Intro to the cell cycle for 2nd year biochemists at Oxford Uni. Does not contain much detail but lots of references to further literature.

1. Tutorial I
Introduction to the Cell Cycle
7th week Hillary Term
Tutorial for 2nd year biochemists
by Christiane Riedinger

2. Christiane Riedinger
2nd Year Biochemists, HT 2010
Tutorial 1: Introduction to the Eukaryotic Cell Cycle
YOUR ESSAY QUESTION
Describe briefly the key players of the eukaryotic cell cycle, the key experiments that
The assignment
lead to its discovery and new insights gained since then.
(Approximately 2 pages for the first part of the question, then 1 page for each other
part of the question. Please keep it short and to the point)
In this essay, I’d like to see that you have understood the most important aspects
of the topic, there is no need go into extreme amounts of detail. Parts of your
work could also be bullet points and/or diagrams/flowcharts. Do not copy the
answer directly from the book, but read and understand the references, then
process the information in your head, read and understand the question, and
then write the best possible answer. • any problems?
READING MATERIAL
Text books:
• favourites on reading list?
• Murray, A. and Hunt, T., The cell cycle.
• Morgan, David. The cell cycle (for the seriously keen)
Reviews:
• Nurse, P., Masui, M. and Hartwell, L., (1998), Nature Medicine 4: 1103-1106.
• your essays
Understanding the cell cycle.
• Nurse, P. (2000), Cell 100: 71-78. A long twentieth century of the cell cycle and
beyond. • aims for today
• Morgan, D.O., (1997), Ann. Rev. Cell. Dev. Biol. 13: 261-291. Cyclin-
dependent kinases: engines, clocks and microprocessors (ATTACHED)
• Murray, A. (2004), Cell 116: 221-234. Recycling the cell cycle: Cyclins revisited.
(In the same volume short papers by T. Hunt and T. Evans on pp S63-S66).
(ATTACHED)
• Ekholm and Reed, Regulation of G1 cyclin-dependent kinases in the mammalian
cell cycle. Curr. Opp. Cell. Biol. 12, 676-684
• Malumbres and Barbacid (2005) mammalian cyclin dependent kinases TIBS 30,
630-41 (ATTACHED)
• Nurse P., Understanding the cell cycle (ATTACHED)
Websites:
• Paul Nurse’s Noble lecture: (ATTACHED)
http://nobelprize.org/nobel_prizes/medicine/laureates/2001/nurse-lecture.html
EXAM QUESTIONS TO KEEP IN MIND:
• "What is the evidence that eukaryotic cells are dependent on
checkpoints to regulate progression through the cell cycle? “
• "There are multiple mechanisms for regulating the activity of the
cyclin-dependent kinases which control passage through the eukaryotic cell cycle
and different mechanisms can be used in the cell to
regulate kinase activation. What is the evidence that this is true?"

3. Essay
• what is the cell cycle
• key players: CDKs/cyclins
• regulation of cell cycle
• discovery:
cyclins originally ID’d from sea urchin eggs
cell cycle controlling genes from temperature sensitive cell division cycle mutants
(CDC’s)
• not all cyclins/CDK’s are essential (redundancy)

4. Contents
• Cell Division - Overview
• The Cell Cycle Engine
• Control of Cell Cycle
• CDKs and Cyclins
• Cell Cycle Phases
• Checkpoints
• Differences Yeast and Mammalian
• Experiments to discover CC
• Recent Insights

5. The importance of cell division
• we have descended from an ancestral cell that lived 3-4 billion years ago
• since then, genetic information has been preserved through cell division
• cell division is FUNDAMENTAL
• generates completely new organisms in single cell organisms
• is responsible for the generation of a multicellular organism from a single “founder cell”
• is key to maintaining a living organisms by renewing damaged cells

6. Simple Cell Cycle
Interphase

7. The Cell Cycle Engine
A cell’s life in-between divisions SIGNALLING PATHWAYS
regulate engine, response
to outside and inside events
ENGINE CYCLE
consists of 4 steps/strokes
with regular fluctuations
of cyclin/CDK activity
DOWNSTREAM EVENTS
driven by engine, such as
DNA replication, mitosis...

8. What controls the cell cycle?
• cell cycle control system (ccc)
• controls timing, coordination, order of events
• high level of speed and accuracy
• most important that genetic information is passed on
correctly : perpetuation and evolution!!!
• how it works: events are dependent on each other,
feedback from cc machinery to ccc
• simple ccc....
• this regulation takes place through phosphorylation
• ccc is based on oscillations in activities of regulatory
proteins: cyclin-dependent protein kinases (CDKs) !!!!
in some early embryonic cc’s: clock
whose rotation sequentially triggers
cc events at certain times, even if
events fail.
not dependent on intra/extracellular
signals to allow rapid division

9. CDKs and cyclins
• (remember kinases catalyse attachment of ATP-phosphate to each cyclin + 1-2 CDK’s
their substrate) most CDK’s + 1-2 cyclins
• cyclins phosphorylate a distinct set of proteins
• cyclins are activating partners of CDKs, the CDK subunit is
inactive as a protein kinase without the cyclin subunit
• CDKs are ser/thr kinases implicated in control of cc
progression
• CDK activity oscillates, which oscillates the activity of cc
machinery through phosphorylation
• cyclin activity depends on their concentration (controlled
through synthesis/degradation)
• different cyclins present at different stages of cc
remember: binding of cyclin confers
• distinct cdk/cyclin complexes basal activation, phosphorylation full
activation.

10. Where CDKs/cyclins occur in CC
CDK11-cyclinL
CDK1-cyclinB
CDK1-cyclinA
G0
RESTRICTION CDK4-CyclinD
POINT CDK6-CyclinD
CDK3-CyclinC (humans)
CAK
(CDK-activating kinase)
CDK7-cyclinH/Mat1
CDK2-cyclinA CDK2-cyclinE
see also Figure 3 of

12. Structure of CDK’s/cyclins
• cyclins vary in molecular mass from 30-45KDa
• homologous region: ~100aa, 30-50% similarity amongst cyclins A, B, D1 and E
• homologous region called cyclin box
• five-helix-bundle, responsible for binding to cdks (and sometimes substrate)
• 2nd-five-helix bundle similar in fold but not sequence
• CDKs more homologous (40-75% similarity CDK1-7)
• conserved catalytic core of 300aa
• same fold and tertiary structure, but differences in catalytic site
• two substrates: ATP and protein to be phosphorylated

13. Structure of CDK’s/cyclins
red/yellow: areas of CDK2 undergoing most conformational changes upon cyclin binding
N-terminal lobe, PSTAIRE helix
ATP
cyclin box
cyclin A
CDK2
cyclin: two helical domains with identical chain topology
phosphorylation site (Thr 160) cdk2: most conserved PSTAIRE sequence at interface
adapted from:

14. Structure of CDK’s/cyclins

15. T-loop, PSTAIRE helix

16. T-loop, PSTAIRE helix

17. G1 cc phases
• Gap-phase
• usually longer than S-phase or M-phase
• cell grows and prepares for DNA synthesis (prepares enzymes required for S phase)
• in presence of sufficient nutrients and appropriate signals, cell commits to completion of CC at
restriction point R, otherwise enters G0
• G0 = prolonged non-dividing state, result of unfavourable growth conditions or inhibitory signals,
or non-dividing terminally differentiated state
• mitotic and s-phase cyclins are degraded
• time for cell to integrate info from environment
• ensure that replication factors are bound to origin so that DNA rep can start successfully
• proteins, RNA and other cellular macromolecules are synthesised continuously and present in
many copies throughout the cytoplasm, hence simply divided by cytokinesis
• Golgi is thought to be fragmented into smaller vesicles during M-phase for even distribution
• centrosome: single copy per cell that is duplicated only once per cc, in S-phase

18. cc phases
S
• S=Synthesis
• DNA is replicated
• chromosome duplication
• CDK2/E is active at beginning, initiation of DNA synthesis
• CDK2/A present throughout S-phase, progression of DNA synthesis (function less defined)
G2
• further growth and preparation for cell division
• protein production, e.g. making microtubules

19. M cc phases
• mitosis = division into two daughter cells that recommence CC in G1
• chromosome segregation
cytokinesis:
distribution of duplicated nuclei
and cytoplasmic components
into daughter cells by division
of mother cell
sister-chromatids are attached
to opposite poles of spindle
sister-chromatids are separated

20. Transition through CC: Checkpoints
• cc is controlled through checkpoints where progression can be blocked
• if no problem: initiation of next phase of cc
• response at checkpoints:
• is previous phase complete?
• also connected to external signals
• conditions not ideal: cc stopped until ok or even apoptosis

21. Yeast and Mammalian CDKs / cyclins
• single cdk, cdc28 in cerevisiae (budding yeast), cdc2 in fission yeast (pombe)
• but multiple cyclins
• only cdk1 and cdk2 are homologous to yeast cdc2/28 and have central cc function

22. Yeast and Mammalian CDKs / cyclins
• in vertebrates: > 10 cdc2-related proteins
• much more complex?
• no, only CDK1 and CDK2 are functionally homologous to Cdc2/28

23. Experiments
• cdk1 (cdc2/cdc28) id’s from genetic screens for s.pombe
and s.cerevisiae mutants with defects in the CC
• cyclins id’s from sea urchin egs
• synthesised and destroyed at each cleavage division, named
‘cyclins’
• biochemical connection made 1989: cdk1/A/B
• using temperature sensitive cell division cycle mutants
(CDC’s)
• ok di ID genes required for S and M phases
• but difficult to ID which genes are involved in CC control
• in fission yeast (pombe) division occurs at a certain cell size
• some mutants divide early, when still small (WEE), -->
control of CC impaired
• all initial mutants mapped to single gene, wee1
• T sensitive mutant, almost normal at low T but wee at high T
• wee1 acted in G2 and controlled onset of mitosis

24. Recent New Insights

25. Recent New Insights
• many cells are able to survive without CDK2
• cdk2 knockout mouse viable (although sterile)
• cyclin E knockout lethal, but embryos developed to mid-gestation
• stable cyclin E-/- mouse embryonic fibroblast cell lines can be generated
• hence: redundancy in CDK2-mediated signalling!
• Tetsu, Cencer Cells 3 (2003), 233-245
• Berthet, Curr. Biol. 13 (2003), 1775-1785
• Ortega, Nat. Genet. 35 (2003) 25-31
• Aleem, Nat. Cell Biol. 7 (2005) 831-836

26. Some more references
Overviews:
Text books
Murray, A. and Hunt, T., The cell cycle.
Nurse, P., Masui, M. and Hartwell, L., (1998), Nature Medicine 4: 1103-1106. Understanding the cell cycle.
Nurse, P. (2000), Cell 100: 71-78. A long twentieth century of the cell cycle and beyond.
*Morgan, D.O., (1997), Ann. Rev. Cell. Dev. Biol. 13: 261-291. Cyclin-dependent kinases: engines, clocks and microprocessors.
*Murray, A. (2004), Cell 116: 221-234. Recycling the cell cycle: Cyclins revisited. (In the same volume short papers by T. Hunt and T. Evans on pp S63-S66).
1. The original observations.
(See also Murray and Hunt for a nice summary)
*1a. Rao, P.N. and Johnson, R.T., (1970), Nature 225: 159-164. Mammalian cell fusion studies on the regulation of DNA synthesis and mitosis.
*1b. Johnson, R.T. and Rao, P.N., (1970), Nature 226: 717-722. Mammalian cell fusion: Introduction of premature chromosome condensation in interphase nuclei.
1c. Matsui, Y. and Markert, C.L., (1971), J. Exp. Zool., 177: 129-145. Cytoplasmic control of nuclear behaviour during meiotic maturation of frog oocytes.
2. Genetics.
2a. Hartwell, L.H. et al., (1973), Genetics 74: 267-286. Genetic control of the cell division cycle in yeast: genetic analysis of cdc mutants.
2b. Nurse, P., (1975), Nature 256: 547-61. Genetic control of cell size at cell division in yeast.
2c. Nurse, P, (1976), Molec. Gen. Genet. 146: 167-178. Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe..
2d Nurse, P. and Thuriaux, (1980), Genetics 96: 627-637. Regulatory genes controlling mitosis in the fission yeast Schizosaccharomyces pombe.
*2e. Nurse, P. and Bissett, Y., (1981), Nature 292: 558-60. Gene required in G1 for commitment to the cell cycle and in G2 for control of mitosis in fission yeast.
*2f. Lee, M. and Nurse, P., (1987), Nature 327: 31-35. Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2.
3. Biochemistry
*3a. Lohka, MJ. et al., (1988), P.N.A.S. 85: 3009-13. Purification of maturation promoting factor, an intracellular regulator of early mitotic events.
3b. Dunphy, W.G. et al., (1988), Cell 54: 423-431. The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis.
3c. Gautier, J. et al., (1988), Cell 54: 433-439. Purified maturation-promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene cdc2+.
Identification and characterisation of cyclins.
*4a. Evans, T. et al., (1983), Cell 33: 389-396. Cyclin: A protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division.
4b. Murray, A.W. and Kirschner, M., (1989), Nature 339: 275-280. Cyclin synthesis drives the early embryonic cell cycle.
4c. Murray, A. et al., (1989), Nature 339: 280-286. The role of cyclin synthesis and degradation in the control of maturation promoting factor activity.
*4d. Glotzer, M. et al., (1991), Nature 349: 132-138. Cyclin is degraded by the ubiquitin pathway.
5. The CDK family in higher eukaryotes-functional redundancy?
Nb parts of the Murray Cell review are also relevant here.
*5a. Reviewed in Sherr, C.J. and Roberts, J.M., Living with or without cyclins and cyclin-dependent kinases. (2004) Genes Develop. 18: 2699-2711.
*5b. Cdk2-/- mice
Berthey, C et al., (2003) Curr. Biol. 13: 1775-85. Cdk2 knockout mice are viable.
Ortega S, et al., (2003) Nat Genet. 35:25-31. Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice.
*5c Cdk2 RNAi expts
Tetsu, O. and McCormick, F. (2003) Cancer Cell 3: 233-45. Proliferation of cancer cells despite CDK2 inhibition.
Comment in: Cell. 2003 114:398-9.
5d. cyclin E -/- mice
Geng Y, et al., (2003) Cell 114:431-43. Cyclin E ablation in the mouse.
Parisi, T et al., (2003) EMBO J. 22: 4794-803. Cyclins E1 and E2 are required for endoreplication in placental trophoblast giant cells.
However see for cyclin E Cdk2-independent function.
Matsumo, Y and Maller, J (2004) Science 306: 885-8. A centrosomal localization signal in cyclin E required for Cdk2-independent S phase entry.
*5e Moore, JD et al., (2003) Science 300: 987-990. Unmasking the S-phase promoting potential of cycin B1.
6. CDK inhibitors
*6a. Sherr, C.J. and Roberts, J.M., (1999), Genes Dev. 13: 1501-1512. CDK inhibitors: positive and negative regulators of G1-phase progression.
7. SCFs and APCs, Cell Cycle E3 Ubiquitin ligases
*7a. Vodermaier HC. (2004), Curr Biol. 2004 14 (18):R787-96. APC/C and SCF: controlling each other and the cell cycle.
7b. Deshaies, R. (1999), Ann. Rev. Cell Develop. Biol. 15: 435-467. SCF and Cullin/RING H2-based Ubiquitin ligases.
7c. Bartek, J. and Lukas, J. (2001), Science 294: 66-67. Order from destruction.
7d. Peter, JM (1998) Curr Opin Cell Biol. 10:759-68. SCF and APC: the Yin and Yang of cell cycle regulated proteolysis.
7e. Nash, P. et al., (2002), Nature 414: 514-521. Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication. (And associated News and views article in the same issue).
And accompanying structural study:
Orlicky, S et al., (2003), Cell 112: 243. Structural basis for phosphodependent substrate selection and orientation by the SCFCdc4 ubiquitin ligase.