SCF in S phase, and APC are related complexes that recognize specific substrates and target for destruction.

• 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

• 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

• 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 (pre-replicative 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:
  • Origin of Replication (yeast ARS: Autonomously Replicating Sequence) on the DNA: site where DNA replication begins.
  • ORC complex, identifying the origin throughout the cell cycle
  • Cdc18 (AKA Cdc6) protein: activates origin, allows loading of additional factors. targeted for destruction by mitotic CDK.
  • MCM proteins: together, form likely helicase. Can only load at origin when CDC18 is there.
  • replication machinery: polymerases, ligases, etc. Require that MCMs are loaded first.
  
  
  
• Example from S pombe. Note: put the cursor over the righthand image to see an animated version!
  • Cdc18 protein is transcribed specifically before S phase, in M/G1 phase.
  • 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
  • Once origin is assembled, CDK activity required to fire it (exactly how remains unclear)
  • premature activation is prevented by CKI Rum1p, which keeps CDK activity low
  • 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.
  • Independent pathways also involved, including another kinase CDC7 (Hsk1 in the figure below), and sequential loading of different replication factors.
  • (See this page for complete description)

10) Dependency of mitosis on S phase

• 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 re-replication include :
  • temperature sensitive mutations of Cdc2 kinase (when returned to permissive temperature), suggesting that the kinase is re-set in some way.
  • Overproduction of the CKI Rum1 (roughly equal to SIC1 in cerevisiae
  • Deletion of the mitotic cyclin Cdc13
  • 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.

  

• Mitotic progression is also directly dependent upon S phase. For animation, move your cursor over the image at the right
  • Cohesion between sister chromatids is established during S phase
  • Absence of cohesion leads to premature chromosome separation and segregation defects
  • 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.
  • Cohesin connections are dissolved by ESP1 (separin) when PDS1 (securin) is destroyed by APC
    

• 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.
  
  
• 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
  • 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
  • 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.
  • 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.
  • 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 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:
  • Rad3 activates the downstream kinases Chk1 and Cds1/RAD53/Chk2 in response to different signals.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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. 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 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. p53 highly unstable, targeted for destruction by binding MDM2. This binding prevented when p53 phosphorylated by checkpoint kinase cascade. p53 induces expression of range of genes including CKIs that block CDK activity Inactivation of 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. 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 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 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.

12). Meiosis

• 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.
  • 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.
  • 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

• 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. cerevisiae	S. pombe	metazoans
CDK	cyclin dependent kinase	CDC28	Cdc2	Multiple CDKs: CDK1-6
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	Cdc13	Cdc2
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 psc3 psm1 Psm3	Rad21 aka SCC1 SCC3 SMC1 SMC3
SCF	Multi-component ubiquitin ligase required for degradation of phosphorylated substrates in G1	SKP1 Cdc53 Cdc4	SKP1 ? Pop1, 2	S is SKP1 C is cullin F is F box protein
CKIs	CDK inhibitors--generally small molecules, not conserved in primary sequence	SIC1	Rum1	p16 p19 p21 p27
ATM/ATR	Master kinase regulators of checkpoint pathways	MEC1 TEL1	Rad3 Tel1	ATR ATM
checkpoint sensor	Complex of proteins consisting of a clamp loader and a clamp that binds DNA and monitors damage	RAD24 MEC3 RAD17 DDC1	Rad17 Hus1 Rad1 Rad9	Rad17 Hus1 Rad1 Rad9
Effector kinases	Downstream of sensor kinase, respond to different challenges	CHK1 (damage) RAD53 (HU)	CHK1 Cds1	CHK1 CHK2
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, 2-component 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	Spg1 Cdc16 Byr4 ? Clp1 ?	?
Tumor suppressors	Negative regulators of the cell cycle, which are not found in fungi	NONE	NONE	p53 Rb
Transcription factors	Regulated transcription; the ones here are active for synthesis of S phase genes	SWI6 SWI4 MBP1	Cdc10 Res1	E2F
preRC	Pre-replication complex, which marks a replication origin as ready to fire	ORC1-6 CDC6 MCM2-7	Orp1-6 Cdc18 Mcm2-7	ORC1-6 CDC6 MCM2-7
Cdc7	Origin-activating kinase, which may play other roles in maintaining genome integrity. Requires a subunit (DBF4) which does not look like, but acts like, a cyclin	CDC7 DBF4	Hsk1 Dfp1	CDC7 DBF4/ASK1

Created 4/00 Last updated 041502
text and original drawings © S. L Forsburg

Made on a Macintosh.