Dr Forsburg's all-purpose Cell Cycle Lecture Notes
These notes were used for my lectures in both BIMM112 (UCSD Division of Biology)
and BMS210 (UCSD School of Medicine, Biomedical Sciences Program).
The 2001 Nobel Prize in Physiology or Medicine was awarded to Lee Hartwell, Paul
Nurse, and Tim Hunt for their ground-breaking work on cell cycle regulation. Starting in
the late 60s, Hartwell used budding yeast to identify mutants that blocked specific stages
of cell cycle progression. Nurse, working in fission yeast in the 70s, went on to isolate
mutants that could also speed up the cell cycle, thus focussing his attention on the
original CDK kinase, cdc2. In the 80s, Hunt identified proteins in sea urchin extracts, the
levels of which varied through the cell cycle hence "cyclins". All three have continued to
make important advances in cell cycle research including the identification of
checkpoints, mechanisms coupling cell morphology to the cell cycle, and identification of
additional classes of kinases, cyclins, and inhibitors. For more information about their
studies that led to the award, see this BBC brief. You can also visit their web pages:
Hartwell, FHCRC Seattle, Nurse (ICRF London), and Hunt, ICRF-Clare Hall.
Background on the pombe cell cycle can be found on our site.
Problems printing? Try printing with graphics turned off to see the text properly.
Additional slides used in lecture are available here.
Reading list of some interesting papers that illuminate the principles discussed here.
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Rao, P. N., Johnson, R. T. (1970) Mammalian cell fusion: studies on the
regulation of DNA synthesis and mitosis. Nature: 225:159-164. A classic paper
indicating tht the cell cycle is regulated by trans-acting fctors.
Nurse P. A long twentieth century of the cell cycle and beyond. Cell. 2000
100:71-8. Review.
Noton E, Diffley JF CDK inactivation is the only essential function of the APC/C
and the mitotic exit network proteins for origin resetting during mitosis.Mol Cell
2000 5 :85-95
Uhlmann F, Lottspeich F, Nasmyth K. Sister-chromatid separation at anaphase
onset is promoted by cleavage of the cohesin subunit Scc1. Nature. 1999 400:3742.
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Hirao A, Kong YY, Matsuoka S, Wakeham A, Ruland J, Yoshida H, Liu D,
Elledge SJ, Mak TW. DNA damage-induced activation of p53 by the checkpoint
kinase Chk2. Science. 2000 287:1824-7.
All students: you are responsible for knowing the UCSD Policy on the Integrity of
Scholarship.
The Cell Cycle | Evidence for Regulation | Genetic Analysis | Cdc2 Regulation | Cyclins | Inhibitors |
Destruction | Mitotic exit | Replication | S to M phase | Checkpoints | Meiosis | Links | What the heck are all
these gene names?
Play the cell cycle game
1) The cell cycle
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A process of division allowing the duplication of cells
a cycle: end products (daughter cells) are the same as starting products (mother
cells)
G1 - S - G2 - M phase, where G1/S/G2 represent interphase; stages of mitosis are
prophase, metaphase, anaphase, and telophase
A chromosome cycle of chromosome duplication and segregation; S phase and
M phase must be coordinated but non-overlapping
Chromosome duplication occurs in S phase. All the DNA must be copied
faithfully exactly once. Must occur in a timely fashion: coordination of individual
origins of replication to fire once and only once. Duplicated chromosomes must
remain attached to allow subsequent cell cycle events: concept of cohesion.
Chromosome segregation occurs during M phase. During prophase, DNA
chromosomes condense or package to allow more efficient movement. Cell must
assemble a mitotic spindle. At metaphase, the chromosomes align upon spindle,
attached via their kinetochores to microtubules. Upon attachment and
organization of all the chromosomes, they are segregated by releasing cohesion
that attaches the sisters together, and reeling in the spindle (anaphase) until they
decondense and form new nuclei (telophase). Click here for more info and
diagrams of mitotic cells
Question of cell cycle regulation: how to ensure the orderly progression of events
so that nuclear cycle is coordinated with cell growth and physical separation.
Replication must occur once per cell cycle and precede chromosome segregation;
segregation must be complete before cytokinesis (cell division)
Question of chromosome dynamics: the cell cycle is also a chromosome cycle
2) Evidence for cell cycle regulation
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Convincing evidence for actual cell cycle regulators came from several key
experiments.
Rao and Johnston: mixing nuclei together in the same cytoplasm (heterokaryon)
to determine whether they could influence one another.
o S + G1 : induces the G1 nuclei to start S phase. Suggests that the S phase
nucleus contains a diffusable factor that will induce replication.
o S + G2 phase: the G2 nucleus does not do S phase. Something about G2
phase is refractory to the diffusible factor from S phase.
o G1 + G2 phase: no S or M phase
o M phase + interphase: induces inappropriate mitosis
o Conclusion: there are diffusible factors that can promote S or M phase.
The S phase promoting factor only works on G1 nuclei. The m phase
promoter works on everything.
Xenopus extracts: Take oocytes blocked before their final division (like M
phase), ask is it possible to induce them to enter M phase by injecting them with
cytoplasm from post-M phase eggs. Answer: Yes. Purify and find two proteins
comprising an activity called MPF.
Sea urchin embryoes. Find when looking at total proteins in a population
undergoing synchronous division that some proteins go up and down with the cell
cycle: cyclin.
Yeast cells: it is possible to isolate yeast mutants that can grow (e.g., synthesize
macromolecules) but cannot divide their nuclei: deficient in proteins required
specifically for cell division.
3) Key work required genetics.
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Genetic analysis in simple cells provides blueprint for biochemical studies
Yeast cells, which are simple, single celled eukaryotes, undergo cell division
cycle like human cells. Yeast is a general term, like "animal", so that it covers
organisms as diverse and humans and worms. Two types of yeast were used. The
key is that yeast cells are (a) simple and (b) haploid, that is one copy of each
chromosome.
Saccharomyces cerevisiae: budding yeast.
o Simple cell that divides by budding. Unusually short G2 phase. Brewer's
or Baker's yeast. Popular model for cell biology problems
Schizosaccharomyces pombe: fission yeast
o pombe means beer in Swahili. More typical cell cycle with long G2. Good
model for studies of growth control.
For both yeasts: key insight was the idea that it might be possible to identify
genes required to regulate cell division by looking for mutants. Key to this:
haploid cells. expect genes for cell division to be essential. Look for mutants that
are conditional: that is mutant only in one condition, not another. Use
temperature. TS (temperature sensitive) mutants are unable to do their job at high
temperature, presumably due to altered protein structure. At low temps, proteins
stay intact and can grow cells. Isolate cells with mutations, ask what happens at
high temp? cells will get "stuck" at a particular cell cycle stage.
o How to determine the stage at which blocked? use landmarks. For
example, did they replicate their DNA or not? For budding yeast: is there a
bud and how big? In fission yeast, cells simply elongate For both: is there
a mitotic spindle? Did nuclei condense? cdc mutants ("cell division
cycle")
o If it is possible to remove a key component, is it also possible to change
regulation/timing? Model: if you slow down the fission yeast cell cycle,
get long. if you speed it up, get short. isolate short or "wee" mutants:
defective in timing or regulation.
using methods of genetics, determine how many genes are mutated. That is, for all
the mutants that arrest cells in G2, how many different genes do they represent?
combine mutants together to see if it is possible to determine pathways between
them.
4) isolation of Cdc2 kinase and determining regulation
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Fission yeast: two different types of mutation in gene called cdc2: elongated
mutant (ie, cell cycle delay), and small mutant (ie, cell cycle speeds up). Other
phenotypes will be discussed later in lectures Suggests that cdc2 may function
in timing: required for division (long mutant) but changing its regulation changes
timing (short mutant).
Mutants and phenotypes: mutant name: means protein not active. OP: means
producing too much of the normal protein: essentially over-active. LOF (loss of
function), and GOF (Gain of function), respectively.
cdc25
long
wee1
short
cdc2-L long
cdc2-w short
OP cdc25 short
OPwee1 long
cdc13
long
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Suggests that changing activity of these proteins can change how they behave.
How to determine order of events? Combine mutants and ask what the double
mutant looks like. Called epistasis. If one protein is required for cell cycle, then
changing activity of protein s upstream won't make a difference if protein 1 is
missing. Requires the mutants have different phenotypes (appearance) to
distinguish which phenotype is displayed. Results:
cdc2-L wee1
long
cdc2-L OP cdc25 long
cdc2-w cdc25
short
cdc2-w OP wee1 short
cdc25 wee1
normal
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Conclusion: wee1 and cdc25 act upstream of cdc2. wee1 inhibits cdc2: because
absence of wee1 speeds things up. But if cdc2 is missing wee1 doesn't matter.
cdc25 activates cdc2, because if cdc25 is missing, things slow. but if cdc2 already
active (cdc2-w), cdc25 doesn't matter. cdc13 works with or downstream of cdc2
(can't do experiment reciprocally to be sure). Important: cdc25 and wee1
antagonize each other, because together everything looks okay.
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These yeast experiments agreed with biochemical data. Showed that cdc2 and
cdc13 bind together. Yeast cdc2/cdc13 turn out to be the same two proteins that
bound together in Xenopus MPF. Also showed cyclin (Cdc13) levels go up and
down as cell goes through cell cycle.
cdc2 is a kinase that is only active if bound to cyclin. Found that it is itself
phosphorylated on Y15 by wee1, and this phosphate is removed by cdc25. Wee1
is a kinase, cdc25 a phosphatase. Additional biochemical and genetic experiments
showed an additional phosphorylation on T161, activating.
o cdc2 alone: not active
o cdc2 cdc13 : not active
o cdc2 (Y15 Phosphorylated) cdc13: not active
o cdc2 (Y15 Phos T161Phos): not active
o cdc2 (T161Phos) cdc13: active
Basic cell cycle regulation is regulation of state of cdc2. requires 2 things: cyclin
partner and appropriate phosphorylation. Cyclin accumulates during cell cycle,
destroyed during M phase. phosphorylation provides additional switch.
Phenotype of cdc2 Y15 mutant: chronically activated. Relies on accumulation of
cyclin. Insensitive to wee1/cdc25 "switch". Dangerously active kinase, cannot
respond to damage (see below)
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5) Cyclin/CDK cycles in the cell cycle
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Also apparent that other major transitions in cell cycle regulated by cyclin/CDK
("cyclin dependent kinases"). other cyclins that peak earlier -- G1, S phase
cyclins.
In yeast, only one CDK, multiple cyclins. Presumed that cyclins make kinase
specific for particular substrates. Periodic activation of kinase via association with
different cyclins. In S. cerevisiae, several overlapping cyclin activities: G1 cyclins
CLN1-3, S cyclins CLB5-6, M cyclin CLB1-4
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In humans, multiple CDKs as well, so overlapping activation of different
complexes.
CDK4/cyclin D -> CDK2/cyclin E -> CDK2/cyclin A -> cdc2/Cyclin B
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How is the transition accomplished, to assure cell cycle moves forward? three
broad methods:
o 1) Phosphorylation/dephos of CDK (as described)
o 2)Specific inhibitors to regulate CDK activity.
o 3) Destruction of cyclin and inhibitors at appropriate time in cell cycle
6) Inhibitors of cell cycle
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CKI, or Cyclin Kinase Inhibitors. prevent accumulated cyclin/CDK from acting.
Probably dosage dependent. p21, p27, p16 in human cells. In yeasts, SIC1 or
rum1.
As yeast cells enter G1, specific inhibitor of B-cyclins is present (SIC1/Rum1).
Keeps the mitotic kinase from acting either too long, or too soon. As cells proceed
into S phase, destruction of this inhibitor is triggered.
In budding yeast, Sic1 active as cells exit mitosis and enter G1. At the same time,
a special set of G1 cyclins is transcribed and synthesized. Sic1 only acts on G2
cyclins (B class). The CDK/G1 cyclin phosphorylates SIC1. The phosphorylated
Sic1 is then bound by a complex called the SCF, which targets it for destruction
(discussed below). Thus, the cycle is set up to (A) prevent premature activation of
the NEXT set of cyclins and (B) ensure that Sic1 is turned off as the current set
gets activated.
In Fission yeast, Rum1 protein works like budding yeast SIC1. Although they do
the same job, these proteins are not related to one another by sequence.
Other inhibitors are regulated analogously. Direct inhibition of CDK activity is
not the only way to block cell cycle progression. For example, the Rb protein in
human cells binds to the E2F transcription factor and prevents its activity. no E2F,
no S phase genes are expressed. Just as in yeast the increasing levels of the G1
cyclins lead to inactivation of SIC1 protein, so in human cells does increase of
CDK4/cycD and then CDK/cycE lead to inactivation of Rb. As CDK activity
increases, Rb more phosphorylated. This causes Rb to release E2F transcription
factor, which then can turn on S phase genes. Rb is dephosphorylated late in the
cell cycle to prevent further division unless appropriate cyclin activity is present.
Thus Rb is DOWNSTREAM of CDK activity, while CKIs are UPSTREAM.
In mammals, two broad classes of CKI: INK4 family (p15/p16/p19) specific for
CDK4/Cyclin D; probably regulates cell cycle entry in response to growth factors
etc. CIP/KIP family (p21, p27) induced by p53, may mediate normal control and
cell cycle response to damage. Interestingly p21 may act both positively and
negatively, depending upon how much is present. CIP/KIPs can regulate all
classes of CDK/cyclins, at least in vitro.
7) Regulated destruction
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A number of proteins are regulated by turnover: proteolysis. Ensures that cell
cycle can't roll backward. This requires that the targets be ubiquitinated by
specific ubiquitn ligases, which targets them to the proteosome for destruction.
o Degradation of cyclin is essential to keep cell cycle moving forward.
Making a cyclin mutant that cannot be degraded traps cells in M phase.
o CKIs, such as Sic1 and Rum1, must also be turned over.
o Cohesion between sister chromatids must be removed: must degrade
"molecular glue"
APC identified by isolation of mutants that affect protein stability in mitosis.
mutants that fail to turn over proteins that must be destroyed for cell cycle
progression, will block at that point in the cell cycle. Expression of a "nondegradeable cyclin" has the same phenotype as mutation of the protein that
degrades cyclin. In this way, isolate components APC (anaphase promoting
complex, or cyclosome) required for ubiquitination of substrates and targeting to
proteasome. Two points of destruction: metaphase to anaphase transition, when
chromosomes separate, and mitotic exit, when cyclin degraded. These were
distinguished because expression of a non-degradeable cyclin did not prevent
chromosome segregation
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APC works through two sets of targets because it has two specificity
factors. First, molecular glue (metaphase to anaphase transition). Protein
PDS1 ("securin") binds ESP1 ("separin") and cohesion between
chromosomes remains intact. If EPS1 is freed from PDS1, chromosomes
will separate. Thus, destruction of PDS1 mediated by APC leads to
chromosome separation. If pds1 is mutant, this will happen prematurely.
APC targeting to PDS1 is provided by CDC20 subunit.
Activation of this APC/CDC20 requires mitotic CDK activity (makes
sense since CDK promotes mitosis!)
mutants in substrates show that APC independently affects meta->ana
transition and cyclin degradation/exit. Target sequence is called
"destruction box". CLB2 lacking destruction box blocks after anaphase;
PDS1 still degraded. However PDS1 affects cyclin turnover by blocking
activation of APC/CDH1: crosstalk between the two APC subunits. PDS1
lacking destruction box inhibits degradation of CLB2 and ASE1. Still
Pds1 is not essential for viability in yeast.
This system is not sufficient to allow mitotic exit and cyclin turnover.
Different specificity factor CDH1/HCT1 required here. Inactivated by
CDK; when APC/CDC20 gets activated, that inactivates CLB5 and thus
allows APC/CDH1 to be activated sequentially. In turn, this inactivates
CLB2, allowing cells to proceed out of mitosis. Additional kinases also
involved in pathway to release cells to G1 phase (see mitotic exit, below).
Polo-kinase (Cdc5 in budding yeast) is required for activation of
APC/CDH1. Note that activation of APC/CDH1 will inactivate the mitotic
kinase--which activated APC/CDc20. This ensures forward momentum.
net effect: cell cycle can't "roll backward"
As ever, system may vary in different cell types. In fission yeast and other
eukaryotes, the CDH1-homologue appears to be responsible for degrading
the mitotic cyclin in G1 phase, NOT in the exit from mitosis. Thus, the
"second" form of the APC may be G1-specific. This may relate to
observations in fission yeast that the mitotic exit network - equivalent
proteins act in septation....which occurs after mitotic exit.
 SCF in S phase, and APC are related complexes that recognize specific substrates and
target for destruction.
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Both are types of ubiquitin ligase (E2) enzymes that covalently attach the small
peptide ubiquitin to targets.
Ubiquitinated proteins are targeted by the proteasome, which degrades them
SCF ubiquitinates proteins in G1 to S phase (e.g., SIC1).
Contains several subunits including "cullin" (CDC53 in yeast), SKP1 linker
protein, and specificity factor containing an "F box sequence" (CDC4 in yeast)
that binds to phosphorylated substrate. These proteins are distinct from APC but
share some motifs. As CKI (SIC1) is expressed, it inhibits the downstream CLB
kinase. But at the same time, the CLN form of the kinase is induced, leading to
the phosphorylation of Sic1, and its recognition by the SCF. This results in SIC1
turnover, and release of inhibition of S phase kinase. F box factor itself is
ultimately targeted for destruction.
Other specificity factors (F box proteins) can association with the cullin-SKP1
base and thus its activity not limited to G1 or even cell cycle regulation. However,
its substrates must be phosphorylated, and thus recognized by F box factors.
8) Regulation of mitotic exit: from one cell cycle to another
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Mitotic exit network (MEN) coordinates APC with inactivation of CDK--one
more layer of complication, one more mechanism for irreversability. The network
consists of a GTPase-activated kinase cascade that ultimately regulates the
activity of a phosphatase, CDC14.
In budding yeast an important part of this regulation is spatial: the MEN is
activated when one of the spindle poles moves to the new bud. (It is also affected
by the spindle checkpoint, discussed below) This leads to the release of CDC14
phosphatase from the nucleolus, where it has been sequestered by NET1 protein.
CDC14 phsophatase antagonizes the mitotic CDK by dephosphorylating the same
substrates. Thus, whether cell will exit from mitosis will be determined by the
relative balance of the kinase and the phosphatase.
The MEN is inhibited by the spindle checkpoint, and by the activity of PDS1.
CDC14 also dephosphroylates CDH1, leading to increased activity of the
APC/CDH1, and further inactivation of CLB2. Because the mitotic form of the
kinase prevents formation of the replication complex called the preRC (see
below), the action of CDC14 not only helps push the cells out of mitosis but
facilitates the next S phase.
CDC14 release leads to the activation of SIC1 by two pathways:first, by
dephosphorylating the SIC1 transcription factor SWI5, which allows SWI5 to
enter the nucleus and activate SIC1 expression, and second, by directly
dephosphorylating SIC1, and thus protecting it from degradation. However, as the
CLN cyclins accumulate, they will eventually overcome the CDC14
dephosphorylation of SIC1, and this will drive the cycle forward.
In fission yeast, the CDC14-equivalent protein is not essential, and the
components of the MEN are not involved in promoting mitotic exit. rather, they
form a septation-inducing network (SIN). As discussed above, the APC/CDH
equivalent complex also appears to be G1, rather than late-M specific. Thus the
same sets of proteins can be used to accomplish subtly different requirements.
9) Regulation of replication onset: downstream of CDK
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Another example of how interplay of CDK, degradation, and CKI work is in the
regulation of the onset of DNA replication.
Problem: Must ensure that replication origins finally only once per cell cycle, and
fire after mitosis is complete.
Solution: couple assembly of structures that fire replication to CDK activity.
Mitotic CDK activity prevents formation of active origin structures (prereplicative complex, or pre RC. Thus, only when mitotic CDK activity (B cyclin)
is low can complex assemble. Thus preRC assembly is limited to late M
phase/G1.
However, firing of this complex, and activation of replicaiton, requires CDK
activity. Thus, CDK activity both activates the origin and prevents it from
reactivating, all at once.
Components required:
o Origin of Replication (yeast ARS: Autonomously Replicating Sequence)
on the DNA: site where DNA replication begins.
o ORC complex, identifying the origin throughout the cell cycle
o Cdc18 (AKA Cdc6) protein: activates origin, allows loading of additional
factors. targeted for destruction by mitotic CDK.
o MCM proteins: together, form likely helicase. Can only load at origin
when CDC18 is there.
o replication machinery: polymerases, ligases, etc. Require that MCMs are
loaded first.
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Example from S pombe. Note: put the cursor over the righthand image to see
an animated version!
o Cdc18 protein is transcribed specifically before S phase, in M/G1 phase.
o But CDK activity is high, e.g. in M or S phase, the origin cannot be
assembled because Cdc18 is targeted for destruction by CDK
phosphorylation
o Once origin is assembled, CDK activity required to fire it (exactly how
remains unclear)
o premature activation is prevented by CKI Rum1p, which keeps CDK
activity low
o Deletion of Cdc18 results in failure initiate DNA replication. OP-rum1
(like SIC1, a CDK inhibitor) leads to inhibition of kinase, failure to
degrade Cdc18, re-initiation of origins.
o Independent pathways also involved, including another kinase CDC7
(Hsk1 in the figure below), and sequential loading of different replication
factors.
o (See this page for complete description)
10) Dependency of mitosis on S phase
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Cell cycle requires sequence of S phase - M phase. Maintained in part by
regulation of CDK. For example, mutations in S. pombe that affect mitotic CDK
activity can lead to "re-replication" in which cells repeat S phase without any
intervening M phase (see diagram of phenotypes, above).
S. pombe is particularly easy to manipulate in this way. Mutations that cause rereplication include :
o temperature sensitive mutations of Cdc2 kinase (when returned to
permissive temperature), suggesting that the kinase is re-set in some way.
o Overproduction of the CKI Rum1 (roughly equal to SIC1 in cerevisiae
o Deletion of the mitotic cyclin Cdc13
o
Overproduction of Cdc18, an S phase inducer. Cdc18
is a CDK substrate, and when overproduced may saturate the CDK
system. Phosphorylated Cdc18 is degraded by the SCF.
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Mitotic progression is also directly dependent upon S phase. For animation,
move your cursor over the image at the right
o Cohesion between sister chromatids is established during S phase
o Absence of cohesion leads to premature chromosome separation and
segregation defects
o Normal cohesion is required to establish tension between the
chromosomes on the mitotic spindle, and also to orient the chromosomes
properly for the mitotic divisions.
o Cohesin connections are dissolved by ESP1 (separin) when PDS1
(securin) is destroyed by APC
11) Checkpoints
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Checkpoints maintain the order of unrelated events by signaling if something
goes wrong, e.g. Damage in G1 or G2, incomplete replication, incomplete
establishment of mitotic apparatus.
Characteristic of checkpoints is that they are usually not essential (pace, Don...).
This is particularly true of the checkpoints in yeast As long as everything works
all right, then no problem. However, if anything is perturbed--damage occurs, S
phase takes too long, spindles fail to assemble, then checkpoint becomes essential
to prevent continuation of the cell cycle. In checkpoint mutants, mechanical
apparatus of division is intact, and the problem is regulatory.
in multi-cellular organisms, some checkpoints are essential for viability of the
organism. Generally, even in cell types where some checkpoints are essential, we
can think of them as extrinsic to normal engine of the cell cycle--an additional
layer of regulation on top of the CDK/cyclin engine.
A checkpoint response consist of three broad components: something to generate
the signal, something to transduce it, and something to receive it. The transducers
are typically what we think of as "checkpoint proteins".
how to identify checkpoint proteins, if they aren't essential? Key is to make them
essential. For example, in response to irradiation, most yeast cells will arrest the
cell cycle, repair the damage, and then continue. A cell that cannot repair the
damage will arrest permanently. A cell that can repair the damage but can't arrest
will go on to divide, with lethal consequences. Difference is arresting as one cell,
or as a microcolony. Similarly, treatment with low levels of a spindle poison such
as benomyl will lead to transient arrest and recovery in wild type. Lethally
sensitive mutant such as tubulin mutants will die as single cells. Checkpoint
mutant cells continue to try to divide--again, lethality as microcolonies.
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DNA metabolism checkpoints. First
identified by mutants that failed to
respond properly to radiation (many are
called "rad" mutants), or treatment with
the drug hydroxyurea, which blocks at an
early stage of S phase by inhibiting
ribonucleotide reductase
o While at first it was thought there
was a single response pathway,
subsequently became apparent
that HU and radiation and other
forms of damage challenge the
cell in subtly different ways. Referred to as replication and damage
checkpoints, respectively
o These pathways overlap and use conserved kinases. General scheme can
be defined as shown at right. However, there are differences in the details
in different species.
o In mammals, two kinases at top of pathway ATM and ATR which
respond to different signals. In cerevisiae, major kinase homologue is
MEC1. Similarly, in pombe, Rad3 is the major player. The yeast kinases
appear to respond to all types of signal. Downstream, the Chk2 kinase is
called RAD53 in budding yeast, Cds1 in fission yeast. Fortunately, Chk1
has the same name in all species.
o Different pathways activated in response to different challenges. The
Chk2/RAD53/Cds1 pathway responds early; the Chk1 pathway is specific
for damage later in the cell cycle. As well as arresting cell cycle
progression, the Chk2/RAD53/Cds1 pathway actively promotes repair of
the damage. In mammalian cells, p53 is an important player but there is no
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p53 equivalent in yeast. Apoptosis is also regulated by this pathway;
again, there is no similar phenomenon in single celled yeast.
Experiments in fission yeast help to elucidate the signal:
o Rad3 activates the downstream kinases Chk1 and Cds1/RAD53/Chk2 in
response to different signals.
o Chk1 is the damage-response kinase. It is activated by DNA damage,
irradiation, or S phase progression (during which cells generate
chromosomal structures characteristic of DNA damage). It phosphorylates
Cdc25 protein which is inactivated and also exported from the nucleus.
Chk1 mutants are viable, but sensitive to irradiation and other forms of
damage.
o Cds1/RAD53/Chk2 is the replication-response kinase. While it may
overlap with chk1, its primary function appears to be in regulating
replication in response to perturbations. For example, in HU treated cells,
Cds1/RAD53 is active, and prevents the cells from firing/extending their
replication forks while HU is present. It is also required for cells to be able
to re-start replication when HU is removed and to repair DNA damage.
Cds1/RAD53 checkpoint mutants are viable, and sensitive to HU. They
are not particularly radiation sensitive.
o Cells that fail to initiate S phase never activate the checkpoint, because
they never send the signal that replication has started. Thus, initiation
mutants such as cdc18-null in S pombe try to divide without replicating,
generating a cut phenotype (see phenotypes, above). In contrast, cells that
initiate, but cannot complete S phase, do activate the checkpoint. Classic
cdc-style arrest of replication mutants is therefore checkpoint-dependent.
o What happens to cells lacking checkpoint? No effect, as long as nothing
goes wrong. BUT cannot arrest mitosis when damaged. Cannot prevent
mitosis when S phase does not occur
o Some mutants are checkpoint-defetive because they do not initiate the
signal, such as cdc18-null. some are bona fide checkpoint mutants, such as
rad3 or chk1 mutants. some are defective in the receiver. For example,
cdc2-3w, version of cdc2 that is independent of cdc25 activation, is
naturally checkpoint deficient. Ditto cdc2-Y15F.
o A conserved group of proteins called the "checkpoint rads" may be
involved in sensing the damage and activating Rad3. These have similarity
to DNA replication proteins. During DNA replication, the PCNA protein
is clamped around the DNA by its loading factor, RFC1-5. Intriguingly,
the checkpoint Rad17 prtoein is structurally related to the RFC1
replication protein), and Rad1, Rad9, and Hus1 form a complex that looks
like PCNA. Current models suggests that Rad17 and the RFC2-5 subunits
form a damage-specific clamp loader protein that loads on the
Rad1/9/Hus1 complex which in some way signals damage to Rad3.
However, not all forms of damage require the checkpoint rads for the
activation of Rad3.

in budding yeast, things
are different.
o damage checkpoint
still regulates
mitosis, but by
modulating activity
of APC and its
targets, instead of
Cdc2. That is,
regulates events in
mitosis rather than
events pre-mitosis.
This may reflect
very short G2 in
budding yeast (S
and M phase
normally tightly
coupled). Thus,
PDS1 is a crucial
molecule in the
replication and
damage checkpoint
o

responses in
budding yeast.
Also, the MEC1
and RAD53
proteins are
essential for
viability, indicating
an essential role for
these proteins.
However, there are
alleles that are
checkpoint
defective but viable,
indicating the
essential and
checkpoint
functions are
genetically
separable.
In humans, these proteins
are also present.
Significantly there are
additional transducers,
including p53 and BRCA
tumor suppressor genes,
which are substrates of the
ATM/ATR (=Rad3) family
of kinases.
o p53 highly unstable,
targeted for
destruction by
binding MDM2.
This binding
prevented when p53
phosphorylated by
checkpoint kinase
cascade.
o p53 induces
expression of range
of genes including
CKIs that block
CDK activity
o Inactivation of
o

CDKs blocks
engine of cell cycle,
including
preventing Rb from
activating S phase
genes.
Visit this site for
more about p53.
spindle checkpoint:
separate mechanism
monitors whether cells
assemble their
chromosomes and
spindles correctly. This
works by affecting
APC activity, in all
systems. Don't want to
divide in absence of
aligned chromosomes,
or might lose one. In
yeasts this pathway is
not essential for life,
but in other organisms,
it is required. This may
indicate that more
complex genomes are
more prone to
problems.
o pathway
bifurcates. One
arm: Mad2
protein binds to
unattached
kinetochores,
and prevents
APC activation
by binding
Cdc20 (APC
activating
complex). Other
arm: Bub2
inhibits
activation of
TEM1 and
blocks activity
o
o
of the Mitotic
Exit network
(MEN). Once
all kinetochores
attached and
spindle is ready,
MAD/BUB
release blocks
of APC/MEN
and mitosis can
proceed.
Unattached
kinetochores are
phosphorylated,
which may lead
to Mad2
binding. Mad1
binds Mad2 and
is a substrate for
the MPS1
kinase. Mad2
binds CDC20,
which prevents
APC activation.
BUB2 is also a
substrate for
MPS1. As
described
above, BUB2
prevents the
GTPase TEM1
from being
activated and
thus blocks
activation of the
Mitotic Exit
Network. This
keeps the
mitotic cyclins
from being
degraded, and
thus keeps
cerevisiae cells
in mitosis. In
fission yeast,
the BUB2
o
12). Meiosis
equivalent
prevents cells
from
undergoing
cytokinesis.
This occurs
during G1.
Thus, the
checkpoint
response, like
the APC
pathways, vary
in different
organisms
although they
use conserved
proteins.
MAD and BUB
proteins
involved in all
these
checkpoints in
yeast are also
involved in
human cells. As
is the case for
DNA
metabolism
checkpoints, in
some cases
there are several
related proteins
where yeast has
one. Ability to
manipulate
spindle
dynamics in
larger cells
allows testing
of theories from
genetical yeast
expts.




Meiosis is a specialized cell cycle that functions to reduce a diploid to a haploid.
This is accomplished by undergoing one round of S phase followed by two
divisions, reducing a diploid to 4 haploids in g1 phase. For basic description of
meiosis including detailed diagrams, go to this page on this site.
Many of the "usual" cell cycle proteins are involved in meiosis as well, including
the CDKs, cyclins, APC, and cohesins. There appear to be meiosis-specific
versions of at least some of these proteins functioning in addition to their mitotic
counterparts.
A central component to meiosis is the recombination between homologous
chromosomes, which is required for proper chromosomal segregation.
Recombination appears to require DNA replication and recombination proteins
may influence the rate of meiotic progression. Additionally, DNA replication in
meiosis may involve different proteins from those involved in vegetative cell, at
least those involved in origin control.
One particular area of interest recently in cohesion. There is a meiosis-specific
cohesin, Rec8, that appears to be important for orienting the kinetchores properly
and allowing the reductional and equational divisions to occur properly.
o In the meiosis I division, the homologous chromosomes separate, but the
sister chromatids remain attached. This is the reductional division, because
it essentially reduces the number of chromosomes in the daughter nuclei
from two to one. Recombination intermediates hold the chromosomes in
synapsis, presumably limiting the access of the spindle to one kinetechore
per homologue. During MI, the cohesion dissolves in the chromosome
arms, but remains around the centromeres. Thus, the homologues remain
attached, but the recombined arms can separate from each other.
o During the MII division, the homologues separate, much as they do in
mitosis in vegetative cells. In MII, the remaining cohesin around the
centromeres is degraded so that the chromosomes separate properly.
Other sites
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Cell cycle slide show
Visit Mitosis World
The Cell Cycle
CellsAlive.com has excellent schematics of animal cell mitosis and the cell cycle
Quiz yourself on mitosis and meiosis
Cell Cycle Chapter of MOLECULAR BIOLOGY OF THE CELL (on line at
NCBI)
Sample topics including CDK and cyclins, from a textbook called The Cell Cycle:
Principles of Control. YOu can get started here
Milestones in the cell division from Nature
More cell cycle notes/study tips
Cell cycle and cell division links
What the heck are all these gene names anyway?
More than many fields, the cell cycle is particularly complicated because of the plethora
of different gene names in different systems. One option in lecture is to use just one
generic name--but then you can't read any papers, because everyone in the literature uses
different gene names. Here is a table that should help negotiate the different species and
different nomenclatures in this lecture.
Factor
what is it
S.
S.
cerevisiae pombe
metazoans
CDK
cyclin dependent kinase
CDC28
Cdc2
Multiple
CDKs: CDK16
G1 cyclin
regulatory subunit of CDK for cell
cycle entry
CLN1,2
and 3
?
Cdk4-cyclinD
S phase
cyclin
regulatory subunit of CDK for S
phase entry
CLB5, 6
Cig2
Cdk2-cyclinE
late S phase
cyclin
regulatory subunit of CDK for S
phase progression
CLB3, 4
?
Cdk2-cyclinA
M phase
cyclin
regulatory subunit of CDK for
mitosis
CLB1, 2
Cdc2<CDK1)Cdc13 CYCLINB<
TD>
APC
Multi-component ubiquitin ligase
required for degradation of
substrates in mitosis and G1
Many
genes
Many
genes
Many genes
APC
specificity
factors
target the APC towards different
substrates
CDC20
HCT1
Slp1
Srw1
Cdc20, fizzy
Hct1, Fzr
securin
An APC target, inhibits sister
chromatid separation
PDS1
Cut2
securin
separase
The securin target, a protease that
degrades cohesin
ESP1
cut1
separase
Cohesin
A complex of proteins that holds
sister chromatids together
SCC1, aka
MCD1
SCC3
SMC1
SMC3
Rad21 aka
Rad21
SCC1
psc3
SCC3
psm1
SMC1
Psm3
SMC3
SCF
Multi-component ubiquitin ligase
required for degradation of
phosphorylated substrates in G1
SKP1
Cdc53
Cdc4
S is SKP1
SKP1
C is cullin
?
F is F box
Pop1, 2
protein
CKIs
CDK inhibitors--generally small
SIC1
molecules, not conserved in primary
Rum1
p16
p19
sequence
p21
p27
ATM/ATR
Master kinase regulators of
checkpoint pathways
checkpoint
sensor
RAD24
Complex of proteins consisting of a
MEC3
clamp loader and a clamp that binds
RAD17
DNA and monitors damage
DDC1
Effector
kinases
Downstream of sensor kinase,
respond to different challenges
CHK1
(damage) CHK1 CHK1
RAD53
Cds1 CHK2
(HU)
MEN
Mitotic exit network, regulates
progression out of M phase in S.
cerevisiae. Similar proteins in S.
pombe regulate septation (called
SIN, for septation initiation
network). Contains a GTPase, 2component GTP exchange factor
(GEF), and a GAP, upstream of a
phosphatase (PPase) Regulated (in S.
cerevisiae) by nucleolar localization
via protein Net1
TEM1
(GTPase)
BUB2
BYR4
LTE1
CDC14
(PPase)
Net1
Tumor
suppressors
Negative regulators of the cell cycle,
NONE
which are not found in fungi
Regulated transcription; the ones
Transcription
here are active for synthesis of S
factors
phase genes
MEC1
TEL1
SWI6
SWI4
MBP1
Rad3
Tel1
ATR
ATM
Rad17
Hus1
Rad1
Rad9
Rad17
Hus1
Rad1
Rad9
Spg1
Cdc16
Byr4
?
?
Clp1
?
NONE
p53
Rb
Cdc10
E2F
Res1
preRC
Orp1-6
Pre-replication complex, which
ORC1-6
ORC1-6
Cdc18
marks a replication origin as ready to CDC6
CDC6
Mcm2fire
MCM2-7
MCM2-7
7
Cdc7
Origin-activating kinase, which may
play other roles in maintaining
CDC7
genome integrity. Requires a subunit
DBF4
(DBF4) which does not look like,
but acts like, a cyclin
Hsk1
Dfp1
CDC7
DBF4/ASK1
The Cell Cycle | Evidence for Regulation | Genetic Analysis | Cdc2 Regulation | Cyclins | Inhibitors |
Destruction | Mitotic exit | Replication | S to M phase | Checkpoints | Meiosis
Created 4/00 Last updated 041502
text and original drawings © S. L Forsburg
Made on a Macintosh.