cell cycle checkpoint

CHECKPOINTS AND EXPERIMENTS IN

PANKAJ KUMAR

15155

IMSc. BiosciencesCELL CYCLE CHECKPOINTS 1

CELL CYCLE

What is cell cycle?

• The cell cycle is the series of events that take place in a cell leading to its division and duplication (replication) that produces two daughter cells.

• Chromosome replication and segregation are conserved events

CELL CYCLE CHECKPOINTS 2


What is cell cycle?

3 MAIN REGULATORS

• 1) Cyclin-cdkcomplexes

• 2) Ubiquitin-protein ligases, and

• 3) Regulatory phosphatases.

CELL CYCLE CHECKPOINTS 5Oregonstate.edu

G1/S checkpointTumour suppressor proteins keep

transcription factors(E2F) in check

Mitogens (TGF, PDGF) lead to synthesis of G1 cyclins ,which along with cdk phosphorylates the TSP

This releases the TFs

Synthesis of G1 cyclins must reach a specific level to cross the START point.

S cyclin cdk’s inhibitor is degraded

S phase ensues.


Nature.com

Unreplicated DNA checkpointG2/M

This is done till the entire is replicated

ATR and chk1 bind to the replication fork

This leads to sequestering of cdc25


As the replication is still on

SPINDLE ASSEMBLY CHECKPOINT• Mad2 binds to kinetochores that have not bound to

microtubules.

• Kinetochore binding activates Mad2, and it in turn inhibits the activity of Cdc20 which controls the APC/C ubiquitin ligase.

• This delays degradation of securin and anaphase.

• Only after all kinetochores have bound to the spindle is Mad2 inactivated, releasing Cdc20 to trigger securin degradation.


Chromosome segregation checkpoint

• Tem1 .GDP is present on the centrosome.

• Tem1-GEF is present in the region of new bud.

• If the segregated chromosomes go correctly GDP is replaced by GTP .

• This activates Cdc14, which in turn activates Cdh1 by dephosphorylation.

• Cdh1 directs APC to ubiquitinate mitotic cyclins.

• This leads to exiting mitosis.



Experiments Cell fusion MicroinjectionIn-vitro cell free

extractConditional

mutants and functional complementation

Fluorescence tagging


DEVELOPMENTs IN CELL CYCLE

Leland H. Hartwell Timothy Hunt


Biochemical studies and

autoradiography

Sea urchin eggs were used.

Fertilization using sperm was carried

out.

35 S Methionine was added.

Timely examination of protein content

by SDS and autoradiography was done.

Rise and fall in levels of cyclin was

observed.1983 , R. Timothy Hunt , Imperial Cancer Research Fund


In-vitro study of cell free extract

Cytoplasm of Xenopuslaevis egg was used.

Shows that it is the B cyclins that lead to MPF activity.

Also that exit from mitosis will happen only if B-cyclins are degraded. A. Murray and T.Hunt,

(1993)

Temperature sensitive mutants


Ts mutants are

identified.

The mutated gene

is identified by

functional

complementation.

Genomic library is

used.

Microinjection • Done in Xenopus

laevis.

• Shows that some cytoplasmic factor of the egg can induce meiosisI in an arrested oocyte even in absence of progesteron.

• Identified cyclin-cdkheterodimer as the MPF factor.

• Purified by column chromatography


NORMALLY

MICROINJECTION

Y. Masui and C. L. Market, Journal Experimental Zoology

Cell fusion experiments

G1 nucleus S phase nucleus G2 nucleus

1 2

1 2

1 2+

+

+

1

G1 cell

2

S-phase cell

1

G2 cell

2

S-phase cell

1

G1 cell

2

G2 cell

G1 nucleus is

competent to replicate.

S-phase cells contain

activator

G2 nuclei aren’t

competent and do not

re-replicate. G2 cells do

not inhibit replication.

S-phase nuclei retard

mitosis in G2 nuclei.

G2 cells do not

suppress S-phase

entry of G1-phase

nuclei.

THE PATH AHEAD


Centrosome life cycle Structure of cohesin ring On –off mechanism for cohesin ring

The centrosome cycle: Centriole biogenesis,

duplication and inherent asymmetries

•Erich A. Nigg

•& Tim Stearns

Nature Cell Biology

KIM NASMYTHDepartment of Biochemistry, University of Oxford

Working out a model of cohesin loading and dissociation from chromosome

Tim Stearns Erich Nigg

Andrew murray Spindle assembly

checkpoint

Post-translational modifications of chromatin proteins following DNA damage

Their potential biological function.

Jiri Lukas Jiri Beerket Claudias Lucas

Let us keep our eyes open.See to what we can contribute to the society.Every bit of knowledge will help devise better therapies and drugs for disease cure.

http://www.nature.com/ncb/journal/v13/n10/full/ncb2345.html

http://www.nature.com/ncb/journal/v13/n10/fig_tab/ncb2345_ft.html#auth-1

http://www.nature.com/ncb/journal/v13/n10/fig_tab/ncb2345_ft.html#auth-2

References• http://cancerquest.org

• https://adapaproject.org (bio book)

• http://openi.nlm.nih.gov

• Pmc- NCBI

• J. A. Bryant and D. Francis (2011). The plant cell cycle. Annals of Botany

• Nature

• NEWS MEDIACOM

• http://www.nobelprize.org

• http://bioscience.jbpub.com

• Molecular cell biology by Lodish et al., 5th edition.


http://bioscience.jbpub.com/

THANK YOU


cell cycle checkpoint

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