VMED 5161 - Cell Cycle

Cell Cycle

What is the the series of events that a cell completes as it goes from one cell division to the next?

daughter

During the CC, the cell grows in size, makes a copy of its DNA, and divides into 2 ____________ cells

G1

S

G2

M

What are the four stages of the typical cell cycle?

M phase

During what phase does mitosis occur?

S phase

During what phase does DNA replication occur?

- Pre-replication protein complexes form at multiple replication sites on DNA

- Transcription of S-phase components is activated

- S-phase inhibitors are inactivated

What are the activities of G1 phase?

9 hr

What is the duration of G1?

DNA replication

What are the activities of S phase?

10 hr

What is the duration of S phase?

- Preparation for M

- Synthesis/activation of M phase control complexes

What activities occur in G2?

4.5 hr

What is the duration of G2?

- Prophase - chromosome condensation

- Pro-metaphase - nuclear envelope condensation

- Metaphase - spindle assembly, chromosome line up

- Anaphase - chromatid separation

- Telophase - chromosome de-condensation, nuclear envelope reforms, cytokinesis

What are the activities (stages) of M phase (mitosis)?

<1 hr

What is the duration of the M phase?

unidirectional

The CC progression is ________________ because specific transitions (G1-S, Met-Ana) are triggered by an irreversible event (proteolysis of specific proteins)?

Feedback

_____________ loops can delay CC progression until critical events are completed with high fidelity and/or enable blocks to be bypassed if necessary

checkpoint

A ______________ acts as an accessory braking system to help fine-tune CC regulation

mutations

Cells with checkpoint deficits often accumulate _______________ associated with DNA replication, DNA repair, or spindle assembly (may be pro-carcinogenic)

is environment favorable?

What is the G1 checkpoint?

Is all DNA replicated?

Is the environment favorable?

What is the G2 checkpoint?

Are all the chromosomes attached to the spindle?

What is the metaphase checkpoint?

Kinases, phosphatases

_____________ and ______________ are vital for control of CC events

post-translational

De/phosphorylation's are ______________________ modifications that increase the number and biologically active range of proteins beyond what would be expected from genome data alone

Cyclin/ CDK (cyclin-dependent kinase)

What complexes are the central agents controlling CC progression?

Cyclin

What is the regulatory part of Cyclin-Cdks, which are synthesized as needed then degraded?

Cdks

Cyclins alone have no enzymatic activity , but they activate ____________

Cdks

What is the catalytic part of cyclin-Cdk complexes, which are generally stable and are active only when bound to cyclins?

4

How many Cdks are there in vertebrates?

cyclin

The particular ___________ in the complex determines which proteins are phosphorylated by that cyclin-Cdk complex

G1

G1/S cyclins bind Cdks at the end of ____________ and commit the cell to DNA replication

G1/S

__________ cyclins activate transcription factors for the expression of enzymes needed for DNA synthesis and of genes encoding S cyclin-Cdk complexes

S

_____Cyclins bind Cdks during S phase & are needed for initiation of DNA replication.

S Cyclin-Cdk complexes phosphorylate regulatory sites in proteins that form DNA pre-replication complexes assembled during G1.This also serves to prevent re-assembly of new pre-replication complexes until the next CC.

What guarantees that a chromosome will replicate only one time per cycle?

inactive

S Cyclin-Cdks are kept ____________ by specific inhibitors. Late in G1, G1 Cyclin-Cdk complexes induce degradation of the S-phase inhibitors

M

____ Cyclins promote mitotic events

anaphase-promoting complex (APC).

After chromosomes line up on the mitotic spindle, M Cyclin-Cdks activate the _______________________

START

G1-Cyclins promote cell passage through ___________, the late G1 restriction point, past which passage through CC becomes mitogen-independent.

G1-Cyclin-Cdk complexes

Early in G1, extracellular growth factors (mitogens) induce synthesis of ____________

Cyclin D--Cdk 4 & Cyclin D--Cdk 6

What complex in mid-late Gi1 phosphorylates pRb to free & initiates transcription

of cyclin E + more E2F?

Cyclin E--Cdk 2

What complex in late G1/early S further phosphorylates pRb which increases expr./activity of E2F &

Cyclin E-Cdk 2?

Cyclin A--Cdk 2

What complex in S phase activates pre-replication complexes to initiate DNA synthesis?

Cyclin A--Cdk 1 & Cyclin B--Cdk 1

What complex in G2 & M (through ANA) promote mitosis?

Cyclins

What are the specific proteins that are broken down at specific transitions in the CC that make it "unidirectional"?

Ubiquitin (U)

What is a small protein involved in regulated degradation of proteins?

1-“U”, activated by an ATP-dependent enzyme (E1), is transferred to one of 30 different conjugating enzymes (E2).

2- That E2 conjugates with one of 100s of accessory proteins (E3). The E2-E3 complex with the activated “U” attached, is a ubiquitin ligase, and recognizes degradation signals that are part of the structure of the protein to be degraded.

What are the steps in "U" mediated protein degredation?

lysine

The U-ligase targets an amino group on a _______________ residue

of the target protein. Subsequent additions of “U” are made to the first “U” that was bound to the target protein.

proteasome

The poly-"U" protein is thereby targeted to the __________________

- Recognize and eliminate misfolded or otherwise abnormal proteins

- Confer short half-lives on specific normal proteins whose concentrations must change promptly with alterations

What are the functions of intracellular proteolytic mechanisms?

9

How many amino acids are in the destruction box of mitotic cyclins?

ARG

What amino acid begins the sequence of a destruction box in a mitotic cyclin?

APC (a ubiquitin ligase) directs "U" mediated proteolysis of ANA inhibitors

In mitosis, what causes the inactivation of protein complexes that connect sister chromatids at MET, allowing ANA to proceed?

APC directing proteolysis of M Cyclins

In late ANA, what causes chromosome de-condensation, nuclear envelope reformation and ultimately, cytokinesis?

PO4--of APC by G1 Cyclin-Cdk complex in late G1 inactivates APC

During the next CC, what causes the M cyclin accumulation in S & G2?

Proteasome

Where are ubiquinated proteins destroyed?

receptors

"U" proteins are recognized by specific ____________ in the proteasome, a large, v. abundant and v. efficient "macromolecular machine"

Hollow, 4-ring core with cap on each end

- Entry end has ATPases to hydrolyze ATP and provide energy for removal of "U"s

Describe the structure of proteasomes

Unfolding of the proteins and transfer of them to the catalytic core where they are digested into smaller units, that are digested by cytosolic peptidases to individual amino acids

What is the energy from the ATPases in the proteasome used for?

A cell moving through the cycle runs the risk of passing the damage to its daughter cells, if stably integrated

What can occur if DNA damage is not repaired?

G1-S

The checkpoint in late G1 where DNA damage that is not repaired with high fidelity or within the appropriate time period can prevent the ______________ transition

mitosis

The checkpoint in mid-late G2 maintains that DNA damage during S or in early G2 will prevent entry into _______________

Unreplicated

In S, ______________ DNA prevents entry into mitosis

polyubiquitylation

In M, the checkpoint states that defects in the M spindle assembly prevent activation of the APC __________________________ system

The ANA inhibitor will not be degraded and cells will not be able to enter ANA

What happens if the activation of the APC polyubiquitylation system is prevented?

pRb and p53

What are two of the major tumor suppressor genes?

cyclin-cdk complexes

What is needed in the control of the TSGs?

pRb

Which TSG is a nuclear phosphoprotein and is the product of the Rb gene found in all nucleated cells?

Rare eye tumor, retinoblastoma, and predisposes the animal to the development of other tumors

What occurs if there is a loss or mutation in the Rb alleles that produce pRb?

Hypophosphorylated

What is the state of active pRb?

Hyperphosphorylated

What is the state of inactive pRb?

G1-S

Active pRb acts as a brake on CC progression, governing the ____________ transition

growth factors

When a cell is stimulated by __________________ (early in G1), pRb is inactivated by phosphorylation, the brake is released and the cell passes through the G1-S transition

E2F

In G1, pRb in the active state, is bound to the transcription factor ______________

upregulated

As a result of growth factor activity in early G1, cyclin production is _______________ and the CyclinD-Cdk4/6 & Cyclin E-Cdk2 complexes are formed

DP1

When inactivated by phosphorylation, pRb separates from E2F, which then forms a heterodimer with the ________ protein

Activation of a number of target genes that are critical for the cell to traverse S phase

What happens when E2F binds the DP1 protein?

The brake on transition through S phase is lost and uncontrolled passage through S phase will result.

Remember that one of the key events during G1 is surveillance of DNA for damage and repair of that damage. If pRb control is removed, the cell may progress into S phase with damaged (mutated) DNA, which can then be passed on to daughter cells.

What happens if the Rb gene is lost or inactivated by mutation?

p53

What is the most commonly mutated gene associated with human tumors (~50%) and is often referred to as the gatekeeper against tumor formation?

one

Inheritance of one defective p53 allele predisposes the individual to increased tumorigenesis (Li-Fraumeni syndrome) in a variety of tissues, because additional loss of only __________ more allele in that tissue is needed for tumorigenesis to proceed

short-lived

p53 is a _______________ nuclear protein that regulates transcription of a number of genes

p53 does not regulate normal CC progression - levels are low in healthy cells and upregulated when DNA damage is present - governs CC arrest and apoptosis

How does p53 differ from pRb?

Late in G1, when DNA damage is detected. p53 up-regulation is enhanced transcription of p21, which inhibits the Cyclin-Cdk-mediated PO4--ation of pRb, as noted earlier, keeping the pRb bound to E2F. By delaying progress to S phase, this allows the cell to repair the DNA damage, if possible, in G1

How does p53 work?