4 cell cycle february 27 2006

Inhibit the activity of the cyclosome/APC

Cells arrest at anaphase


M267 lecture 4 cell cycle february 27 2006

Inhibit the activity of the cyclosome/APC

APC directed degradation of some other protein – before cyclin is degraded – must be necessary for sister chromatid separation; i.e., for the metaphase to anaphase transition to occur.

Degradation of cyclin B must be necessary for a subsequent step; for the transition from telophase to G1.

?

Cells arrest at anaphase


M267 lecture 4 cell cycle february 27 2006

Cut 2 accumulates in anaphase in APC mutants

Expression of a non-degradable form of Cut 2 causes arrest in anaphase.


M267 lecture 4 cell cycle february 27 2006

Cohesin, holds sister chromatids together

Separase, a protease that cleaves cohesins, permitting chromosome separation (i.e., anaphase) to occur

When securins are destroyed, separase is activated, the cohesins are cleaved, and the sister chromatids can separate in anaphase.

Securins; inhibitors of the separase enzyme.


M267 lecture 4 cell cycle february 27 2006

securin

Cohesins, separase and securins


M267 lecture 4 cell cycle february 27 2006

Pds1 (securin)

Clb2


M267 lecture 4 cell cycle february 27 2006

Hct1

Cdc20

securin

Cohesins, separase and securins


M267 lecture 4 cell cycle february 27 2006

Pds1/Cut2/securin


M267 lecture 4 cell cycle february 27 2006

1 2 3 4 5 6

Time (min)

0 15 30

0 15 30 60

1, 3, 5: w.t. cdc20.

2, 4, 6: mutant cdc20.


M267 lecture 4 cell cycle february 27 2006

APC

APC

cdc20

cdh1

proteasome

proteasome

Substrate 1

Substrate 2

AT M/G1

AT ANAPHASE

D-BOX (CUT2)

Target

RXXLXXXXN

Target

KENXXXN

Target


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