cell division cycle and checkpoints in eukaryotes

1. Cell division cycle in Yeast
• Submitted by-
Seema Mehra
Msc. Microbiology
2nd semester

2. Introduction
• The cell cycle is the succession of events whereby a cell grows
and divides into two daughter cells that each contain the
information and machinery necessary to repeat the process.
• Between one cell division and the next, all essential
components of cell must be duplicated, the most important
component is genetic material which must be accurately
replicated and the two copies carefully segregated to two
daughter cells.
• Division cycle of most cells consist of four coordinated
processes-
 Cell growth
 DNA replication
 Distribution of the duplicated chromosomes to daughter cells
 Cell division.

3. Contd…
• In eukaryotes cell cycle is more complex.
• Although cell growth is usually a continuous process, DNA is
synthesized during only one phase of the cell cycle and the
replicated chromosomes are then distributed to daughter
nuclei by a complex series of events preceding cell division.
• Progression between these stages of the cell cycle is
controlled by a conserved regulatory apparatus.

5. Phases of cell cycle
 Cell cycle is divided into two basic parts-
 Interphase
 Mitosis
INTERPHASE
Chromosomes are decondensed and distributed
throughout the nucleus, so nucleus appears
morphologically uniform.
At molecular level interphase is the time during which
both cell growth and DNA replication occur in an orderly
manner in preparation for cell division.
 Cell grows at steady rate throughout interphase

6. Contd…
 It consist of 3 phases-
1. G1 phase(gap 1)
- Corresponds to interval (gap) b/w mitosis and initiation of DNA replication.
- Cell is metabolically active and continuously grows but does not replicate its DNA.
2. S phase (synthesis)
- DNA replication takes place.
3. G2 phase (gap 2)
- cell growth continues and proteins are synthesized in preparation for mitosis.
- The duration of these cycle phases varies considerably in different kinds of cells.
- In budding yeast, all 4 stages of cell cycle gets completed in approx. 90 mins.

8. Mitosis
• The cell cycle is traditionally divided into several distinct
phases, of which the most dramatic is mitosis, the process
of nuclear division, leading up to the moment of cell
division itself.
• In mitosis the nuclear envelope breaks down, the contents
of the nucleus condense into visible chromosomes, and the
cell's microtubules reorganize to form the mitotic spindle
that will eventually separate the chromosomes.
• As mitosis proceeds, the cell seems to pause briefly in a
state called metaphase, in which the chromosomes,
already duplicated, are aligned on the mitotic spindle,
poised for segregation.
• The separation of the duplicated chromosomes marks the
beginning of anaphase, during which the chromosomes
move to the poles of the spindle, where they decondense
and re-form intact nuclei.
• The cell is then pinched in two by a process called
cytokinesis, which is traditionally viewed as the end of the
mitotic phase, or M phase, of the cell cycle

9. Fig..

10. • Some cells in adult animals cease division altogether (eg.
Nerve cells) and many other cell division only occasionally, as
needed to replace cells that have been lost because of injury
or cell death( eg. Skin fibroblast, liver).
• These cells exit G1 to enter a quiescent stage of cycle called
G0, where they remain metabolically active but no longer
proliferate unless called on to do so by extracellular signals.

11. • Identification of cells at mitotic stage can be done
microscopically while cells in other phases of the cycle (G1, S
and G2) must be identified by biochemical criteria.
• Cells in S phase can be readily identified because they
incorporate radioactive thymidine, which is used exclusively
for DNA synthesis.

12. Regulation of cell cycle by cell growth
and extracellular signals
 Progression of the cells through the division cycle is regulated
by extracellular signals from the environment as well as by
internal signals that monitor and coordinate the various
processes that take place during different cell cycle phases.
 Cell cycle progression is accomplished by a series of control
points that regulate progression through various phases of the
cell cycle.
 A major cell cycle regulatory point in many types of cells
occurs late in G1 and controls progression from G1 to S.

13. Contd…
 This regulatory point was first defined by studies of budding
yeast (Saccharomyces cerevisiae) where it is known as START.
 Once cells have passed START, they are committed to enter S
phase and undergo one cell division cycle.
 Passage through START is highly regulated event in yeast cell
cycle where it is controlled by external signals, such as
availability of nutrients, mating factors, as well as by cell size.

14.  Eg. If yeasts are faced with a shortage of nutrients, they arrest
their cell cycle at START and enter a resting state rather than
proceeding to S phase.
 Polypeptide factors that signal yeast mating also arrest the
cell cycle at START, allowing haploid yeast cells to fuse with
one another instead of progressing to S phase.

16. Contd…
• Also, START is a point at which cell growth is coordinate with
DNA replication and cell division.
• It is evident in budding yeast in which cell division produce
progeny cells of very different sizes- a large mother cell and a
small daughter cell.
• In order for yeast cell to maintain a constant size, the small
daughter cell must grow more than the large mother cell does
before they divide again.
• The cell size must be monitored in order to coordinate cell
growth with other cell cycle events.

17. • Some cell cycles are controlled principally in G2.
• Eg. In Schizosaccharomyces pombe , cell cycle is
regulated primarily by control of the transition from
G2 to M, which is a principal point at which cell size
and nutrients availability are monitored.

18. Cell cycle checkpoints
• The events that take place during different stages of
cell cycle must be coordinated with one another, so
that they occur in appropriate order.
• Cell cycle checkpoints prevent entry into the next
phase of the cell cycle until the events of the preceding
phase have been completed.
• Several cell cycle checkpoints function to ensure that
incomplete or damaged chromosomes are not
replicated and passed onto daughter cells .
• These checkpoints sense unreplicated or damaged DNA
and coordinate further cell cycle progression with the
completion of DNA replication or repair.

19. Various checkpoints are-
1. G2 phase checkpoint
– Checkpoint in G2 prevents initiation of mitosis until DNA replication
is completed.
– It senses unreplicated DNA, which generate a signal that leads to cell
cycle arrest.
– Thus operation of G2 checkpoint prevents initiation of M phase. It
also senses DNA damage, such as that resulting from irradiation.
2.G1 phase checkpoint
– Arrest at the G1 checkpoint, allows repair of damage to take place
before cell enters S phase, where the damaged DNA would be
replicated.

20. Contd…
3. S-phase checkpoint
• Provides continual monitoring of the integrity of DNA to ensure that
damaged DNA is repaired before it is replicated.
• It also provides a quality control monitor to promote repair of any errors
that occur during DNA replication.
4.Spindle assembly checkpoint
• Maintains integrity of genome, occurs towards the end of mitosis.
• Monitors the alignment of chromosomes onto the mitotic spindle,
ensuring that a complete set of chromosomes is distributed accurately to
daughter cells.

22. Contd..
• Cell cycle arrest at G1, S and G2 checkpoints is
mediated by two related protein kinases, ATM
and ATR that recognize damaged or
unreplicated DNA and are activated in
response to DNA damage.
• ATM and ATR activate a signalling pathway
that leads not only to cell cycle arrest, but also
to activation of DNA repair and in some
programmed cell death.

23. Restriction of DNA replication to once
per cycle
 It is important to ensure that the genome is replicated only once per cell
cycle.
 Thus once a segment of DNA has been replicated in S phase, control
mechanisms must exist to prevent re-initiation of DNA replication until cell
cycle has been completed.
 Thus this mechanism involves action of MCM helicase proteins that bind
to replication origins together with the origin recognition complex(ORC)
proteins.
 MCM proteins act as ‘licensing factors’ that allow replication to initiate.
 MCM proteins bind to replication origins during G1, allowing DNA
replication to initiate when cell enters S phase.
 Once initiation has occurred, MCM proteins are displaced from the origin,
so replication cannot initiate again.

25. Regulation of cell cycle progression
• Cell cycle of all eukaryotes is controlled by a conserved set of
protein kinases, which are responsible for triggering major cell
cycle transitions.
Protein kinases and cell cycle regulation
I. Maturation promoting factor(MPF)

27. Contd..
2. Second approach was genetic analysis of yeasts.
• Pioneered by Hartwell and colleagues in early 1970s.
• Investigators identified temperature sensitive mutants of
Saccharomyces cerevisiae that were defective in cell cycle
progression.
• These mutants called cdc mutants underwent growth arrest at
specific ponts in the cell cycle.
• Eg. Cdc28 mutants in S.cerevisiae caused the cell cycle to
arrest at START, indicating cdc28 protein is required for
passage through this critical regulatory point in G1.

28. Contd…
• cdc2 mutant of Schizosaccharomyces pombe arrest cell cycle
both in G1 and at G2 to M transition.
• Further studies demonstrated that cdc2 and cdc28 encoded a
protein kinase-first indication of prominent role of protein
phosphorylation in regulating cell cycle.
• The protein kinase encoded by the yeast cdc2 and cdc28
genes has since been shown to be a conserved cell cycle
regulator in all eukaryotes, which is known as cdk1.

30. Contd..
3. Third line of investigation
• It converged with identification of MPF and yeast genetics.
• Studies with protein synthesis inhibitors had revealed that
entry into M phase of these embryonic cell cycle requires new
protein synthesis.
• In 1983, Tim Hunt identified 2 proteins. These proteins
accumulate throughout interphase and rapidly degraded
toward end of each mitosis.
• Hunt called these proteins cyclins ( cyclin A and B) and
suggested that they might function to induce mitosis, with
their periodic accumulation and destruction controlling entry
and exit from M phase.

31. Contd…
• When MPF was purified from frog eggs in 1988, its molecular
characterization showd that this conserved regulator of cell cycle is
composed of 2 key subunits: cdk1 and cyclinB.
• Cyclin B is a regulatory subunit required for catalytic activity of cdk1
protein kinase.
• MPF activity is controlled by periodic accumulation and degradation of
cyclin B during cell cycle progression.
• Once activated, the cdk1 protein kinase phosphorylates a variety of target
proteinsthat initiate the events of M phase.
• cdk1 activity triggers the degradation of cyclin B (ubiquitin mediated
proteolysis). This proteolytic destruction of cyclin B then inactivates cdk1,
leading the cell to exit mitosis, undergo cytokinesis and return to
interphase.

33. Families of cyclins and cyclin-
dependent kinases
• cdk1 controls passage through START as well as entry into
mitosis in yeast. It does so, however in association with
distinct cyclins.
• G2 to M transition is driven by cdk1 +mitotic B type cyclins
(clb1, clb2, clb3 and clb4).
• Passage through START is controlled by cdk1 +G1 cyclinsor
cln’s.
• cdk1+clb5 and clb6- required for progression through S
phase.
• These associations of cdk1 with distinct B typeand G1 cyclins
direct cdk1 to phosphorylate different substrate proteins.
• Required for progression through specific phases of cell cycle.

35. Activity of cdk’s during cell cycle progression is
reglated by four molecular mechanisms
1. Association of cdk’s with their cyclin partners.
2. Activation of cdk/cyclin complexes require phosphorylation
of a conserved cdk threonine residue. Phosphorylation of
cdk’s is catalyzed by an enzyme called CAK (cdk activating
kinase).
3. Inhibitory phosphorylation of tyrosine residues near cdk
amino terminus, catalyzed by wee1 protwin kinase.
4. cdk’s activities are also controlled by binding of inhibitory
proteins( called cdk inhibitors or CKIs).

37. DNA damage checkpoints
• DNA damage checkpoints play a critical role in maintaining
the integrity of genome by arresting cell cycle progression in
response to damaged or incompletely replicated DNA.
• They allowtime for the damage to be repaired before DNA
replication or cell division proceeds.

39. Mitosis phase
• During mitosis, the chromosomes codense, the nuclear envelope of most cells
breaks down, the cytoskeleton reorganizes to form the mitotic spindle and the
chromosomes move to opposite poles, chromosomes segregation is then
usually followed by cell division(cytokinesis).
Stages of mitosis
1. Prophase
- marked by appearance of condensed chromosomes, each of which
consists of two sister chromatids.
- daughter DNA molecules produced in S phase.
- these newly replicated DNA molecules remain intertwined throughout
S and G2, becoming untangled during process of chromatin
condensation.
- end of prophase corresponds to breakdown of nuclear envelope.
- but in yeast, closed mitosis occurs in whih the nuclear envelope
remains intact.

40. Contd…
• In closed mitosis, daughter chromosomes migrate to opposite poles of
nucleus, which then divides in two.
• In these cells, the spindle pole bodies are embedded within the nuclear
envelope and nucleus dividesin two following migration of daughter
chromosome to opposite poles of the spindle.
2. Prometaphase
• Transition between prophase and metaphase.
• Microtubules of mitotic spindle attach to kinetochores of condensed
chromosomes.

41. Contd…
3. Metaphase
• Chromosomes shuffle back and forth until they eventually align on
metaphase plate in the center of the spindle.
4. Anaphase
• Breakage of link between sister chromatids which then separate and move
to opposite poles of the spindle.
5. Telophase
• Nuclei reform and chromosomes decondense.
6.Cytokinesis
• Begins during late anaphase and completed by the end of telophase.
• Resulting in formation of two interphase daughter cells.

44. Cdk/cyclinB and progression to
metaphase
• Events of M phase are initiated by activation of cdk1/cyclinB protein
kinase(MPF). Cdk1/cyclinB not only acts as a master regulator of M phase
transition, phosphorylating and activating other downstream protein kinase
but also acts directly by posphorylating some of the structural proteins
involved in cellular organization.
• Chromatin condensation is driven by protein omplexex called condensins,
which are members of a class of structural maintenance of chromatin(SMC)
proteins that play key roles in organization of eukaryotic chromosomes.
• Another family of SMC proteins, called cohesins, contribute to chromosome
segregation during mitosis.
• Cohesins bind toDNA in S phase and maintain linkage between sister
chromatids following DNA replication.
• As cells enter M phase, condensins are activated by cdk1/cyclinB
phosphorylation.
• Condensins replace cohesins along the length of chromosomes except at
centromere.

47. Spindle assembly checkpoint and
progression to anaphase
• Spindle assembly checkpoint monitors the alignment of
chromosomes on metaphase spindle.
• Progression from metaphase to anaphase results from
ubiquitin- mediated proteolysis of key regulatory proteins,
triggered by activation of an E3 ubiquitin ligase called
anaphase promoting complex.

49. Cytokinesis
• Cytokinesis of yeast and animal cells is mediated by a
contractile ring of actin and myosinII filaments that forms
beneath the plasma membrane.
• Cleavage proceeds as contraction of the actin-myosin
filaments pulls the plasma membrane inward, eventually
pinching the cell in half.

51. Meiosis
• It is a specialized kind of cell cycle that reduces the
chromosome number by half, resulting in theproduction of
haploid daughter cells.
• Unicellular eukaryotes, such as yeasts, can undergo meiosis as
well as reproducing by mitosis.
• Diploid S.cerevisiae undrgo meiosis and produce spores when
faced with unfavourable environmental conditions.

52. Process of meiosis
• Meiosis results in the division of a diploid parental cell into haploid progeny,
each containing only one member of the pair of homologous chromosomes
that were present in the parental cell.
• This is accomplished by two sequential rounds of nuclear and cell division
(called meiosis I and meiosis II), which follows a single round of DNA
replication.
• Like mitosis, meiosis I initiates after S phase has been completed and the
parental chromosomes have replicated to produce idenical sister chromatids.
• During meiosis I, homologous chromosomes first pair with one another and
then segregate to different daughter cells.
• Sister chromatids remain together, so completion of meiosis I results in the
formation of daughter cells containing a single member of each chromosome
pair (consisting of 2 sister chromatids).
• Meiosis I is followed by meiosis II, which resembles mitosis in that the sister
chromatids separate and segregate to different daughter cells.

54. Thank you