Lecture 12: Cell Cycle

Question 1

S-phase

Answer 1

DNA synthesis phase; Genome is replicated

Question 2

M-phase

Answer 2

mitotic phase; chromosomes are segrageted to daughter cells

Question 3

Monitoring S phase: DNA replication can be monitored by the incorporation of [3H]thymidine into newly synthesized DNA. Explain the process:

Answer 3

• The duration of G2 is revealed as you continue to take samples of cells from the culture until labeled mitotic chromosomes are observed. The first cells with mitotic chromosomes that are labeled must have been in the last stages of DNA synthesis at the start of the incubation with
  [3H]thymidine. The length of time between the start of the labeling period and the appearance of cells with labeled mitotic figures corresponds to the duration of G2.
• Cells that are actively synthesizing DNA, such as those in the S phase of the cell cycle, take up thymidine from their environment. By using [^3H]thymidine instead of regular thymidine, the incorporated tritium label (^3H) becomes part of the newly synthesized DNA molecules. This radioactive label allows the detection and quantification of DNA synthesis. The level of radioactivity detected corresponds to the extent of DNA replication occurring in the cells. Higher levels of radioactive signal indicate increased DNA synthesis, which is characteristic of cells actively replicating their DNA during the S phase of the cell cycle.

Question 4

Experimental demonstration that cells contain factors that stimulate entry into mitosis:

Answer 4

• entry of a cell into M phase is initiated by a protein called maturation-promoting factor (MPF).
• MPF consists of two subunits: (1) a subunit with kinase activity that transfers phosphate groups from ATP to specific serine and threonine residues of specific protein substrates and (2) a regulatory subunit called cyclin.

Question 5

When the cyclin concentration is low, the kinase ……..

Answer 5

lacks the cyclin subunit and, as a result, is inactive.

Question 6

When the cyclin concentration rises, the
kinase is —-, causing the cell to —–.

Answer 6

• activated
• enter M phase

Question 7

Cell cycle regulation in yeast

START

Answer 7

• cell cycle is controlled primarily at 2 points: START and G2-m transition
• The first transition point, which is called START, occurs in late G1. Once a cell has passed START, it is irrevocably committed to replicating its DNA and, ultimately, completing the cell cycle.1
• Passage through START requires the activation of cdc2 by one or more G1/S cyclins

Question 8

A simplified model for cell cycle regulation in fission yeast. The cell cycle is controlled primarily at two points, START and the G2–M transition. Passage of a cell through these two critical junctures (purple arrowheads) requires the ——-. A third major transition occurs at the end of mitosis. Cells make a third commitment during the middle of mitosis, which determines
whether they will complete cell division and reenter G1 of the next cycle. Exit from mitosis
and entry into G1 is triggered by a ——

Answer 8

• activation of the same cdc2 kinase by different classes of cyclins, either G1/S or mitotic cyclins
• rapid drop in concentration of mitotic cyclins

Question 9

E2F and pRB relationship (4)

Answer 9

• E2F binds to specific transcription region that makes cyclin E used for cycle cycle progression
• In G1 E2F is bound to pRB on promoters which is gene repression, cell cycle progression is paused
• Activation of Cdk phosphorylates pRB and it releases E2F turning E2F into an activator. Seperation makes cell cycle resumes
• G1-S

Question 10

P 27 (3)

what it does+ forms+elimination of this causes

Answer 10

• progression through the S phase is inhibited by the action of p27. a CDK inhibitor
• P27 forms a trimeric complex with CycA and Cdk2 preventing their activity
• elimination of this cell cycle progression inhibitor causes excess cell proliferation and organ growth in p27 knockout mice

Question 11

The response to single strand DNA breaks

Answer 11

• The ATM gene encodes a protein kinase that is activated by certain DNA lesions, particularly double-stranded breaks. Remarkably, the presence of a single break in one of the cell’s DNA molecules is sufficient to cause rapid, large-scale activation of ATM molecules, causing cell cycle arrest. A related protein kinase called ATR is also activated by DNA breaks as well as other types of lesions, including those resulting from incompletely replicated DNA or UV irradiation. Both ATM and ATR are part of multiprotein complexes capable of binding to chromatin that contains damaged DNA. Once bound, ATM and ATR can phosphorylate a remarkable variety of proteins that participate in cell cycle checkpoints and DNA repair

Question 12

p21

Answer 12

inhibits the kinase activity of the G1 Cdk. This prevents the cells from phosphorylating key substrates and from entering S phase.

Question 13

ATM a protein kinase

activatedby+activates

Answer 13

• activated by ionizing radiation induced double strand breaks and activates CHK2

Question 14

ATR protein kinase

activatedby+activates

Answer 14

• activated by UV radiation induced DNA damage (SSDNA breaks) and activates CHK1

Question 15

G2/M checkpoint (3)

prevents+ activation is required for G2/M arrest…

Answer 15

• prevents cells from entering M phase if there is DNA damage
• activation of CHK1 by ATR results in phosphorylation and inactivation of CDC25 (phosphatase)
• CDC25 inactivation is required for G2/M arrest

Question 16

G1/S checkpoint with double strand DNA break (4)

Answer 16

• double stranded DNA break leads to ATM activation and CHK2 phosphorylation/activation
• phosphorylation by Chk2 stabilizes P53 and it transcribes p21
• p21 inhibits the G1 CDK when DNA is damaged at the G1 to S transition (no PRB phosphorylated, no transition to S phase)
• P53 can also transcribe cell death proteins if repair is not possible

Question 17

CHK1 or CHK2 can pause the cell cycle : G1/S via —– or G2/M via —— to allow for dna repair

Answer 17

• P53 and P21
• cdc25