Definition of checkpoints

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Definition of checkpoints
Biochemical pathways that control the order and
timing of cell cycle transitions and ensure that
critical events such as replication and
chromosome segregation are completed with high
fidelity.
From Elledge, S. 1996. Science, 2741664-71
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What was the evidence for checkpoints?
DNA replication is always completed before
mitosis begins
Chromosome congression is completed before
separation
Cell cycle delay exhibited by irradiated cells
Cell cycle delay exhibited by cells treated with
drugs such as HU and MT destabilizing drugs
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Regulation of the umuDC operon by RecA and LexA
E. Coli Cell cycle arrest in response to DNA
damage
Cell cycle arrest
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Three major checkpoints

• DNA replication checkpoint

• DNA damage checkpoint

• Spindle assembly checkpoint

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DNA damage checkpoint
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radiation sensitive mutants
(rad mutants)
DNA repair deficient
Deficient in cell cycle delay
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Time lapse of cells irradiated with X-rays
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Evidence providing the basis for a DNA
damage checkpoint screen in budding yeast
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Screens for checkpoint mutants
cdc13-1 cells were mutagenized and plated as
single colonies at the permissive temperature
(23)
Screens for cdc13-1 rad9 type mutants
Plates were replica plated and grown at the
restrictive temperature 36 for 6 hours then 23
overnight, followed by the same regime the
following day.
1) Rapid death
2) Microcolony formation
Presumptive double mutants were screened
individually for Microcolony formation at the
restrictive temperature 36.
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DNA damage checkpoint genes are evolutionarily
conserved
Function
S. cerevisiae gene
S. pombe gene
Human gene
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1) Replication checkpoint
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2) DNA damage checkpoint
allows time for DNA repair
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Subcellular location of Cdc25 and Cdk- cyclin-B1
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One mechanism of checkpoint control
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A model for upstream activation of the DNA damage
checkpoint
Mec1

P
RAD53
RAD9
P
Anaphase
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Conseved checkpoint pathways in yeast and mammals
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3) Spindle assembly checkpoint
Microtubule defects
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Chromosome congression
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The screen for spindle checkpoint components
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Spindle checkpoint components identified
genetically
21
Mad2 is maintained on unattached kinetochores
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Molecular marker of spindle checkpoint engagement
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Spindle assembly checkpoint
(a protein kinase cascade)
Mad and Bub proteins
This cascade has been conserved throughout
evolution and is present in
humans
24
How does the spindle checkpoint halt anaphase
onset?
25
Activation of Mad2 at kinetochores is regulated
by microtubule attachment
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Checkpoints govern the coordination of cell cycle
events
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Model for how the spindle checkpoint
regulates cell cycle progression
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Methods for cell synchronization

• drugs

1) excess thymidine - blocks cells prior to DNA
replication
2) aphidicolin - inhibits DNA polymerase and
blocks cells prior to DNA replication
3) hydroxyurea - blocks ribonucleotide reductase
and blocks cells in S phase
4) microtubule destabilizing drugs (nocadazole,
benomyl, colchicine) - blocks cells in mitosis
since they cannot make a spindle

• mating pheromones

• cdc mutants can be used in block and release
  experiments

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Methods for cell synchronization

• centrifugal elutriation - takes advantage of
  size changes during
• the cell cycle

• mitotic shake-off - takes advantage of shape
  changes during
• the cell cycle

• serum deprivation - arrests cells in G0

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Monitoring synchrony

• Flow cytometry using FACS
• (fluorescence activated cell sorter)

• label DNA (look for S phase)

1) 3H-thymidine
2) BrdU (bromodeoxyuridine)
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Molecular mechanisms controlling the activation
of Cdc25 and Cdk1/cyclin-B1