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M267 Lecture 4, Cell Cycle February 27, 2006

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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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slide2

Cohesins

cyclin deg.

cdc2/28

G2

Prophase

Metaphase

Anaphase

Telophase

G1

slide3

-p34cdc2cyB

+p34cdc2cyB

0 15 30 50 0 15 30 50

min

min

interphase

mitotic

slide4

M extract

I extract

buffer

Time 0 6 12 20 25 30 35 40 60

slide5

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

slide6

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

slide7

Ub

Ub

Ub

Ub

p

p

APC

proteasome

Cyclin B

Cyclin B

Cyclin B

Ub

Ub

Ub

Ub

slide8

Cln1-

Cln2-

Cln3-

G1

M

G2

S

slide9

Presence of methionine

Cln1-

Cln2-

Cln3-

G1

M

G2

S

Met-cln2 (met repressible)

Gal-clb2 (gal inducible)

slide10

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

slide11

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

slide12

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

slide13

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

slide14

pGAL-CLB2

-GAL

+GAL

WT

Cdc16-123

Cdc23-304

Cse1-22

slide15

Cse1

cdc16

cdc27

cdc23

The cyclosome, the “machine” that

ubiquitinates cyclin B, and leads to

cyclin B desruction

slide16

Express a non-degradable Cyclin B

Cells arrest here

Cells do not arrest

at anaphase, even

though cyclin B is

present.

slide18

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

slide19

Cut 2 accumulates in anaphase in APC mutants

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

slide20

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.

slide21

securin

Cohesins, separase and securins

slide24

Hct1

Cdc20

securin

Cohesins, separase and securins

slide26

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.

slide27

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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