Chapter 15 Flashcards
What is a highly ordered sequence of events through which a cell progresses?
cell cycle
Where is the gap or growth phases in most cells?
between S and M phase
In what phases are tremendous amount of growth occur?
G1 and G2
Other than growth, what does G1 and G2 ensure?
ensure that each of the major cell cycle events is competed properly before the next is begun
Cells are sensitive to their surroundings such as what 2?
a. resource availability for unicellular organisms
b. neighbor constraints for multicellular organisms
An elaborate cell cycle control system is sensitive to what?
checkpoints
Checkpoints serve to do what 2?
a. ensure that a cell cycle event does not start before the previous one has been successfully completed
b. impose independence of the initiation of the cell cycle on the cell’s surroundings
What 3 cell populations are inherently synchronous?
sea urchin, frog, and clam oocytes
When group of cells that are inherently synchronous are induced to undergomeiotic maturation synchronously by treatment with appropriate hormones, they go from what state to what state?
interphase-arrested state to a metaphase-arrested state
Interphase-arrested state
immature oocyte
metaphase-arrested state
awaiting fertilization
Other than meiotic maturation, what is also synchronous?
early embryonic cell divisions of oocytes
The synchronous of early embryonic cell division of oocytes allows study of what behavior?
The behavior of a large population of cells all in the same point of the CDC
Why does the size of the oocytes or eggs make them amenable to study?
because they can be micro-injeted with biomolecules
Budding yeast
Saccharomyces cerevisiae
Fission yeast
Schizosaccharomyces pombe
What are single-celled eukaryotes that are easy and fast to grow, well-characterized genetically, and synchronized fairly readily?
Yeast cells
What cells can be grown as haploid cells?
yeast cells
What mutants can be isolated in yeast cells?
conditional loss-of-function
Temperature-sensitive lethal mutations grow normally at _____ growth conditions and arrest when at _______ growth conditions.
permissive; restrictive
For S. cerevisiae, what is an indicator of the stage of the CDC that the cell is in?
bud size
For S. pombe, what is the good indicator of the CDC stage?
length of the cell
What are 3 experimental organism systems given?
Aspergillus nidulans, Drosophilia melanogaster, and mammalian tissue
Why is the study potential of normal primary cells for mammalian cells limited?
because they will stop dividing in culture after 25-40 cell divisions.
What cells are immortalized?
cancerous cells
What can be formed between normal and cancerous cells and is immortalized?
hybrid cells
Fusion of what cells accelerates the G1 phase nucleus into DNA replication?
S-phase cell with G1 cell
Fusion of what cause their chromosomes to prematurely condense?
mitotic cells with interphase cells
Fusion of G2 cell with an S phase cell results in what?
S-phase cell continue to replicate
G2 cell did NOT begin to re-replicate its DNA
CDKs
Cyclin-depedent protein kinases
What is the key components of the central cell cycle control system?
Cyclin-dependent protein kinases
Structure of cyclin-dependent kinase (what it binds to)
two polypeptides, bound to ATP, has an active site
When is the cyclin-dependent protein kinase active?
when bound to a second polypeptide, a cyclin
What 3 things do active CDK need?
- a cyclin bound
- any inhibitory P removed by cdc25 phosphatase
- active P by CAK to them
What protein was first identified as proteins whose [ ] in the cell rose and fell coincidental to the onset of mitosis?
cyclin proteins
What promotes mitosis by phosphorylating the lamin proteins so the nuclear envelope can break down?
Cdk1
Cdk1 also phosphorylates other proteins to regulate what?
assembly of the mitotic spindle
How many cyclins does baker’s yeast and humans have?
baker’s yeast - 9
humans - at least 12
Metazoan cells have cyclins that have a similar region that enables them to bind to what?
catalytic CDK subunit
Metazoan cells have ______ that is involved in substrate recognition
hydrophobic patch
What helps to make the transitions between the phases of the cell cycle sharp and irreversible?
Regulatory layers
What pauses or advances cell cycle?
Regulatory layers
Regulatory layers help pause or advance while being sensitive to what?
intrinsic or extrinsic signals
What family adds inhibitory phosphates to specific tyrosine and threonine residues?
Wee1 kinase
Wee1 kinase adds inhibitory phosphates to where?
specific tyrosine and threonine residues
What family of phosphatases removes inhibitory phosphates?
Cdc25
Activating phosphorylation occurs on a threonine residue by what?
CDK-activating kinase
CAK
CDK-activating kinase
What regulates CDKs?
cyclin kinase inhibitors
CKI
cyclin kinase inhibitors
What family of CDIs interacts with the CDK subunit and prevents cyclin association?
P16 family
What family of CKIs binds and inhibits CDk-cyclin complexes?
Cip/Kip family
What is accomplished by segregating CDKs in different regions of the cell from their activators, inhibitors, or substrates?
Regulation
Where are the cytoplasmic retention signal?
on cyclin B1 and NES
What is a key mechanism of CDK regulation?
regulation via periodic availability of cyclins
Where do the cyclins become highly unstable and irreversibly destroyed?
cell transition points
Abrupt instability of cyclins is due to what?
activation of specific ubiquitin ligase complexes
What covalently attaches a multi-ubiquitin chain?
Ubiquitin (Ub) ligase (E3)
Ubiquitin (Ub) ligase (E3) covalently attaches a multi-ubiquitin chain with the help of what enzymes?
Ub-activating (E1) and Ub-conjugating (E2) enzymes
Once the cyclin is tagged, it is recognized and degraded by what?
proteasome
Cdks program their own inactivation by activating cyclin ubiquitin ligases that lead to their destruction. This is called?
feed-forward control
Metozoan cells enter ____ state if they do not receive a signals that they should divide
quiescence
When conditions change, most cells can get out of G0 and reenter cell cycle but others enter a permanent G0 ______.
senescence
Apoptosis is stimulated by extracellular factors such as signals like what?
TNF-alpha
TNF-alpha
tumor necrosis factor-alpha
Where does the cell become irreversibly committed to a round of cell division regardless of extracellular cues? (Yeast and multicellular eukaryotes)
START in yeast
restriction point in multicellular eukaryotes
Reentry is primarily via what?
ubiquitin-mediated destruction of CKIs
Purpose of reentry via ubiquitin-mediated destruction of CKIs?
To relieve the inhibition of G1 CDK-cyclin complexes
What are class of peptides and hormones that stimulate proliferation?
growth factors
Many of the growth factors are present in where?
serum
PDGF
platelet-derived growth factor
PDGF is released by what?
platelets upon blood clotting
PDGF contributes to what
rapid cell proliferation required for wound healing
Growth factors bind to receptors on target cells to initiate what?
intracellular signaling pathway
Intracellular signaling pathway leads to what?
cellulat proliferation
Steps of signaling pathway activation
- PDGF binds to PDGF receptors
- PDGF receptor dimerizes
- Autophosphorylation
- Adaptor proteins and the Ras GTPase are recruited
- Kinase signaling pathway is activated
What are early response genes?
those expressed very quickly after serum addition to cell cultures
What is one example of delayed early response genes?
cyclin D
Tumor suppressor that is phosphorylated for CDKs with cyclins to get through the restriction point
Rb (retinoblastoma)
When Rb is unphosphorylated, Rb is bound to and inhibiting what transcription factor?
E2F
Rb
retinoblastoma
When phosphorylated, what happens to Rb?
dissociates from E2F
Rb dissociates from E2F when phosphorylated by what?
cyclin D associated with either Cdk4 or Cdk6
E2F stimulates its own expression and expression of other genes such as what?
cyclin E
Cyclin E is a protein for what?
replication initiation
Steps of phosphorylation of Rb
- Rb binding inactivates E2F
- Cdk4, 6-cyclin D phosphorylates Rb, causing dissociation from E2F
- E2F upregulates expression of itself and cyclin E
- Cdk2-cyclin E phosphorylates Rb
Positive feedback loop of cdk2-cyclin E
When enough Cdk1-cyclin E accumulates, the cell traverse the restriction point
Loss of normal Rb function plays an important role in what?
tumor development role
ARSs
autonomously replicating sequences
What are ARSs in yeast?
DNA sequences
What can replicate independently of the context of a chromosome and gave insight on the origins of replication?
ARSs
In yeast, ARS is part of what?
an origin
Some organisms have small consensus sequenes in _______ region and _______ region?
origin; A/T-rich
Organisms with more than one chromosome have what?
multiple origins per chromosome
Several layers of regulatory issues must be dealt with what 3?
a. firing origins only during S phase
b. making certain replication is complete before proceeding into mitosis
c. firing each origin only once
Prior to DNA replication initiation, what must be assembled on the origin?
pre-replication complex (pre-RC)
ORC
original recognition complex
structure of ORC
6-protein complex
ORC serves as a platform for binding of _____ and ____
Cdc6; Cdt1
Cdc6 is a member of what family?
AAA+ ATPase family
MCM
minichromosome maintenance complex
Structure of MCM
6 proteins
When does Cdc6 and Cdt1 bind ORC?
during late mitosis to G1
Where is the pre-RC assembled?
in the end of M phase to early S phase
Cdc6 is only available during ____, so no replication other than s phase
G1
Cdt1 is negatively regulated by _____
geminin
Pre-RC assembly is restricuted by what?
mitotic CDK-cyclin activity
CDK phosphorylation of _____ inactivates pre-RC assembly
Cdc6
CDK phosphorylation of ___ dring S phase helps get them removed from DNA
MCM
Cell trasitions from pre-replicative to replicative states under the control of what 2 kinases?
CDK-cyclin and DDK
DDK
Dbf4-dependent kinase
Function of CDK-cyclin
couples replication to CDC progression by preventing pre-RC assembly and by promoting origin activation
Function of DDK
initiate DNA synthesis
How is the DNA synthesis initiated by DDK?
phosphorylation of MCM proteins changes their structure to allow initiation
Binding of ____ is rate-limiting for initiation at individual origins
Cdc45
Binding of Cdc45 is dependent upon ____ and ______.
CDK-cyclin; DDK activity
Binding of Cdc45 plus ____ results in unwinding of DNA
GINS
Unwinding of DNA by binding of Cdc45 and GINS causes what?
activation of MCM
Activation of MCM converts from an ______ to _____, which will remain as part of the elongation complex
assembly factor in the pre-RC; helicase
Unwinding of DNA exposes what?
ssDNA
ssDNA exposed is bound by _____
ssDNAbp
Exposing of ssDNA results in?
RPA loading of primase/DNA pol alpha complex and DNA synthesis is initiated
_____ and ___ move with replication frok as part of a large replisome
MCM; Cdc45
Large replisome include MCM, cdc45, and primarily what?
DNA pol delta
As S phase proceeds, what happens to MCMs?
dislodged from the chromatin
As the replication fork moves, it helps what?
to establish cohesin proteins to connect the newly synthesized sister chromatids until mitosis
Growth in G1 and G2 is necessary to maintain what?
an ideal nuclear/cytoplasmic ratio following cytokinesis
Error in DNA replication is detected and corrected in
G2
Major mitotic kinase that promotes the G2 to M transition
Cdk1-cyclin B
CDK1 is kept inactive by
phosphorylation by Wee1 kinase
To activate Cdk1, _____ removes inhibitory phosphates
Cdc25 phosphatase
To activate Cdk1, cdc25 needs what?
Polo-like Kinase (PLK)
To activate Cdk1, _____ phosphorylates an activating phsophate on Cdk1
CAK
Cdk1 helps to inactivate ____
Wee1
What are the 2 differnt CDK-cyclin complexes in multicellular eukaryotes that govern G2-M and mitotic progression?
Cdk1-cyclinA and Cdk-1-cyclin B
5 roles that PLK family is inolved in
a. entry into mitosis
b. spindle fomation
c. cytokinesis
d. centrosome maturation
e. chromosome segregation
3 things that all Plk family members have
a. N-terminal kinase domain
b. C-terminal domain
c. Polo boxes
What are C-terminal domain with one or more polo boxes?
intracellular targeting domains for PLKs
C-terminal domain with one or more polo boxes direct them to where?
docking sites at centrosomes, kinetochores, mitotic spindle, etc
What binds to proteins that have previously been phosphorylated by Cdk1 or other kinases?
polo boxes
NEK
NIMA-like kinase
Kinase activity of NIMA parallels that of ____ during mitosis
Cdk1
What is NEK family involved in?
chromosome condensation and centrosome separation
Aurora family of kinases are involved in what 5?
a. chromosome condensation and segregation
b. kinetochore function
c. centrosome maturation
d. spindle formation
e. cytokinesis
3 classes of Aurora in humans
Aurora A, B, or C
When does Aurora A levels peak and fall?
peak early in mitosis and fall at onset of anaphase
Why does Aurora A levels fall at the onset of anaphase?
because of ubiquitin-mediated proteolysis
Aurora A activity is regulated by
phosphorylation
Example of involvement of the four maor mitotic protein kinase
Lamins contains Cdk1 phosphorylation sites, if they are mutated, the lamina doesn’t dissolve
What 4 family members localize to to centrosomes and regulate centrosome duplication and separation?
Cdk1-cyclin, Plk, Aurora, and NIMA
Aurora B moves from ___ to ___ during anaphase
kinetochores; central spindle
Aurora B work with other proteins like
Borealin
CPPs
chromosom passenger proteins
CPPs are necessary for what 3?
a. chromosome to condense properly
b. align at metaphase plate
c. make connections to both centrosomes
How do CPPs function?
by dismantling kinetochore-spindle microtubule connectionsthat do not lead to tension across the kinetochroes
What do CPP not result in?
bipolar attachment
What happens in the prophase?
a. disruption of the nuclear envelope, dissolving of the nuclear lamina
b. cytoskeleton changes to allow formation of mitotic spindle
c. centrosomes finish separating and a bipolar spindle form between them
d. kinetochores attach to microtubules
What helps position the centrosomes?
astral ray of microtubules
Astral rays of microtubules help position the centrosomes, thus helping what?
determination of the orientation of the plane of cell division
What is made of microtubules anchored at opposite centrosomes that do not contact kinetochores?
central spindle
In what manner is the central spindle bundled?
in an antiparallel manner
What is the remnants of the central spindle as the cell undergoes cytokinesis?
midbody
How does the actin cytoskeleton change?
actin concentrates in the medial cell region in preparation for cytokinesis
What forms the cleavage furrow or cytokinetic ring?
actin cytoskeleton
A multiprotein complex that belongs to the SMC family proteins and is made of 2 coiled-coil proteins with ATPase domains plus other proteins.
Condensin
Function of condensin
mediates chromosome condensations and help pull different DN regions together to compact DNA
When is condensin allowed to bind chromosomes?
during mitosis
CDK phosphorylation of a condensin subunit allows ____ during mitosis
nucelar entry
Phosphorylation of ____ and ____ is indicative of chromosome compaction
histon H1 and H3
______ phosphorylates histone H3
Aurora kinase
Sister chromatid cohesion is mediated by ____.
cohesin complex
When is cohesin removed from chromosome arms?
in prophase
Cohesin is removed from chromosome via __
Plk1 phosphorylation of a cohesin subunit
What part of cohesin persists until anaphase?
around centromeres
Protease that celaves the cohesin protein at centromeres to allow sister chromatid separation
Separase
Separase is kept inactive by binding of ___
securin
What happens to securin at the metaphase-anaphase transition?
ubiquintated and proteasomed
What target securin for destruction?
E3 ubiquitin ligase
What complex is the E3 ubiquitin ligase?
APC (anaphase-promoting complex)
APC
Anaphase-promoting complex
APC helps triger what?
proteolysis of securin and cyclin
When is APC active?
during mitosis and G1
APC is regulated via ___ involving what?
phosphorylation; Cdk1 and Plk1
Why do APC remain active during G1?
to keep Cdk1 activity low, permitting formation of the pre-RC
Once the chromosomes are well-separated and Cdk1 activity has fallen, what ensues?
cytokinesis
The process of return to interphase
mitotic exit
Mitotic exit involves the activation of Cdk1 by what?
Ub-dependent proteolysis of cyclin B
Mitotic exit requires the reversal of the phosphorylation events triggered by ____
Cdk1
What reverses much of Cdk1’s phosphorylation?
Cdc14 family of phosphatases
Other than the reversing Cdk1 phosphorylation events, what other mitotic kinases are reversed?
Plk, NEK, and Aurora
Other mitotic kinases like Plk, NEK, Aurora are reversed by protein phosphatases such as ___ and ___
2A (PP2A); 1 (PP1)
What cleaves cohesin
Separase
Separase triggers the release of _____ from its anchoring compartment in early anaphase
Cdc14 phosphatase
What anchoring compartment does separase trigger the release of Cdc14 phosphatase?
the nucleolus
Cdc14 is maintained in its active form by a signaling cascade called ___
MEN (mitotic exit network)
Where is MEN discovered?
S. cerevisiae
MEN
Mitotic exit network
SIN
septation initiation network
What is the analogous signaling cascade in S. pombe?
SIN
MEN and SIN are regulated by?
GTPase
MEN and SIN signaling promote activation of what?
Cdc14 family of phosphatases and cytokinsesis
Active Cdc14 causes APC to?
mediate cyclin proteolysis and Cdk1 inactivation
Central cell cycle control system receives what?
feedback throughout the cycle
What monitors the proper completion of the separate cell cycle events?
checkpoints
Checkpoints control ___.
transitions
What prevents the control system from allowing the next event to initiate?
Inhibitory signals
What are the signals that activate a checkpoint to prevent the cell from entering mitosis until the problem is corrected?
Incompletely replicated or damaged DNA and improper spindle fiber attachmetn
_____ are signaling pathways that inhibit cell cycle progression.
checkpoints
Checkpoints are activated when a problem arises like ___?
DNA damage or incompletely replicated DNA
What are 3 distinct components of CDC checkpoints?
a. sensor
b. signaling module
c. CDC target
Sensor function
detects an event defect
Signaling module function
transmit signal when an error is detected
CDC target function
controlled to halt CDC progression
Which regions are recognized by the DNA damage checkpoint?
regions of ssDNA (single-stranded DNA) or DSBs (double-stranded breaks)
What response does ssDNA elicit?
ATR-dependent response
What response do DSBs elicit?
ATM-dependent response
ATM kinase binds to?
DNA termini of a DSB
ATR kinase binds to?
UV-induced DNA structures
Structure of ATM
homodimer
DNA damage triggers what?
autophosphorylation that dissociates the dimer
DNA damage releases what?
active monomers that phosphorylate many targets
What is loaded onto damaged DNA?
9-1-1 complex
9-1-1 complex loaded onto damaged DNA, forming what structure?
ring-like structure
Major transducers/mediators in the DNA damage/DNA replication checkpoints are?
Chk1 and Chk2 kinases
Chk1 and Chk2 function
directly phosphorylate components of the CDC engine to block them
3 Distinct checkpoints that delay CDC progression in response to DNA damage or replication delays
a. G1/S
b. S-phase
c. G2/M
G1/S delay function (7)
a. activates and load 9-1-1 complexes
b. activate ATM/ATR kinases
c. phosphorylate/active Chk1 and Chk2
d. phosphorylate Cdc25A
e. Cdk2-cyclin E remains inactive
f. phosphorylate p53
g. p53 turnover is prevented, it transcribes more target genes, including CKI
A transcription factor and a tumor suppressor?
p53
CKI inhibits
G1 CDK-cyclin
S-phase checkpoint function (5)
a. activate and load 9-1-1 complexes
b. activate ATM/TR kinases
c. phosphorylate/active Chk1 and Chk2
d. phosphoryalte Cdc25A
e. Cdk2-cyclin E remains inactive
G2/M checkpoint function (2)
a. activation of ATM/ATR - Chk1/Chk2 pathways
b. inhibition of CDK1-cyclin B
4 Outcomes of the DNA replication checkpoint
a. prevention of further origin firing
b. slowing of replication elongation
c. maintenance of stalled replication forks
d. inhibition of mitotic entry
4 Types of kinetochore attachement to MT-spindle fibers
a. monotelic
b. amphitelic
c. syntelic
d. merotelic
Amphitelic arrangement
two kinetochores, each attached to different poles
Which arrangement is correct attachment?
amphitelic (bipolar) arrangement
Monotelic arrangement
one kinetochore to one pole
Syntelic arrangement
both kinetochores to one pole
Merotelic arrangement
one kinetochore to both poles
Which kinase is important for destabilizing incorrect attachments?
Aurora B
If some chromosomes are not attached to spindle at all or are attached incorrectly, which signal is generated?
wait-anaphase signal
What generates wait-anaphase signal?
SAC (spindle assembly checkpoint)
SAC
spindle assembly checkpoint
SACs are activated by (2)
a. kinetochores which are unattached or improperly attached, to the spindle MTs
b. lack of physical tension acoss each kinetochore
SAC signaling involves
phosphorylation cascade
SAC components are ___ and ____.
protein kinases; phosphorylated
What is one target of SAC activity?
Cdc20
Cdc20 is the activator of?
APC
Binding of SAC comonenets to ____ inhibits ____ activity.
Cdc20; APC
Binding of a SAC component of Cdc0 inhibits APC activity, stabilizes ____, keeping sister chromatin cohesin intact by inhibiting ____.
securin; separase
Mutations in SAC function result in
a. cell death
b. aneuploidy due to nondisjunction
Mutations in genes that control ____ can result in cancer
the fidelity of cellular proliferation
Mutations in 2 types of genes that lead to unrestrained cell proliferation
a. proto-oncogenes
b. tumor suppressor genes
Proto-oncogenes result in?
signaling proliferation inappropriately or constitutively
Proto-oncogenes can become ___
oncogenes
Tumor suppressor genes function (2)
a. restrain cellular proliferation
b. ensure genome stability
If both copies of tumor suppresor genes have loss-of-function mutations, it can lead to
loss of growth control and be involved in tumor formation
Key tumor suppressor gene
p53
function of p53
to restrain cell cycle progression
p53 is also a ____ gene
checkpoint gene
Most human tumors contain?
inactivating mutations in p53
In absence of p53 function, what happens to cells?
escape cell cycle regulatory control and proliferate in the presence of DNA damage
