L18&19: Cell Cycle and its Control

Question 1

Ouline the phases of the cell cycle

Answer 1

-G1: metabolic changes prepare the cell for division. At restriction point cell is committed to division and moves into S phase. Lasts ~10 hours
- S phase: DNA synthesis replicates genetic material. Each chromosome now consists of 2 sister chromatids. Lasts ~6 hrs
- G2 phase: metabolic changes assemble the cytoplasmic material necessary for mitosis and cytokinesis. Lasts ~3/4 hrs
Interphase now occurs (PMAT)
- M phases: mitosis (nuclear divsion) followed by a cell division (cytokinesis). Lasts ~2 hrs

Question 2

How does the cell cycle process work?

Answer 2

• process is unidirectional, only other point is cell death
• driven by series of cell cycle kinases (CDKs)
• regulated by checkpoints to prevent erros
• spindle assmebly checkpoint occurs at end of M phase
• restriction point is just before G1

Question 3

Explain the strucuture of chromosomes

Answer 3

• centrosome is a constricted region of chromosme containing specific DNA sequence, to whivh is bound 2 discs called kinetochores
• kinetochores serve as points of attachment for microtubules that move the chromosomes during cell division

Question 4

Explain the use of sea urchins in cell cycle research

Answer 4

Sea urchin eggs were used in the first demonsration of periodic protein degradation, a concept whihc is fundamental to the cell cycle

Question 5

Explain the use of yeast in cell cycle research

Answer 5

Yeast via genetic mutation analysis has been used to identify nearly all cell cycle regulatory genes

Question 6

Explain the use of xenoupus eggs in cell cycle research

Answer 6

Xenopus eggs and embryos extracts have been used to make biochemical discoveries
- one discovery being of mutation promotion factor (MPF), now commonly referred to as CDK1 activity and is the key protein kinase that controls cell cycle

Question 7

Explain the use of drosphila in cell cycle research

Answer 7

Drosphila embryos can be used for knockin/out genes of interset and mammalian cells in culture

Question 8

What drives the cell cycle?

Answer 8

The rise and fall of several protein kinase activities

• cdk activity is dependent on 4 hroups of cell cycle specific cyclin:
• cyclin D1,2,3 (cdk4&6): G1
• cyclin E (cdk2): G1/S
• cyclin A (cdk2,1): S
• cyclin B (cdk 1): M

Question 9

How is the rise in cdk governed?

Answer 9

• rising cyclin levels (affects activation of cdk partner): cyclin txn & translation result in production of cyclin proteins which partner with relevant cdk and drive that phase of cell cycle
• dephosphorylation of cdk-cyclin complex
• specific inhibitor proteins of the cdk

Question 10

What molecules usually phosphorylate residues in cdk?

Answer 10

Wee1 and Myt1
- to remove thesse we have cdk25 (phosphotase protein) which has 3 forms
cdc2A: CONTROLS G1/S & G2/M
cdc25B: controls G2/ M

Question 11

What is the function of cks1 protein

Answer 11

Adaptor to target cells to phosphoproteins and mediates APC interaction with cyclin A and B

• enhances phosphorylation of cdk substrates
• has 2 forms: cksHs1 & 2

Question 12

What is cdk1 activation based on?

Answer 12

+ve feedback: trigger activates cdc25 resulting the +ve feedback loops

• pushes cyclin kinase into active state, removing phosphorylation
• once complex is activated it further activates cdc25

Question 13

Name and explain a key mechanism in cell cycle and mitsosis

Answer 13

Protein degradation and is acheived by ubiquitin- mediated protein destruction, which is a multist
* refer to notes for image

Question 14

What is the function of SCF?

Answer 14

Its an E3 ubiquitin ligase which controls the G1/S transition
- formed Cu11, skp1 & Rbx1/ Roc1 subunits

Question 15

What is the function of anaphase promoting complex?

Answer 15

Controls the metaphase- anaphase transition\

- targets are cyclin A&B plus securin

Question 16

Explain how cycle control occurs in the S phase

Answer 16

Specifc cdk inhibitors (cdk 4&2: cyclin d&e and p21) holds cycle at G1

• in this period GF and mitogenes promote cell division and increase in concentration
• when they get to a certain level they can break this inhibition, resulting in activation cascade of 2 cdks
• this phosphorylates protein RB/ p107 releaseing key txn factor E2F which causes expression of genes needed for S phase
• synthesis of cyclin A (cdk2) and cell goes into S phase

Question 17

Explain the synthesis of the leading and lagging strand

Answer 17

Synthesis is asymmetrical

• leading strand follows direction of unwinding at replication fork
• lagging strand has to be synthesised in opposite direction
• helicase causes unwinding and in its wake replication protein A molecules bind, at which point the primase can come along and synthesise primer for new DNA

Question 18

How is the synthesis of the leading and lagging strand made more efficient?

Answer 18

Replication complex, including polymerase, is held in place by sliding clamp
- primer is loaded to make fragments, process extends and when everything has been replicated DNA ligase joins up okazaki fragments

Question 19

Explain the assembly of prereplicative complexes

Answer 19

1. We have origin recognition complex, Orc1-6 binding at origin
2. Then cdt1 and cdc6 join and then so does helicase
   cdt1 and cdc6 joining is mediated by germinin, which binds to cdH preveting it from causing the bnding of helicase so unwinding can no longer occur
   - germinin accumulates in S phase

Question 20

How does telomerase synthesise DNA at chromosome ends?

Answer 20

• they add several repeat DNA regions on lagging strand to serve as priming region, so lagging strand can be completed
• resulting strucuture at end of each chromosome known as telomere
• shortening of telomere is associated with aging

Question 21

How are nucleosomes packaged in the final stage of S phase?

Answer 21

• nucelosomes have ~147bp of DNA wrapped around each octamer to make a nucleosome
• then tightly packed into a 30nm fibre and further packaged by contraction

Question 22

Histones have protuding N-terminal tails. True or false?

Answer 22

True, these are modified post translation and involved iwth gene expression
- can also involve non-histone proteins (and acetylation to modify chromatin structure)

Question 23

Explain the process of nucleosome assembly

Answer 23

1. Acetylated H3-H4 tetramer complexes with assembly factor CAF-1
2. This is recruited to replication fork and incorprated into new DNA
3. H2A& B dimers loaded on the H3-H4 tetramer via another assembly factor NAP-1 to form octomer

Question 24

Name particular histone modifications associated with differnt functions

Answer 24

• histone acetylation: active genes
• H3K4me3: active promoters
• H3K9me3 and H3K27me3: heterochromatin
• H3T3ph and H3S10ph: mitosis
• H2AX.S13ph (gamma H2AX): DNA damage

Question 25

What can be found on histone tails?

Answer 25

Markers which can have

• writers: enzymes which coe along and deposit phosphorylation
• erasers: gets rid of phosphorylation thus marker
• reader: recognises specific phosphorylation and bind