Checkpoint Control of Cell Cycle Progression

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Checkpoint Control of Cell Cycle Progression

• Systems for quality control have evolved to
  arrest cell cycle advancement if jobs are not
  done right

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A cell cycle description that fits data now
available
MPF goes down
MPF goes up, using S-phase cyclin
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These feedback systems were first clearly noted
for DNA damage and pointed out by Weinert and
Hartwell

• Some yeast mutants were unusually sensitive to UV
  or ionizing radiation.
• These RAD mutants identified lots of genes,
  though not so many as CDC genes
• Careful study showed that some RAD mutants failed
  to delay mitosis after considerable radiation, so
  they entered anaphase with damaged DNA and killed
  themselves
• These observations led to the proposal of
  checkpoint control of cell cycle progression

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Checkpoints are Defined Empirically
When the occurrence of an event B is dependent
on the completion of a prior event A, that
dependence is due to a checkpoint when a loss of
function mutation can be found that relieves the
dependence.
Lee Hartwell and Ted Weinert (1989) Science
246629
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Mutational analysis demonstrated that checkpoint
delay is a result of genes distinct from those
required for repair
Delay is not caused simply by the damage -wild
type cells delay in response to damage
-checkpoint mutants do not delay DESPITE damage
Typically, checkpoint mutants can repair
damage but they cannot DELAY the cell cycle in
response to damage, so they proceed to do
something lethal
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What types of Checkpoints Exist?
There is a checkpoint that
Prevents Replication in the presence of DNA damage
LOF mutations allow cells with DNA damage to
continue S-phase, which permits the replication
of faulty DNA
Prevents Mitosis in the presence of DNA damage
LOF mutations allow cells with DNA damage to
enter Mitosis
Prevents Mitosis in the presence of unreplicated
DNA
LOF mutations allow cells into Mitosis with
unreplicated DNA
Prevents anaphase before chromosomes are aligned
LOF mutations allow anaphase with unaligned
chromosomes
Recently, even more Checkpoints have been
discovered
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Components of the DNA Damage Checkpoint
SIGNALS
SENSORS
TRANSDUCERS
EFFECTORS
TARGETS
Adapted from Zhou and Elledge (2000) Nature
408433
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We will start with the checkpoint(s) that monitor
mitotic spindle assembly and delay the onset of
anaphase

• The existence of such a CP demonstrated by the
  delay of anaphase onset in cells with lagging
  chromosomes
• Its mechanism was suggested by inference from
  Nicklas work on the role of tension in the
  stability of chromosome attachment let the
  source of stability be the source of the signal
  to turn off the wait-anaphase signal
• Mutant screens for strains of yeast that failed
  to delay cell cycle progress in the absence of a
  spindle provided the key information for
  identification of pathway components

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The effectors of this pathway are the factors
that induce anaphase onset

• Sister chromatids are held together by cohesins
• At anaphase onset, cohesins are degraded
• Their degradation is a proteolysis, mediated by a
  specific protease, called Separase (Nature
  40037-43, 1999)
• Separase is kept inactive by Securin, so anaphase
  doesnt start
• Securin is degraded by a ubiquitin-dependent
  proteolysis, induced by the E3 ubiquitin ligase,
  the APC (Nature Cell Biol. 3E12-14, 2001)
• Anaphase onset is initiated by activating the APC

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Search for Genes Responsible for Feedback Control
of Mitosis
The productive screens explored budding yeast for
mutants that failed to display appropriate cell
cycle restraint when spindle formation was
blocked by a drug
Hoyt Budding Uninhibited by Benomyl Bub1,
Bub2, Bub3
Li Mitotic Arrest Deficient Mad1, Mad2,
Mad3
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Spindle Assembly Checkpoint Components
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Spindle Assembly Checkpoint Pathways ordered by
epistasis, analyzed by molecular biology
checkpoint kinases phosphorylate
checkpoint proteins at kinetochores
kinetochores release Mad2 to inhibit APCCdc20
Securin
Separase
From Susan Forsburg, Salk Institute
http//pingu.salk.edu/forsburg/cclecture.html
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Many checkpoint proteins are localized to the
kinetochores

• Mad2p found there first
• Then Bub1 and others
• Then motor enzymes that interact with components
  of the checkpoint system
• Then components of the APC/C
• So how can a kinetochore component control the
  behavior of the whole spindle?

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IN OVERVIEW Mad2p can be activated to Mad2p
in this state it works with Cdc20p to turn off
APC/C
Mad2p is inactive until it has visited a
free kinetochore. There it becomes activated
and is released to go and inhibit APC/C.
Its activity is short- lived, so it always has
to be re- activated by return to the kinetochore
Adapted from Shah and Cleveland (2000) Cell
103997
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When a kinetochore becomes occupied by MTs,
it loses its ability to activate Mad2p. APC/C
then becomes active, degrades Securin
Separase becomes active, and anaphase starts.
securin
Adapted from Shah and Cleveland (2000) Cell
103997
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Spindle Assembly Checkpoint includes A
regulation on mitotic exit pathway
Mitotic Exit Network
Securin
Separase
Adapted From Susan Forsburg, Salk Institute
http//pingu.salk.edu/forsburg/cclecture.html
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Bub2 Prevents Cdc14 Release/Cdh1 Activation
Bub2
Bub2
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Now, the DNA Damage Checkpoint DNA damage
DNA structure checkpoint
DNA repair
Cell cycle arrest
apoptosis
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Types of DNA Damage and Repair
Point Mutations can result from altered
bases which require Excision Repair Base
Excision Repair (BER) Nucleotide Excision Repair
(NER) Mismatch Repair (MMR, utilizes BER and
NER) Radiation, sheer, or enzymes can lead to
double strand breaks Double Strand Break Repair
(DSB) Homologous Recombination
Non-Homologous End Joining (NHEJ)
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DNA structure
Checkpoint DNA damage is detected by one of the
DNA Checkpoint systems. - Different different
checkpoint proteins respond to types of DNA
damage. - Cells monitor DNA integrity at all
stages of the cell cycle - All DNA Checkpoint
systems ultimately lead to an arrest in cell
cycle progression, initiation of repair, and
and if the problem is bad enough, apoptosis.
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Generalized checkpoint response
pathway
(signal transduction)
signal
DNA damage
ATM, ATR kinases
sensor
9-1-1 complex
M-R-N complex
adaptor
Claspin/BRCA1
Chk1, Chk2 kinases
effector
DNA repair
Cell cycle arrest
apoptosis
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signal
DNA damage

• Mismatches (replication errors)
• Altered bases (oxidative, chemical damage)
• Pyrimadine dimers (UV radiation)
• Single strand nicks (replication errors)
• Double strand breaks (ionizing radiation)

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sensor
ATM, ATR kinases
ATM Ataxia telangiectasia mutated ATR
Ataxia-Rad related

• phosphatidylinositol 3-kinase (PI3K) like
  kinases.
• bind directly to the damaged DNA.
• required Rad26 as a co-factor
• substrates include
• themselves (autophosphorylation)
• Rad26 (co-factor for themselves)
• each component of the 9-1-1 complex
  (PCNA-like complex)
• Nbs1 or the M-R-N complex (double strand
  breaks)
• claspin (adaptor)
• p53 (cell cycle arrest apoptosis)
• BRCA1 (DNA repair, transcription)
• Chk1 Chk2 kinases (cell cycle arrest)

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sensor
ATM, ATR kinases
ATM Ataxia telangiectasia mutated ATR
Ataxia-Rad related

• ATM is activated by double strand breaks.
• (in co-operation with the Mre11-Rad50-Nbs1
  complex)
• -ATR is activated by other forms of DNA damage,
• and stalled replication.
• Both associated with specific cancer-prone
  syndromes.
• Is there a role for inositol 6-phosphate?

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sensor
9-1-1 complex

• - Rad9, Rad1, Hus1 (Rad17).
• - PCNA-like complex toroidal homotrimer that
  encircles
• the DNA.
• (PCNA proliferating cell nuclear-antigen,
  DNA polymerase processivity)
• Rad17 complex binds to the 9-1-1- complex,
  opens it up, and
• loads in onto the DNA.
• (Rad17 is homologous to Replication Factor C1
  RFC1)
• (the Rad17 complex shares four components
  (RFC2, RFC2, RFC4, RFC5)
• with the replication complex)

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sensor
9-1-1- complex

• 9-1-1 complex
• - Loaded onto DNA upon DNA damage.
• All components become phosphorylated by the
• ATM/ATR kinases to become activated.
• RNA primer/primase required??
• assemble components of the DNA
  replication/repair system?

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Rad17/RFC1
ATM/adaptor
9-1-1
Chk2
BRCA1
Chk1
Melo Tockski, 2000
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sensor
M-R-N complex

• heterotrimer of Mre11, Rad50, Nbs1.
• binds directly to Double Strand Breaks.
• interacts with components of the DNA replication
  machinery.
• assemble components of the DNA repair system.
• binds components of the telomere maintenance
  machinery.

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adaptor
Claspin/BRCA1

• promotes the activity of the ATM ATR kinases.
• recruit the Chk1 Chk2 kinases to the damage
  site.
• associate with many proteins involved in DNA
  replication,
• repair, chromatin structure, and transcription.
  
• - show cell cycle DNA damage type specificity.

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effector
Chk1, Chk2 kinases

• Serine/Threonine kinases.
• Chk1 is activated (phosphorylation) by ATR in
  response to many
• different forms of DNA damage (ssDNA).
• Chk2 is activated ATM in response to Ionizing
  Radiation (DSB).
• Chk2 is activated by ATR in response to stalled
  replication.
• Both ATM/ATR initiate a cell cycle arrest
  recruit DNA
• repair/replication machinery through
  phosphorylation of
• many different targets.

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effector
Chk1, Chk2 kinases
Chk1 Chk2 substrates include DNA
replication/repair proteins. Chk1 Chk2
checkpoint targets include p53
(activates) CDC25 phosphatases
(inactivates) Wee1 kinase (activates) 14-3-3
proteins (activates) BRCA1 (activates) Polo
kinases 1 3 (activates) Nbs1 (M-R-N complex)
(activates)
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Nyberg, et al., 2002.
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DNA checkpoint activation of p53
ATM
Inhibit the inhibitor
Activate the activator
Chk2
MDM2
p53
Cell cycle arrest
apoptosis
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G1 DNA Checkpoint

• Delays cell cycle prior to S phase.
• Allows time for repair prevents replication of
  mistakes from damage.
• Two different stages
• First response occurs within minutes, but
  only lasts a few hours.
• p53-independent
• ATM activates CHK2 inhibits
  CDC25A prevents S-entry
• Second response takes several hours, but
  may be irreversible (?)
• ATM/ATR activate CHK2
  activates p53 activates p21 inhibits cyclin/CDK

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rapid, transient response
slow, sustained response
Activate the inhibitor
Inhibit the activator
G1 ? S
Nyberg, et al., 2002
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S phase DNA checkpoint

• Stalling of replication forks upon encountering
  damage.
• System requires DNA helicases polymerase
• Results in activation of ATR, and then CHK2.
• Requires adaptors claspin and BRCA1.
• CDC25A may contribute.
• Signal may be the ssDNA found in stalled
  replication forks.

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G2 DNA Checkpoint

• primary target of pathway is CDC25, and its
  regulation of
• CDK1 to prevent entry into Mitosis.
• Both ATM and ATR are activated and then activate
  CHK1 CHK2.
• CHK1 CHK2 inhibit CDC25 directly.
• CHK1 activates Wee1, which antagonizes CDC25
  activity.
• CHK1 also activates 14-3-3- proteins, which
  inhibit CDC25 CDC2,
• and promotes Wee1 activity.
• Polo-like kinases (PKL1 3) inhibit CDC25, and
  may be activated
• by ATM/ATR.

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ATM/ATR
Polo-like kinases
Nyberg, et al, 2002
G2 ? M
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Direct links between DNA checkpoints DNA damage
repair?

• BRCA1 substrate for ATM/ATR
• associates with DNA helicases, PCNA, MRN,
• ORC(?), and BCL (apoptosis).

• ATM/ATR also phosphorylate ssDNA binding
  proteins,
• topoisomerases, and helicases.
• CHK1/CHK2 phosphorylates p53, PML, E2FL which are
  positive
• regulators of Apoptosis.

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Zhou Bartek, 2004