Lecture 9: Cell Cycle

Cell Cycle: series of events that produces two identical cells from one cell. Two most events of the process:
DNA replication and Cell division (Mitosis & Cytokinesis).
Cell Cycle Phases: Events:
G0 Phase Cell exit cell cycle. Two types: permanent or temporary
G1 Growth phase: cell grows to prepare for the division
S Synthesis phase, in which DNA replicates
G2 Growth phase
M Mitotic Phase:
Prophase: DNA condense, nuclear envelope breaksdown and spindle assemble
Prmetaphase: spindle attaches to the chromosome
Metaphse: chromosome line up at the centre of the metaphase plate
Anaphase: sister chromatid separate
Telophase: DNA decondenses, spindle disassemble and nuclear envelope forms

- Cell Cycle control has two components:
o Regulated phosphorylation: by cyclin cdk complex: G1, S and Mitotic cyclin-CDK
o Regulated degradation: by ubiquitinylation of cyclins and proteolysis by E3 ligase complexes
e.g. APC, SCF
Cyclin CDK
complex
regulation model
Events/ Importance Targeted Phosphorylation Targeted
Degradation
1.G1 cyclin
CDK
Prepares the cell for DNA
synthesis by degradating the S
phase inhibiting factors and
phosphorylating the initiation
factors
-transcription factors to initiate
the synthesis of proteins for S
phase
-S phase inhibitors (for
degradation
-S phase
inhibitors
2. S cyclin CDK -activates the pre-replication
complex: activates the initiation
of DNA replication
-controls the amount of
replication by preventing the
reassembly of the pre-replication
complex so that DNA is
replicated only once
-pre-replication complex
-transcription factors for mitotic
phase

3. Mitotic cyclin
CDK
Initiates all the events in the M
phase
-for chromosome condensation
-for spindle assembly
-for nuclear breakdown
-for chromosome alignment along
the metaphase plate
-for APC (Anaphase promoting
Complex): degrades MPF and
securin
-cyclins
-Anaphase
inhibitors

Identification of cyclin CDK in the model organisms (Xenopus Laevis): using two assays:
- To monitor the MPF activity: Histone H1 Phosphorylation
- To monitor the cyclin B activity: antibody to cyclin
Type of assay Components in the
assay
Observation Conclusion
Untreated
assay
Everything Increase in cyclin B during M phase
entry: during DNA condensation
and decrease in cyclin B activity
during M phase exit, during DNA
decondensation. MPF activity cycle
mimics the cyclin B activity pattern
Cyclin B activity regulated MPF
activity
RNAase
treated extact
All the mRNA is gone No activity Therefore, some kind of mRNA
must be present for M phase
entry and exit
RNAase
treated extract
+wildtype
cyclin B
mRNA
Only cyclin B mRNA Similar observation to untreated
assay
Therefore, only cyclin B mRNA
synthesis is required to enter of
M phase
RNAase
treated extract
+
nondegradable
cyclin B
mRNA
only cyclin B mRNA;
however it cannot be
degraded
Increase in the cyclin B and MPF
activity during entry into M phase
but no decrease is observed during
M phase exit
Cell dont decondense due to
constant activity of cyclin and
MPF; therefore, Mitotic arrest is
observed:
Therefore, cyclin B must be
degraded to exit M phase.
Genetic screening: to screen for mutant with desired phenotypes
Temperature sensitive (TS) mutants: are conditional mutants that depict wildtype behaviour at normal
temperature ( 25 C) and disruptive behaviour at elevated temperature (35 C). Better than loss of function models
because yeast only contain one copy of the gene and if that gene is vital for survival, its disruption or loss of
function will lead to death. However, with these mutant wildtype behaviour and mutant behaviour can be
studied at variant temperatures. Two phenotypes:
- Recessive: absence of the wild type function
- Dominant: overexpression of the wild type function.
Genetic screening for TS mutants: create mutants using radiation populate them grow at 25C
screen for elongated or small cells at 35C.
Model
Organism
Mutants + Functions Conclusion:
S.pombe:
nuclear
envelope
does not
breakdown
Recessive Mutant Dominant mutant Types of cdc mutant:
Cdc2, cdc13 cdc25, wee1
-cdc2(cdkin) + cdc13(cyclin B): form a MPF complex
-cdc25 (cyclin B)stimulated mitosis by activating MPF
activity. It encodes for a phosphtase, that phosphotases
try 15 and activates MPF
-wee1 (cyclin B): inhibits mitosis by inhibiting MPF
activity. It encodes for tyrosine kinase and phosphates
tyr 15; inhibiting MPF activity.
-Regulation of MPF: mitotic cyclin + CDK forms MPF
inactive complex with two sites of phosphorylation
Y15, T161 wee1 phosphorylates Y15 CAK
phosphorylates T161 cdc25 dephorlates Y15 and
opens up the substrate binding site.
Elongated: recessive
mutant, absence of cdc
(cell division cycle
mutant) causes the cell
to continue to grow
without dividing; thus,
the elongated
phenotype

-Due to absence of
cdc2 or cdc13 or cdc25
or overexpression of
wee1

Wee: overexpression
of cdc caused cell to
prematurely enter
cell division; thus,
the small cell
phenotype.

-due to
overexpression of
cdc2/cdc13/cdc25
or absence of wee1
S.
cerevisiae
Phenotype: arrested at budding because
cytokinses begins before the cell enter cell M
phase.
To identify the type + function of these cdc,
rescue mutant technique on TS mutants were
done, in which gene (cDNA gene) was added
and grown at regular temperature and then the
temperature was elevated. If the growth was
observed at elevated temperature (cdc 28),
then the gene inserted is the missing gene and
if not (cdc28
TS
), then the gene inserted is not
required for the job. This process allows you
to identify the gene involved in the process,
and thus can be analysed by sequencing the
gene.
Types of cdc`s (cell division cycle) mutants:
Cdc28, cdc28
TS
(only one type of Cdk kinase but
various type of cyclins). Cyclins: CLN1,2,3(can survive
with only one type of CLN but not without all three;
CLB1,2,3 (B-type cyclin)
-Cdc28 is analogous to cdc2; however cdc2 mutants is
arrest in G1 phase while cdc28 mutants arrested in G2,
as it is required to garner entry into M phase while
cdc28 is required to gain entry in S phase. However,
they both have the same function and are
interchangeable. Therefore, S.cerevisiae have SPF
instead of MPF.
These complexes are inactivated by degradation of
them.
-Regulation of second ubiquitin complex to enter the S
phase: sic1on the S-phase cyclin+ CKD inhibits it
G-cyclin-cdk complex phosphorylates Sic1 which is
then recognized by SPF and is degraded, which allows
the cell to proceed into the S phase.

ANAPHASE PROMOTING COMPLEX ( APC): Cdc20 and cdh1. Cdc20 binds to APC (apc-cdc20)
and assists in degrading anaphase inhibiting factor while cdh1 binds to APC (apc-cdh1) to help degrading
cyclins at the end of M phase. B-cyclins that are to be degraded contain a signal sequence at its N terminus
called destruction box (D-box): RxxLxxxxN/Q (R: Argnine; L: Leucine; N:Asparagine; N:glutamine)
- Occurs in both S.pombe and S.cerevicae; in response to high MPF levels.
- Regulation cycle of APC: when MPF is high, MPF phosphorylates APC and activates it APC binds to cdc20 which then
degrades the anaphase inhibitor after the cell proceeds to anaphase and telophase activated APC also bind to cdh1 and
degrades the cyclin low cyclin level, low MPF levels less APC complex activated and the process is halted.
YEAST REGULATION CYCLE:
- G1 cyclin cdk complex: inactivates cdh1; activates the S-cyclin cdk complex by phoshphorylating the
inhibitor (sic1)
- SPF degrades the inhibitor and cell goes into S phase
- S cyclin cdk complex: activates pre-replication complex
- M cyclin cdk complex: activates earl mitotic events. High MPF levels activate APC which then binds to
cdc20 to degrade the anaphase inhibitor and binds with cdh1 to degrade cyclin at the end of mitosis this cdh1
is then inactivated by the G1 cyclin cdk complex and the process repeats itself.
MAMMALIAN CELL CYCLE:
- Has various Cdk`s: 1,2,4,6: but 1 is similar to cdc2 while 2 is similar to cdc28. The similarities and differences
are identified by comparing the sequence between the model organisms and the acquired sample.
- Cyclins: A,B,D,E
- Mammalian cell cycle also has a G0 phase, in which cell are arrested and in this phase, neither cdks or cyclins
are expressed.
o Mitogens are required for the transition of cells from G0 phase to G1 phase; these mitogens initiate the
entry to cell cycle by inducing synthesis of G-cyclin-cdk.
o These mitogens need to expressed until the restriction point and after which, even if they are removed,
cell proceeds to cell cycle.
Evidence: in the quiescent cells in which the mitogen was removed before reaching the restriction
point, no growth was observed and cell were observed to be arrested in the G0 phase.
o Initiation factors can induce the G0 cells to transfer to G1 phase. Two kinds of factors:
Early response genes: encode for transcription factors for the synthesis of late response genes.
These transcription factors are called c-fos transcription factors that target the MAPkinase. These c-fos transcription
factors phosphorlyate TCF and SRF; allowing them to bind to SRE; which initiates the synthesis of genes required
for delayed response genes. These are not blocked by protein synthesis inhibitors, because the proteins
for this are already present in the cell and are not synthesized; however, they remain inactivated
until phosphorylated to become activated. It`s declined response initiates the delayed response;
however, if the protein synthesis is inhibited, then no delayed response is generated and constant
early response is observed because no proteins will be there to down-regulate this response.







Delayed response genes: this response is blocked by protein synthesis inhibitors. These encode
for cyclins and cdk`s. This response regulates the restriction point, as it codes for E2F,
transcription factor that activates the transcription of cyclin E, which is used to transition from
G1 phase to S phase. However, E2F is inactivated by the Rb protein.
Process: cyclin D - cdk 4/6 complex is synthesis is initiate by mitogens which upon synthesis
phosphorylate Rb protein, rendering it inactive and freeing E2F transcription factor E2F
synthesises cyclin E, cdk2 and E2F. therefore, restriction point is just keeping Rb protein inactivated
long enough to generate cyclin E, cdk2; as these perform the same function, of keeping Rb protein
inactivated by phosphorylating it, so that E2F is free to activate cyclin A cdk2 for DNA synthesis.
Therefore, if after restriction point mitogen is removed, it has no effect as Cyclin E cdk2 takes over the
function of Cyclin D-cdk4/6 of keeping Rb protein inactivated.

Wildtype response Response observed when protein synthesis is inhibited