LEC50: Cell Cycle Progression and Checkpoints

Question 1

what determines entry into S phase?

Answer 1

phosphorylation of pRb

Question 2

what are the 3 kinds of signaling/feedback in the cell cycle?

Answer 2

1) the previous cyclin-CDK is responsible for activating the next step
2) negative feedback, the next step turns off the previous step - if the 2nd complex is active, it causes phosphorylation and inhibition of the previous step
3) amplifying signal: previous complex starts the next complex to go, but there are steps that cause amplification fo that 2nd complex to cause a signal

Question 3

what upregulates cyclin D?

Answer 3

Erk pathway:

Growth factors bind to cell surface receptor

Receptor is a RTK on the membrane

It auto-phoshporylates

Activates Ras

Ras > Raf

Raf > Mek

Mek > ERK

ERK upregulates Cyclin D

Question 4

what do cells need to proliferate? why?

Answer 4

cells need growth factors b/c they need upregulation of cyclin D, and cyclin D is what causes proliferation

Question 5

what does Rb mean

Answer 5

retinoblastoma

Question 6

how is Rb normally? what does it do?

Answer 6

Rb normally bound to E2F

When Rb bound to E2F, Rb acts as a repressor, prevents E2F from functioning; E2F expression is **off **in Rb presence

Question 7

what is E2F?

Answer 7

a transcription factor that regulates gene expression

turned OFF when Rb binds it

turns ON when Rb gets phosphorylated

E2F’s target = Cyclin E

Question 8

what disrupts Rb-E2F complex? resulting cascade?

Answer 8

Cyclin D-Cdk4 and Cyclin D-Cdk6 phosphorylate Rb

this releases Rb from E2F

E2F can now act as a transcription factor; regulates Cyclin E

Cyclin E levels increase from E2F transcription activation

Cyclin E-Cdk2 become active; Rb is further phosphoryalted as Cyclin E-Cdk2’s substarte; and E2F is totally released

Question 9

what are the 2 kinds of E2F targets

Answer 9

1) high affinity target: low levels of E2F will turn it on
2) low affinity targets: dihydrofolate reductase, thymidylate synthase, DNA pol-Alpha

Question 10

describe signal cascade of pRb, E2F, leading into S phase?

Answer 10

Rb bound to E2F means no gene expression that’s E2F dependent occurs

Growth factors upregulate Cyclin D

Cyclin D-Cdk4 or 6, active now, phoshporylates Rb enough that E2F turns on Cyclin E

Activated Cyciln E-Cdk2 phosphorylates Rb further

E2F now fully active

Now can enter into S phase

Question 11

what regulates Cyclin A gene expression?

Answer 11

E2F

Cyclin A is a low affinity E2F target; so Cyclin A is only turned on when E2F is fully free of Rb

THEN Cyclin A is upregulated, and Cyclin A-Cdk2 can initiate origin firing for DNA synthesis

Question 12

what triggers DNA synthesis?

Answer 12

Cdk phosphorylation

Question 13

what are the stipulations for origin firing?

Answer 13

1) origin must fire for DNA synthesis to occur
2) EACH origin must fire ONLY ONCE to get a 2N to 4N DNA synthesis

Question 14

what assembles to initiate DNA replication? when?

Answer 14

pre-replication compres (pre-RC)

occurs during G1

Question 15

what activates polymerase to do DNA replication/start origin firing?

Answer 15

Cyclin E-Cdk2 and Cyclin A-Cdk2 phosphorylate polymerase

polymerase is then active

ONLY CAN OCCUR when the poylmrease is sitting ON THE ORIGIN - it is a** localized ability, **kinase is localized to the origin, polymerase gets phosphorylated and fires

Question 16

what ensures initiation of replication occurs only 1x each round?

Answer 16

Cdk phosphorylation of proteins involved in DNA replication includes licensing factors which ensure initiation occurs only 1x each round

factors then are degraded

Question 17

what are the dual effects of phosphorylation at the origin of replication?

Answer 17

1) phosphorylation fires the origin
2) phosphrylation removes the marks of an origin that’s ready to be fired- ensures firing doesn’t occur again once it already has

Question 18

what mediates entry from G2 to M?

Answer 18

Cyclin B/Cdk1 phosphorylation

Question 19

what is stimulated by mitotic Cdk-cyclin activation?

Answer 19

1) nuclear envelope breakdown
2) chromosome condensation
3) mitotic spindle formation
4) targeted protein degredation

Question 20

what causes nuclear membrane to break down?

Answer 20

Loss of a pressor means Cyclin B levels increase, due to transcriptional upregulation

Cdc25 becomes phoshprylated in response to completion of DNA synthesis

this activates Cyclin B/Cdk1

a component of the nuclear membrane is a substrate for Cyclin B/Cdk1; when phosphorylated, Lamins A and C break down, thus nuclear membrane breaks down

Question 21

what makes up nuclear membrane?

Answer 21

lamins, which polymerize w/ each other

Question 22

is nuclear envelope breakdown reversible? how?

Answer 22

yes

When Cyclin B/Cdk1 activity decreases, phosphorylation of Lamins is reversed by a nonspecific phosphatase which removes a phosphate from Lamin, and Nuclear membrane spontaneously will reform

Question 23

what is condensin?

Answer 23

protein that mediates condenstion of DNA into chromsome

when ACTIVE, mediates condensation of DNA into chromsomes

Question 24

when / why does chromoatin condensation occur?

Answer 24

condensin condensation of DNA occurs as a subsrate of Cdk1/Cyclin B complex, like nuclear membrane breakdown

when nuclear membrane breaksdown due to phosphorylation of Lamins by Cdk1/Cyclin B, Condensins come together and chromatin condensation occurs

Question 25

how does sister chromatid separation occur? when?

Answer 25

anaphase of mitosis Cohesin holds sister chromatids together Separase, enzyme, is bound to an inhibitor, Securin, is Inactive when the APC is active, it Ubiquitinates Securin, making Separase active Separase thus cleaves Cohesin, and sister chromatids can separate

Question 26

how does APC become activated?

Answer 26

b/c it's associated w/ a co-factor, either Cdc20 or Cdh1 when activated, Ubiquitinates and degrades Securin; this results in Separase activation thus Sister chromatids Separate

Question 27

what are APC's dual functions?

Answer 27

APC responsible for sister chromatid separation AND active in degredation of Cyclin B, which is necessary for exit from Mitosis

Question 28

G1: Kinase? Cyclin? Mechanism of upregulating cyclin transcriptionally? Substrate of the kinase? Effect of phosphorylation?

Answer 28

Question 29

G1-S: Kinase? Cyclin? Mechanism of upregulating cyclin transcriptionally? Substrate of the kinase? Effect of that phoshporylation?

Answer 29

Question 30

S phase: Kinase? Cyclin? Mechanism of upregulating cyclin txnly? Substrate of the kinase? Effect of that phosphorylation?

Answer 30

Question 31

what causes mitotic exit? what events occur as result?

Answer 31

Cyclin B degradation -\> Mitotic exit Chromosomes decondense, nuclear membrane reforms

Question 32

checkpoint?

Answer 32

an ordered series of events dependent on completion of a previous step senses a problem, causes cell cycle to stop

Question 33

why doesn't cyclin de-upregulation exist?

Answer 33

because checkpoints do exist

Question 34

what is restriction point?

Answer 34

mediated by pRb phoshporylation in response to growth factors lack of growth factors causes cell to arrest in early G1 at this restriction point provides connection btwn extracellular signaling & cell cycle control after restriction pt, cell is committed to DNA replication

Question 35

what does loss of checkpoint control cause?

Answer 35

uncontrolled proliferation aka cancer

Question 36

how does growth factor signaling contribute to proper cell cycle vs cancerous?

Answer 36

growth factor signaling results in transcriptional upregulation of cyclin D this triggers phosphorylation of pRb and release of E2F this regulation is lost in cancer

Question 37

what does DNA damage activate?

Answer 37

2 checkpoints: G1-S and entry into M

Question 38

purpose of each DNA damage checkpt?

Answer 38

1. prior to S phase, so cell doesn't replicate w/ damage 2. prior to M phase, so cell doesn't undergo cell division w/ damaged chromosomes

Question 39

describe the normal state of the G1 checkpoint

Answer 39

normally, the tumor suppressor p53 binds to Mdm2, a ubiquitin ligase that polyubiquitinates p53 and keeps p53 low in absence of DNA damage

Question 40

in presence of DNA damage, describe activation of G1 checkpoint

Answer 40

kinase cascade is triggered, Mdm2 and p53 become modified p53 also is acetylated this disrupts Mdm2-p53 interaction, p53 is no longer ubiquitinated p53 levels increase; p53 binds to DNA and turns on gene expression, mainly via p21, the CIP inhibitor thus CELL CYCLE ARRESTS b/c p21 binds to Cyclin E/Cdk2 and prevents entry into S phase

Question 41

what are p53's effects in regulating cell cycle

Answer 41

cell cycle arrest and apoptosis, depending on if damage is great in cell (apoptosis) or reparable (cycle arrest)

Question 42

if DNA damage is present, what prevents entry into Mitosis?

Answer 42

DNA damage here triggers kinase cascade resulting in inhibitory phoshprylation of Cdc25 thus, no activation of Cyclin B/Cdk1 occurs

Question 43

how does unreplicated DNA prevent entry into mitosis?

Answer 43

Cdc25 must be phosphorylated to be active this phosphorylation doesn't occur until DNA synthesis is complete, and full 4N DNA is achieved

Question 44

what is spindle checkpoint function?

Answer 44

arrests cells in mitosis until all chromosomes are properly aligned in metaphase

Question 45

when spindle checkpoint is OFF, what happens?

Answer 45

APC is active; it ubiquitinates securin this means separase is active cohesin is cleaved sister chromatids are separated

Question 46

when spindle checkpoint is ON, what happens? what causes this?

Answer 46

APC is inactive do not get separation of sister chromatids APC inactivity maintained if there's a microtubule attached to every kinetochore; if each kinetochore has a MT, they align the sister chromatid, and those MTs are responsible for pulling the sister chromatid apart so an unoccupied kinetochore, unbouned by a MT, can send a signal to APC to inhibit it

Question 47

what determines APC activity in the spindle checkpoint?

Answer 47

kinetochore occupany if there isn't a MT at any kinetochore, then APC activation is DELAYED so checkpoint is ON

Question 48

how could spindle checkpoint be exploited therapeutically

Answer 48

a MT disrupting agent will prevent a cell from dividing b/c it depolymerizes all of the MT in a cell, as if any MT are depolymerized, and even 1 kinetochore unoccupied, you trigger the spindle checkpoint and cause same cellular response as you would by depolymerizing all of the MTs

Question 49

how can chemotherapeutic drugs use cell cycle to their advantage?

Answer 49

if disrupte cell cycle by disrupting DNA damage or disrupting spindle formation, or have any DNA breaks, then cell will arrest at a checkpoint