Cell Cycle2

 

Cell cycle and cell Cycle genes:

 

Cell Cycle Check Points:

 

The major checkpoints lie in between G1 and S phase and G2 and M-phase and another control point exists within the M-phase events at anaphase.  Not withstanding the said checkpoints, DNA damage can introduce a checkpoint, where until the damage is repaired, cell does not enter M-phase, this can happen at S-phase or at G2 phase; if the damage is beyond repair the cell is signaled for Apoptosis. 

 

 

Checkpoints act at transition points where all the earlier events have to be completed before it progresses to the next stage.  They also act as surveillance systems.  It is a regulatory loop where initiation of one event depends on the completion of the earlier event, so progression through a checkpoint is strictly controlled.

 

 

www.moodle2.rockview.ab.ca

 

www.youtube.com

 

   

There are three major check pints; G1, G2 and M; G1checkpoint is at the end of G1. Here, the cell evaluates if the ecell has all thr inputs to continue with the division process. The G2 is a very important checkpoint for the cell. At this time, the cell must evaluate if it has properly duplicated all of its chromosomes. If it has not, it may attempt to carry on with the division, or it may simply self-destruct. The third and the last major checkpoint occur during M phase. At this checkpoint the cell evaluates whether the spindle apparatus has properly attached itself to each of the chromosomes, and whether the rest of the cell is ready for cytokinesis, or physical cell division. If something is wrong at this stage, the cell will often simply die. http://moodle2.rockyview.ab.ca/

 

www.mun.ca

 

http://www.fattuesdayproductions.com/

 

Figure 1A, B and C

Simple representations of the cell cycle; (A) a typical (somatic) cell cycle, which can be divided in four sequential phases: G1, S, G2 and M. M phase consist of nuclear division (mitosis) and cytoplasmic division (cytokinesis) (B) variant cell cycles in which specific phases are omitted. (C) approximate time of activity for different combinations of cyclins and CDKs, based on studies of mammalian cyclins and CDKs. C. elegans family members are indicated between brackets. Shapes outside the cycle indicate increase and reduction of corresponding CDK/cyclin activity. http://www.wormbook.org/

 

 

 figure 2

 Model illustrating general aspects of CDK regulation; CDK activation requires cyclin (CYC) expression and association. Cyclin/CDK complexes are kept inactive through association with CDK-inhibitory proteins (CKIs) and inhibitory phosphorylation by Wee1/Myt1 kinases (black circles). Activation requires ubiquitin-dependent proteolysis of the CKI, phosphorylation of the CDK by a CDK-activating kinase (CAK; red circle), and removal of the inhibitory phosphates by a Cdc25 phosphatase. Cyclin destruction leads to inactivation. Ubiquitin-dependent proteolysis of cell cycle regulators in late G1 and S involves cullin-based E3 ligases such as SCF, while in M phase and early G1 the anaphase-promoting complex (APC) is active. The exclamation figure denotes the active kinase complex, the large arrow indicates time; www.wormbook.org

Interplay between SCF and APC/C complexes. (A) During G1, APC/CCdh1 ubiquitinates Skp2, contributing to the post-mitotic down-modulation of SCF. (B) At G1/S boundary, SCFSkp2 activity increases, and CKIs substrates are ubiquitinated and degraded. This, in turn, activates cyclin/CDKs complexes that phosphorylate Cdh1, contributing to the down-modulation of the APC/C complex activity. (C) At the onset of mitosis, SCFβ-TRCPinduces the degradation of the APC/CCdc20 inhibitor Emi1, increasing APC/C activity.;http://cardiovascres.oxfordjournals.org/

Structural similarities between SCF and APC/C E3 Ub-ligase complexes. Both complexes are composed of a catalytic RING protein (blue), a scaffold protein (red), and an adaptor protein (green). Variable components (yellow) give substrate specificity to the complexes: about 70 F-box proteins have been identified in humans, whereas APC/C is modulated by Cdh1 or Cdc20. http://cardiovascres.oxfordjournals.org/

 

 

 

 

This diagram illustrates the levels of cyclins as mentioned above in graphic mode where mitosis promoting activity peaks at specific stage of the cell division. www.mun.ca

 

The concentrations of cyclin proteins change throughout the cell cycle. There is a direct correlation between cyclin accumulation and the three major cell cycle checkpoints. Also note the sharp decline of cyclin levels following each checkpoint (the transition between phases of the cell cycle), as cyclin is degraded by cytoplasmic enzymes. (credit: modification of work by "WikiMiMa"/Wikimedia Commons)www.cnx.org; www.wiki.org

This illustration shows a cyclin protein binding to a Cdk. The cyclin/Cdk complex is activated when a kinase phosphorylates it. The cyclin/Cdk complex, in turn, phosphorylates other proteins, thus advancing the cell cycle.

Cyclin-dependent kinases (Cdks) are protein kinases that, when fully activated, can phosphorylate and thus activate other proteins that advance the cell cycle past a checkpoint. To become fully activated, a Cdk must bind to a cyclin protein and then be phosphorylated by another kinase.www. http://cnx.org/

 

 

 

 

 

 

 

 

The G1 stage, as said earlier, is the stage where cell prepares for DNA replication.  The cyclins required at this stage are cyclins D.  The required components for DNA replication, besides a large nucleotide and histone pools, are of DNA replication machinery, such as DNA polymerase, helicases, SSBs, and many other factors.  It is during this stage or at the end of this stage transcription of genes required for DNA replication is activated.  Transcription of the said genes requires transcription factors and their activation is sine quo non for the entry of the G1 to S-phase.  If there is a damaged DNA at G1 stage entry into S-phase is prevented by the mediation of p53 and its associated components.  If and only if all the required components for replication are provided then cell enters into S-phase.

 

 

The above diagram shows the major checkpoints and the major cyclins and Cdk involvement at specific stages, which are required for the progression of the phases. www.users.rcn.com

 

 

 

The diagram depicts different stages of cell cycle regulated by different sets of Cdc-Cdks and RB proteins; www-bcf.usc.edu

 

 

 

 

The G2 stage is again a preparatory stage for M-phase, which requires a whole set of proteins and organization of cellular components for chromosomal separation and cytoplasm division.  If the DNA damage is not repaired in the S-phase and even in G2 phase the cell won’t enter into M-phase.  Cells have in built sensory system.

 

 

Mutations that exert cell cycle control in yeast are:  Cdc 28 at G1-ŕ S; Cdc 28 at S phase; cdc24 at G-2 and Cdc 34 M-ŕ G1

 

Genetic analysis of single cell systems such as Saccharomyces cerevisiae (budding yeast) and Saccharaomyces pombe (fission yeast) has yielded a wealth of database information.  Combined with genetic data, Proteome search has provided information about the number of genes and gene products involved in cell cycle control.  The database search to 95% accuracy shows that Homo sapiens have 99 gene products [28-protein for G1/S, 28 proteins for -G2/M, 23 proteins for -M, 41-Sphase, 24-others], Mus musculus contain 68 gene products and   S.cerevisiae 87 to name only few.  Important factors that operate cell cycle control system are cell cycle specific kinase complexes and few cell cycle kinase inhibitors.  Among many of the kinases cyclin dependent kinases  (CDK) are important; for their activity cyclins are required, hence the name cyclin dependent kinases.  Kinases are effector molecules and cyclins are required for kinase effector function.  Perhaps the first such kinase discovered was from yeast and it was called Maturation promoting factor (MPF), later it turned out to be Mitotic Promoting Factor (MPF) or mitosis Promoting Kinase (MPK).  This protein complex turned out to be a serine-threonine protein kinase but dependent on specific cyclin.  From a variety of sources many such cyclin dependent kinases and cyclins have been discovered their nomenclature has not been strictly adhered to International nomenclature rules, so there is confusion in identifying, which is which.

 

Cyclin dependent kinase (Cdk):

There are several CDKs, at least 11 or more. In general CDKs have a molecular weight of 34 kd, and they are monomer and function as kinase subunit i.e. as effector domain.  It consists of N-terminal beta-sheet containing ATP binding site and an alpha helix with PSTAIRE sequence.  The C-terminal has helical domain.  When Cyclin binds the PSTAIRE region fits into cylin structure.  CDKs are constitutively synthesized and found in reasonably higher concentration.  When cyclin is not bound to CDK, the C-terminal loop can fold back and mask the ATP binding site and block access to protein kinase site.  CDK is a protein kinase and responsible for phosphorylating several target proteins, thus activate several components that leads to the progression of M-phase and S-phase.

 

 

 

The N-terminal region contains phosphorylating sites at threonine 14 or at tyrosine 15; this depends upon the organism.  These sites are adjacent to the substrate-binding site.  Another site for phosphorylation is Threonine 160 in (CDK2) and Threonine 161 in CDC2 (it is also a CDK) from Schizosaccharomyces pombe.

 

The daigrsmic representation of 3-D ribbon model of Cyclin-A and Cdk2 proteins; lmb.bioch.ox.ac.uk

 

Cyclins:

Cyclins are protein kinase activators. There are several cyclins at least 30, which are synthesized and degraded (by ubiquitination and proteosome mediated) in stage specific manner.  The molecular weight ranges form 35 to 90 KD.  Cyclins are made up of helices into which CDK snugs in.  The 100aa long five-helix domain called cyclin box (shared by all cyclins).  The C-terminal contains sequence of 9 aa, called destruction boxes, which is recognized by ubiquitination enzyme complex.  However cyclins-C, F, G and H have structural relationship but not involved in cell cycle regulation.  Example cyclin-H/Cdk 7 dimers are associated with eukaryotic TFII-H

 

 

Cyclin destruction box:

Cyclin-A:   RTVLGVIGD,

Cyclin-B:    RTVLGVIGN,

Cyclin-B2:  RAVLGVIGN.

Ubiquitination by E1, E2, and E3 target mitotic cyclins of anaphase Promoting Complex (APC) at the end of anaphase

 

CDK activity:

CDK is activity is regulated.  The CDK has at its N-end has a Threonine 14 or Tyrosine 15.  Adjacent to kinase site there is another phosphorylating site i.e. Thr 160 or Thr 161. If these hydroxyl amino acids are phosphorylated (by Wee 1; and Wee 1 is active when it is unphosphorylated and become inactive if it is phosphorylated by nim 1 enzyme) the enzyme remains inactive, it will be active only when cyclin is bound (binding of cyclin opens up Thr160 site) and unphosphorylated Thr 14 or Tyr 15, but CDKs has to be phosphorylated at Thr160 or Thr 161.  Dephosphorylation performed by activated cdc25 ( a phosphotase enzyme, becomes active when it is phosphorylated otherwise inactive).  Phosphorylation of Thr 160 (161) is a must.  This phosphorylation is believed to be by the enzyme called Cdk-activating kinase (CAK).  Phosphorylation of Thr160 (161) can also be achieved by autophosphorylation once the CDK is active.  The Cdk-cyclin dimmer protein is a serine and tyrosine protein kinase.

 

 

 

A list of CDKs and Cyclins:

 

G1/S Regulatory Components:

 

 

 

CDKs

Cyclins

Mammalian & Frog

Cdk2,  4

Cyclins D1, D2, D3,  E

S.cerevisiae (budding)

Cdc  28

Clb B-1-4 (B-like)

S.pombe (fission)

Cdc 2

Cdc13 (B-like)

 

G2 / M Regulatory Components:

 

 

Cdks

Cyclins

Mammals/frogs

Cdk2, (cdc2,)

Cyclin A, B1, B2

S.cerevisiae

Cdk 28 (cdc28)

Cyclin 1-4 (B-like)

S.pombe

Cdk2 (cdc2)

Cyclin13 (cdc13) (A-like)

 

*** Paul Nurse (UK), Thomas hunt (UK) and Leland Hartman (USA) were awarded Nobel prize for their work on Cdc cyclins.

 

Note that the cdc2 (cdk2) of mammalian system is equivalent to cdc28 (Cdk 28) of S.cerevisiae, which is equivalent to Cdc 2 (Cdk 2) of S.pombe.  The Cdk term is used because each of them acts as Cyclin Dependent Kinase.  The Cdk has a molecular weight of 34 KD.

Cdc 13 is a homolog of cyclin-B.

 

Combination of Cyclin-Cdks; Their Functions at Different Stages:

 

S. cerevisiae:

 

 

cyclin

Cdk

 

 

G1>>S phase

Cln 1,2,3

Cdc 28

 

 

S-phase

Clb 5, 6

Cdc 28

 

 

Replication origin firing

Dbf4

Clb 5

Cdc 7

Ccdc28

Firing replication origin

 

M-phase entry

Clb 3,4

Cdc28

 

 

M-phase progression

Clb1,2

Cdc28

 

 

M-phase exit

Clb destruction

 

 

 

 

 

 

 

 

 

 

Human and Vertebrates:

 

Cyclins

Cdk (protein kinase)

Cyclin level

Note

Cyc-D1, D3

Cdk-4, 6

Increase

START- G1 phase progression

Cyc-E

Cdk-2

E-Increase, D-decrease

Onset of S phase, G1 >S

Cyc-A

Cdk-2

A-increase, E-decrease

S-phase progression

Cyc-A

Cdc-2 (cdk-1)

A-decrease,

S through G2

Cyc-B

Cdc2 (cdk-1)

B-increase

M-phase progression

Cyc 13(Mr 45-47KD)

Cdc2 (Mr 34KD)

 

Prevents S-phase before M-phase

Cig 2

Cdc2

 

Prevent the start of M-phase before S-phase completion

 

The figure presents a vivid picture of different signal has an effect or an affect on the activity of CDKs; www.streaming.cineca.it

 

 

 

 

 

 

 

It is important to note that the effector i.e. the protein kinase subunit, often called Cdc-28 in S.cerevisiae, or cdc2 in S.pombe, and CDK in other systems, is more or less same at all stages of cell cycle, but the cyclin, the partner varies from stage to stage, such as G1-cyclins, S-cyclins, G2-cyclins, M-cyclins and so on.  When such cyclins combine with the cyclin dependent protein kinase, when active, for it becomes active, when the kinase subunit is phosphorylated at Thr.160 (or Thr 161 in other systems), they act on different targets at different stages of the cell cycle.  Most of the cyclins are synthesized in temporal fashion, starting at the beginning of G1 and build up to M-phase and then they are degraded (by proteosome in ubiquitination mode), again in temporal fashion; and the timing of degradation is critical; so cyclins act as regulators of the protein kinase, where kinase subunit is same and the cyclins are different.  Thus the regulation of cell cycle is regulated by the synthesis and timely destruction of the said cyclins.

 

Accessory factors:

 

Though cyclins and Cdks are considered as the prime factors in controlling cell cycle events, there are other factors, which are as important as cyclin-CDKs.  There are several kinases (some are cyclin dependent and some are cyclin independent) and several phosphatases.  They are-

 

Nim 1(Never In Mitosis):  is a signal mediated protein kinase Inhibits wee1 by phosphorylation.  Nim-1 pathaway pathway links to cdc2/cyclin system to external signals.

 

Wee 1:  Is a kinase; inactivated by nim-1 by phosphorylation, Dephosphorylation makes it active’ when active it phosphorylates Cdk’s threonine 14 9 (or Tyrosine 15) and makes it inactive.  Wee-1 activity is determined by signal input and signal transduction across the membrane.

 

CDK kinase (CAK): Phosphorylates Cdk’s active site threonine 160 (or Threonine 161).

 

Cdc25:  It is a phosphotase,( counter part of this in the fly is “string” gene). Its Mr. is 80KD.  It is active when phosphorylated and inactive when dephosphorylated.  When Thr 14 (or Tyr 15) is phosphorylated the Cdk is inactive, but Cdc 25 dephosphorylates these sites; when this Dephosphorylation is coupled with the phosphorylation of Thr160 (Thr161) by CAK, Cdk-cyclin becomes active.  The level of cdc25 reaches a threshold at M-phase, perhaps marks the end of S-phase.

 

 

 

 

CDK Inhibitors (CkIs):

Though cyclin-Cdks play critical regulatory roles in cell cycle, there is another set of molecules that regulate the regulators; in yeasts they are Cdk inhibitors or generally they are cell cycle kinase inhibitors (CKIs).  There are different types of CKIs, such as far1p, Sic1p.  The inhibitor binds to Cyclin-Cdk complex and prevents their activity.  In metazoans, such molecules are called inhibitors of Kinase or Ink family of inhibitors.  They bind to Cdk and exclude cyclin binding, they are called kinase inhibitors called ‘Kips’ and those that bind to cyclins and inhibit kinase activity are called ‘Cips’ (cyclin inhibitors).

 

MPkinase inhibitors (in the form of dimers) bind to kinases to form inactive complexes.  Thus they prevent phosphorylation Retinoblastoma proteins.  So the cell cycle is checked at G1 or Go stage.  CKIs classified into two classes-Inks and Kips.

 

 

INKs (Inhibitor of Kinase): 

They are Cdk inhibitor proteins:  INK 4 family is specific to Cdk4 and Cdk 6.  Ink4 has four members- p15 (INK-4B), p16 (INK 4A), p18 (INK4C), and p19 (INK4D).  They contain ankyrin repeat sequences.  P16 and P19 bind next to ATP binding site, so prevents its catalytic activity.  It also induces conformational changes so cyclin cannot bind.  They act on cyclin D complexed either to Cdk4 or Cdk6.

Another class of inhibitors such as Sic I binds to Cdc28-clb2, in S.cerevisiae, inactivates the kinase at G1.  So entry of cell cycle into S-phase requires the degradation of Sic-I by ubiquitination mode.  SCF acts as E3 ligase system.  The Skp1-Cullin factor (SCF complex) consists of cdc53, Cdc4, Skp1 and Cdc34.  These are involved in G1 cyclin destruction.

 

Kips:  Another class of inhibitrs consists of P21, p27 and p57, they are identified by their molecular weights.  They in general act on G1/S class Cdks.  P21 binds to all Cdks- Cdk2, 4 and 6, thus block progress through all stages of G1/S.  Increase in p21 concentration is inhibitory.  Many a times in cultured cells one finds PCNA is also complexed with CDK-cyclin along with p21. so it controls G1/S stage progression.  P27 also binds to Cdk-cyclin and blocks progression into S-phase, but its over expression leads the cell to go into Go stage.

 

A representative diagram shows various Cdc-Cdk inhibitors that act at different stages

 

CyclinH/cdk-7: it is associated with TF-II H and involved in phosphorylation of CTD tail of RNA polymerase II; TF-II B also contains cyclin like helix bundles.

 

Cdc7-cyc-DBf4 kinase: It is serine/Thr protein kinase required for the onset of S-phase.  The cyclin Dbf4 is constitutively synthesized but rapidly degraded from late M to G1.  Activity peaks at the onset of DNA replication.  Human homolog is Hsk (Homolog of Cdk Seven Kinase, however CDK lacks PSTAIRE sequence.   The target of this complex is Mcm2.  Loading of Mcm2 on to ORE region is important in triggering the firing replication origin.

 

Cdk Activating Kinase (CAK):  Cdk 7-cyclin H has CAK activity.  Cyclin-A binding to cdc2 (homolog iscdc28) exposes active site and ATP binding site in Cdk protein, where Thr 160 (Thr 161) is made available for CAK to act upon.  CAK phosphorylates Thr 160 (161) of Cdk to make Cdk-cyclin A to be active.

Positive regulation of Cdk by cyclins is often counterbalanced by negative regulation by Inks, Cips and Kips.

 

Rum 1 protein:  Cdc2/cdc13 MPkinase is influenced by Rum-1 factor.  When rum-1 is over expressed cell does not enter M-phase, but s-phase goes through multiple cycles.  When rum1 is deleted the cell enters M-phase prematurely.  This is expressed between G1 and G2 and keeps the MPK inactive.  So this is essential for the S-phase to proceed.

 

 

Dependence: S-phase on M-phase, which requires Cdc2 regulators.  Till S-phase is over M-phase will not start, and till M-phase is over another round of S-phase won’t begin

 

 

Nucleophosmin:  It is a protein, unphosphorylated form binds to centrosome at the end of M-phase and prevents duplication of centrosome.  But Cdk2/cyclin E phosphorylate Nucleophosphomin, at M-phase.  The phosphorylated Nucleophosphomin then dissociates from centrosome.  This is further augmented by Calcium mediated calmodulin dependent kinase II activity at G1-S boundary facilitates the duplication of centrosome.  Centrosome duplication is essential for the organization mitotic apparatus.

 

APC complex: 

It is called Anaphase Promoting Complex:  This is multisubunit complex made up of eight proteins; the complex is also called Cyclosome.  Such complexes are found in yeast and animal tissues.  APC becomes active during M-phase.  It functions as E3-ligase in ubiquitinated proteosome mediated protein degradation.  First the proteins Cdc20 and then Cdh-1 displaces cdc20 and binds to APC and activate its ubiquitination activity.  Cdc20-APC is essential for the degradation of Securin, which paves the way from Metaphase to Anaphase

 

·         Once activated, MPKs initiate M-phase, the progress of it takes its own course and it does not require active MP kinase any more, so to exit from M-phase MPkinase has to be inactivated.  One way to inactivate is to block the catalytic site by inhibitors, or dissociate Cyclin from the kinase, or phosphorylate Thr14 (or Tyr15) or destroy cyclin the CDK partner. Actually, as the M-phase sets in the first cyclin to be destroyed is cyclin-A at Metaphase.  Then little later i.e. at Anaphase, Cylin-B is degraded by ubiquitination mode, making MPkinase inactive.  This type of degradation mediated by Cdh1 activated APC complex (Cdh1 is essential for the degradation of Clb 2 which are B-like cyclins); this paves the way for the cell to exit from Mitosis.

 

When chromosomal DNA replicates, single stranded chromosome becomes double stranded, for reasons of stability, a protein complex called cohesins glue the two strands to each other.  But when they reach equatorial region or little earlier, the tightly held chromosomal strands release from one another, yet they are still held at centromeric region.  For equal segregation of chromosomes, the kinetochore complex has to split and free chromosomal strands from one another, so the strands can move to their respective poles.  For the chromosomal strands to free from one another, glue called Cohesin complex that holds chromosomal strands, is degraded, so at Anaphase chromatin strands separate.  In some systems chromosomal strands are freed at the end of prophase itself, but centromere is still held together, in such cases kinetochore complex splits by the protease activity induced by APC.

 

·         The APC complex that targets cohesin complex and kinetochore complex is activated by cdc20.  It is activated at M-phase or little earlier and performs destruction of Securin (pds-1p), which triggers the release of two chromosomal strands from one another.  This process is critical for the separation of chromosomes at anaphase, so the complex is called Anaphase Promoting Complex.

 

Cohesins and Condensins:

Cohesins and condensins are heteromeric proteins made up of smc proteins (Structural Maintenance of Chromosomes) and non-Smc proteins.  Cohesins are made up of two smc proteins, Smc-1p and Smc-3p and two non-Smc proteins, Sec1p and Sec3p; where as condensins are made up of Smc2p and Smc4p and Sec2p and Sec4p.  Cohesins are responsible for adhesion of two sister strands together as parallel strands all along the length including kinetochore region.  But condensins are responsible for the condensation of chromatin from long convoluted threads into short and stable threads at metaphase.

Models ofCondensins and Cohhesins; www.nature.com

 

Cohesins:   They are complex of proteins, made up of Smc1 & 3 and Scc1P & Scc3p.  Smc1 and Smc3 of cohesins are coiled coils with a flexible hinge.  In Smc1 and Smc3 coiled coil protein pairs show V-shaped bending with 86 ^o apart.  But the Smc2 and Smc 4 proteins when dimerizes the flexible angle is steep of 8^o. 

When Smc1 and Smc3 dimerize parallel to each other they are oriented in antipolar fashion.  At either ends they have a DNA binding domain and a domain for the binding of ATP.  One end of the coiled coils bind to the DNA and the other end of the protein dimerizes with another Smc protein pair.  Two such Smc pairs can hold on to the same DNA at one site and at the other end is free for dimerization with another Smc protein pairs that is anchored on to another DNA.  If two sets of Smc protein pairs, one holding one strand and another holding the opposite strand.  When the free ends are paired and linked by Scc1p and Scc-3p, two chromosomal strands will be held parallel to each other.  Several protein pairs all along the length of chromosomes provide such links, thus chromosomal strands are paired and glued.  Here the glue is Scc1p and Scc3p, perhaps one more protein pds5 may also be present.  Degradation of this makes chromosomal strands to separate from one another.

 

Condensins:  They are also made up of a complex of proteins, such as Smc 2 and Smc4 and two non-Smc proteins called Scc3 and Scc4.  Here the Smc proteins 2 & 4 coil to each other in antiparallel fashion.  The flexible portion can generate an 8^o angle.  These proteins bind to the same chromosomal DNA at two sites i.e., one pair at one site and the other pair at another site, perhaps at a distance.  The free ends can dimerize.  When Scc2 and Scc4 proteins dimerize the other ends, which are bound to chromosomal DNA; the DNA found in between is looped out.  Many such condensins all along the length of chromosomes act on at different sites and condense chromosome to a maximum at metaphase.  Whether or not there is any relation between Histone-1 phosphorylation induced condensation and condensin operated condensation, is not clear; sure there should be a relation

 

 

 

Operation of Cell Cycle:

 

Cells, irrespective of their ploidy divide either during growth or during reproduction.  During reproduction, a diploid gamete-producing cell undergoes reduction or meiotic division.  But a similar diploid or haploid cell during growth and development goes through a series of mitotic cell divisions.  Even a fully grown organism, where most of the cells at all times are in resting or what is called Go state, occasionally undergo cell division in order to compensate cell loss.  In culture condition cells also undergo cell division to multiply in numbers.

 

·         In general cells in an environment provided with rich nutrients divide and redivide e.g. yeast, but cells under culture conditions initiate cell division when they are stimulated by mitogens.  Cells in a tissue require stimulation for division.  At that time cell size increases and when the cell mass reaches an optimum level to its volume, it initiates cell division, if it is somatic it is called Mitosis.

 

Mitosis goes through several physical and biochemical changes in the form of stages or phases.  Mitosis has several stages such as M-phase and Interphase.  Between two M-phases there exist an intervening phase called Interphase.  The M-phase its self consists of sequential steps like Prophase, Metaphase, Anaphase and Telophase and finally Cytokinesis culminates in producing two daughter cells, which have inherited their genetic material equally.  Interphase, in general occupies longest time in cell division.  It can extend to 10-12 hrs in a 24 hr cell cycle.  But the M-phase takes just 30 minutes or 1 hr.

 

·         Interphase when in resting phase exists in what is called Go stage, where all cell cycle processes are shut down.  But when the cell is stimulated, either by nutrient supply or by mitogens, they renter from Go stage and enter into G1 stage.  The G1 is a preparatory stage for the next phase called S-stage.  In the S-stage the chromosomal DNA replicates and generates two copies of them.  Then the cell enters into another stage called G2 stage; which is again another preparatory stage for M-phase.  G1, G2 are called so scientists did not know what exactly happen at these intervening stages, so they called it G1 and G2, which is a Gap in the knowledge about them.  Though these stages are sequential and temporal, they don’t enter to the next stage until and unless each of the stages has completed their requirements and functions.  To prevent any such precautious entry into the next stage, they use checkpoints, which act as control loops where cellular events should be completed at the earlier stage to move to the next stage, other wise they remain in the same stage till all the events required are completed.

 

G1 Stage:

The G1 occupies approximately 10 to 12 hr where the cell prepares for S-phase.  Important check point here is called START point or restriction point, once it passes through there is no going back.

 

www.journals.cambridge.org

 

Once cells are stimulated they measure cell mass and cell volume.  There are genes, which do this function.  Once cell mass to cell volume is measured and full filled, it launches into a series of molecular events that sets the stage to next stage.  In general at G1 stage, inputs for DNA replication are shut off.  This is achieved by sequestering all those required Transcriptional Factors (TF-IIs) required for activating genes that are essential as inputs for initiating and executing DNA replication to completion.  The factor that block is RB protein, which was identified as a mutant gene causing Retinoblastoma disease (a cancer).  In its native state RBs sequester all those transcriptional factors -E2Fs.  These are required for activating genes for cyclins.  Once cyclins are synthesized, they activate certain cyclin dependent kinases (Cdks).

 

RB binds to transcription factors like E2Fs and some non-E2Fs, which are sequestered when RBs are non-phosphorylated, but when, phosphorylated they are released for activating S-phase specific gene expression.www.images.1233.tw

 

A simple diagram showing the possible phosphorylation of RBs by different combination of Cdc-Cdks at different stages

 

·         Earlier signal transducing cellular events triggered by mitogens

or nutritional factors do cause specifc phosphorylation and dephosphorylation reactions.

 

All most all cells have a battery of thousand or more Kinases and equal number or more phosphotases, which act specifically on their targets; thus they activate or inactivate certain substrates, which can be a protein or a carbohydrate or any other target cellular component.

 

 

 

 

When the cell is still in earlier G1 stage the Cdks are phosphorylated at Thr 14 (tyr15) and rendered inactive by wee1 protein kinase (wee1 is active when it is dephosphorylated and inactive when it is phosphorylated; this is controlled by nim1 ‘never in mitosis’ protein).  The Cdk has another site for phosphorylation, i.e. is Thr 160 (Thr161), which is located next to Kinase active site, and ATP binding site at the C-terminal part of the Cdk, which actually folds back over kinase active site when its Thr 160 is not phosphorylated.  If this Thr 160 is not phosphorylated cyclin cannot bind and make Cdk-cyclin complex active.  At earlier G1 stage the Cdk is rendered inactive.  As the cyclins build up, another protein level increases; it is cdc25 and it is a phosphotase specific to Cdk Thr 14 (Tyr15). The build up of cyclin synthesis starts at early part of G1 stage and continues to build till M-stage, at which time it is degraded abruptly.  Cyclin protein have rapid turn over, their half life is just 15 minutes or so.

 

Thus, RBs and similar proteins bind to E2F factors, thus transcription of genes required for DNA replication are kept in active state.  Another event that keeps Cdk inactive is by phosphorylation of Thr14 (Tyr15) by Wee1.

 

·         After cell stimulation for mitosis, cyclins (mostly cyclin-D) build up, Cdc25 also builds up.  At this stage Cdc25 dephosphorylates Cdks Thr-P14 (Tyr15-P).  At the same time another protein kinase called Cdk activating enzyme called CAK phosphorylates Thr160 (Thr161).  Almost at the same time Cdk inhibitor proteins that are bound to Cdk-cyclin complex are also released.  These biochemical events facilitate the binding of G1 cyclins (cyclin-D) to Cdk properly and the complex becomes fully active.

 

As the Cdk-cyclin complex in its fully active state phosphorylate RBs and its associated proteins, thus make E2F factors released free.  These transcription factors with their associated DPs (Dimer proteins) bind to their respective promoter elements and activate the transcription of genes, whose products required for more cyclin synthesis and factor and components for DNA replication.  Once the said factors synthesized in sufficient amounts cell enters into S-stage. At this stage several cyclins are synthesized, such as cyclin E and others for M-phase activity

 

S-Stage:  S-phase is of short duration of 6-8 hrs.  This is most precise and exact process and its execution should be error free.  S-phase initiation is again contr0olled by another set of factors.  Until and unless they are made available DNA replication is not initiated.

For the replication of DNA, replication origins have to be fired, that is they have to open into replication bubbles.  In eukaryotes DNA is compacted by nucleosomal organization into higher order of compaction i.e. chromosome.  Chromosomes at this stage have to be relaxed and origins should be made available. 

 

Entry into S-phase is critical, but it is governed by and regulated by the Cdc-Cdk inhibitors and RBs.  The release of E2Fs and other non E2F TFs is critical for the entry of the cell into S-Phase; teach.med.ncku.edu.tw

 

·         Eukaryotic DNA is long (~100 million bp or so), and linear, unlike E.coli, which is circular.  The origins are located in what is called replication initiator zones, which contain several origins.  On the basis of yeast’ ARS sites, most of the origins contain a 11 bp long ORE (Origin Recognition Elements) with specific sequences.  Next to it there are DNA unwinding elements called DUE.  On either side of these two elements there can be auxillry sequences. 

 

At the time of initiation of replication the ORE should bound by a complex of proteins called ORC- origin recognition complex (> 400KD).  For firing the replication origin it requires Mcms (mini chromosome maintenance proteins; they are hexamers; they are acquired when the nuclear membrane is dissolved.  This happens only once in one cell cycle at M-phase.  Cdc6 proteins are also acquired at this phase; together they act as licensing factors.  At the same time, two more factors are acquired; they are Cdt1 and Geminin.  The Geminin prevents the loading Mcms second time before completing M-phase.  Loading of Mcms is crucial for it actually opens the origin region into replication bubble.  Cdc6 performs this only when it is phosphorylated, which is performed by Cdk-S1 cyclins?  Once replication is initiated Mcm and Cdc6 are released from the origin, but CDC complex remains bound to the origin.

 

·         When all the inputs are made available DNA replication progresses to completion; then only the cell enters G 2 stage.  If during replication any error or damage is done cell cycle halts and starts repairing the damage.  Even the cell enters to G2 it still waits till it is repaired.  If the damage is beyond repair, p53 protein, which act as a sensor for DNA damage activates the genes for the synthesis of P21 and such proteins, which bind to Cdk-cyclin complexes and halt the cell cycle progress.  Progress of S-phase is greatly facilitated by s-phase cyclins or cyclin-E, but once the S-phase is initiated s-cyclins get degraded.  The next cyclin expressed is Cyclin-A from S to G2 stage.

 

G2 Stage:  Again it is phase waits for all the inputs required for M-phase.  If the DNA is damaged it waits in G2 stage till the damage is repaired, otherwise the cell is signaled to suicidal death or Apoptosis.

 

M-phase:  this stage shows lot of physical changes like disassembly of nuclear membrane and pore complexes, duplication of centrosome, appearance of mitotic apparatus emanating from MOTC at poles, chromosomal strand separation from one another that are bound by cohesins, this includes splitting of kinetochore, chromosomal condensation, attachment of tractile fibers on to kinetochore complexes, movement of sister strands to opposite poles headed by kinetochore tractile fibers, , dissolution of all membrane components into vesicles, depolymerisation of microtubules into tubulins, reorientation of actin filaments, destruction of many proteins including cyclin-A and cyclin-B.

 

·         M-phase is activated by Cdk-cyclin-B complex.  During G1 and S-phase cyclin accumulates in cytoplasm, when it reaches a threshold, gets activated and moves in to the nucleus, where Cdk subunit undergoes dephosphorylation at Thr14 (Tyr15) by cdc25 phosphotase, phosphorylation of Thr160 (Thr 161) by CAK.  Now cyclin-B binds to Cdk; this complex is often called MPF, or MPK.  This activated Cdk-cyclin-B complex targets many proteins for phosphorylation such as chromosomal H1, nuclear lamins, centrosome, microtubules and yet many unknown structures.

 

It is at this stage the MP-kinase activates Anaphase promoting complex (APC).  Activated APC combines with specific adaptors such as cdc20 and targets cohesin complex where it targets Securin and degrades Securin.  The Securin always keeps Separin sequestered.  Separin is an endopeptidase or call it endo-protease.  Degradation of Securin releases Separin, which now acts of Scc1 and Scc3 which are the components of Cohesin protein complex, thus the glue is dissolved and chromatin separate from one another, also the kinetochore by some unknown sensing mechanism also splits and facilitates the anaphase movement of chromosomal strands.  APV complex as described earlier, is ligase system targets proteins for ubiquitinated proteosome mediated digestion

 

·         The APC system gets replaced with another adaptor protein called Cdh1; this then targets; first cyclin A and then Cyclin-B.  Mitotic Cdk-cyclin complexes prevent reinitiation of S-phase DNA replication and also prevent second round of Mitosis before DNA replication.

 

The molecular biology of head and neck cancer

Cell cycle deregulation; The molecular biology of head and neck cancer;

The cell cycle is regulated by complexes of cyclins and cyclin-dependent kinases (CDKs), some of which are indicated. In addition, there are various important inhibitors of these cyclin–CDK complexes. To allow cell cycle progression, cells have to pass the G1 restriction point (red bar) that is controlled by the retinoblastoma pocket proteins, RB, p107 (also known as RBL1) and p130 (also known as RBL2). Only RB is indicated, but the other pocket proteins have similar activities. These normally bind to and inactivate the E2F transcription factors, which induce the expression of S phase genes. In response to a mitogenic signal, the cyclin D1–CDK4 and cyclin D1–CDK6 complexes are activated. These phosphorylate the Rb pocket proteins, causing release (and therefore activation) of E2Fs. Induction of cyclin E by E2F and subsequent additional phosphorylation of RB by the cyclin E–CDK2 complex initiates entry into S phase. The inhibitor for the cyclin D1–CDK4 and cyclin D1–CDK6 complexes is p16INK4A, which is encoded by CDKN2A, a gene in theINK4A locus at chromosome 9p21. The expression of p16INK4A mediates senescence and differentiation. The interplay between the cyclins, CDKs and their inhibitors determines whether the restriction point can be passed, and a growth factor stimulus is usually required. A second important control mechanism of the cell cycle occurs during G2 phase, when the DNA has been replicated and replication errors are repaired. The key protein involved in the response to replication errors and other DNA damage is p53, which is usually maintained at low concentrations by MDM2-mediated degradation (not shown). DNA-damage sensors, including ataxia-telangiectasia (ATM) and ataxia-telangiectasia and Rad3-related (ATR), phosphorylate the checkpoint kinases CHK1 and CHK2, leading to increased p53 activity by phosphorylation of various downstream molecules, including p53 itself (not shown). The p53 tetramers act as a stress-induced transcription factor and induce the expression of p21CIP (also known as CDKN1A), which inhibits several cyclin–CDK complexes and halts the cell cycle. Besides its crucial role in cell cycle control, p53 is also a master regulator of apoptosis and many other stress-associated cellular functions, and is therefore one of the main targets for inactivation in many cancers. The human papillomavirus (HPV) genome contains various early and late open reading frames and encodes two viral oncoproteins: E6 and E7. The E6 protein binds p53 and targets the protein for degradation, whereas the E7 protein binds and inactivates the Rb pocket proteins. The molecular consequence of the expression of these viral oncoproteins is cell cycle entry and inhibition of p53-mediated apoptosis, which allows the virus to replicate. In a 'productive infection' the expression of E6 and E7 is confined to the differentiating layers of the squamous epithelium of the cervix and virions are produced. An oncogenic infection is associated with E6 and E7 expression in the basal layer (where the stem cells reside) and causes abrogation of the cell cycle checkpoints. C. René Leemans, Boudewijn J. M. Braakhuis & Ruud H. Brakenhoff http://www.nature.com/

 

 

Genetics of Cell Cycle

 

Regulation of Cell Cycle:

 

As in prokaryotes, Eukaryotic DNA replication is restricted to either Mitosis or Meiosis stages.  Mitosis is used for growth and development and in some lower forms it is one of the modes of reproduction.  But meiosis is mostly involved in reproductive stages.  Whether Mitosis or Meiosis, cell division is highly regulated and precise and exact.  Mitosis goes through several stages such as Prophase, Metaphase, Anaphase, Telophase and cytokinesis (not always) and then enters Interphase, which is an intervening stage at which the cell prepares for the next division or goes into resting phase where the cells undergo differentiation to specific cell type.

 

The above photomicrograph shows yeast S.pombe is going through cell division.

 

www.biology.kenyon.edu

 

The diagram shows the time required by each of the phases.

www.uic.edu

www.pha.jhu.edu

 

 

 

 

 

Interphase consists of sub-stages such as G1, S and G2; where, G at earlier times stands for gap in the knowledge about these stages.  In 24 hr cell cycle events G1 occupies 10-12 hrs, S-stage about 6-8 hr and G2 stage 4-4.5 hr.  The G1 phase is considered as preparatory phase for DNA replication, but the Cells escape from G1 phase in terminally differentiating cells into what is called Go stage, where cells assume specific shape, structure and function. But some of the cells remain embryonic and such cells can be stimulated to become dividing cells by some mitogens, and they can be differentiated depending upon the kind of stimulus they get, or stimulus provided.  They are called STEM cells. Such cells are found not only in animal tissues but also in plant tissues, in fact plant cells have greater potentiality to be mitotic.   Such cells can be stimulated by mitogens to enter into cell division mode, where they enter again into G1 phase.  The S-phase is for DNA replication and G2 stage is a preparatory phase for M-phase, where the nucleus disassembles, chromatids separate and mitotic apparatus assembles, the centromere split, sister chromatids are pulled to their respective poles, daughter nuclei reform and cytokinesis leads to division of cytoplasm into two cells.  This is a simplistic description of cell division.

 

 

www-bcf.usc.edu

 

In classical experiments as shown above certain molecular events during each of the stages generate a set of factors and they are responsible for executing the stage and perhaps provide signals for the next stage.  For example when a cell in S-stage is fused with G1 stage, the cell in G1 stage is stimulated to proceed into S-phase.  But if a cell in S-stage is fused with a cell at G2 stage, nothing happens, which means the components found in S-phase cells have no effect on G2, because the cells at G2 cells have already achieved what the S-phase components can provide.  Fusion between G1 and G2 does not result in any changes in each of them.  But if an Interphase cell is fused with a cell at M stage the Interphase cells directly enter into M-phase with disastrous consequences.  The Interphase cell is not yet competent to enter into M phase, but M phase cells have all the components for chromosomal separation.  So there is regulation at each of the entry points called check points, which is tightly regulated.

 

www.personalpages.manchester.ac.uk

 

The major checkpoints lie in between G1 and S phase and G2 and M-phase and another control point exists within the M-phase events at anaphase.  Not withstanding the said checkpoints, DNA damage can introduce a checkpoint, where until the damage is repaired, cell does

not enter M-phase, this can happen at S-phase or at G2 phase; if the damage is beyond repair the cell is signaled for Apoptosis. 

 

 

 

Checkpoints act at transition points where all the earlier events have to be completed before it progresses to the next stage.  They also act as surveillance systems.  It is regulatory loop where initiation of one event depends on the completion of the earlier event, so progression through a checkpoint is strictly controlled.

 

 

 

 

 

 

 

 

Chekpoint regulation by the DDR. ATM and ATR orchestrate a transitory delay of the cell cycle in response to DSBs and ssDNA, respectively. Wherease direct phosphorylation of of cdc25a and wee1 allow a rapid establishment of the G1/S and G2/M checkpoints, p53-dependent regulation contributes to checkpoint maintenance at later timepoints. During S and G2 phases DSBs can be resected leading to the generation of ssDNA, which also activates ATR-signaling.The diagram is an elaborate depiction of various components assembling and disassembling at specific stages of G1 and S-phase.  The critical components are ORC, MCMCdc6 and Cdt1. Components like SCF, p21 and p53 have controlling power over the said events. www.intechopen.com

 

 

The G1 stage, as said earlier, is the stage where cell prepares for DNA replication.  The cyclins required at this stage are cyclins D.  The required components for DNA replication, besides a large nucleotide and histone pools, are of DNA replication machinery, such as DNA polymerase, helicases, SSBs, and many other factors.  It is during this stage or at the end of this stage transcription of genes required for DNA replication is activated.  Transcription of the said genes requires transcription factors and their activation is sine quo non for the entry of the G1 to S-phase.  If there is a damaged DNA at G1 stage entry into S-phase is prevented by the mediation of p53 and its associated components.  If and only if all the required components for replication are provided then cell enters into S-phase.

 

 

 

The diagram shows the kind of cyclin-cdks involved in specific stages.

The retinoblastoma protein (Rb) is also a famous tumore suppressor protein. Rb binds to the activation domain of E2F and then actively represses the promoter by a mechanism that is poorly understood. It has been recently reported that Rb associates with a histone deacetylase, HDAC1, through the Rb 'pocket' domain. Rb cooperates with HDAC1 to repress the promoter of the gene related to cell-cycle. Active transcriptional repression by Rb may involve the modification of chromatin structure.www.cyclex.co.jp

 

 

 

 

 

 

 

 

 

The diagram depict different stages of cell cycle regulated by different sets of Cdc-Cdks and RB proteins; fmc.med.univ-tours.fr

There is a short window in the mammalian cell cycle during which cells can respond to extracellular cues by withdrawing temporarily from the cell cycle. When these cells re-enter the cell cycle, they require several extra hours in the G1 phase before they replicate their DNA compared with their cycling counterparts. More than 20 years after this initial observation, we still do not understand what is taking so long. Hillary A.Coller; www.nature.com

www.streaming.cineca.it

 

 

 

The figure though shows many of the components involved in critical S-phase, the diagram is very illustrative and self-explaining.

 

The G2 stage is again a preparatory stage for M-phase, which requires a whole set of proteins and organization of cellular components for chromosomal separation and cytoplasm division.  If the DNA damage is not repaired in the S-phase and even in G2 phase the cell won’t enter into M-phase.  Cells have in built sensory system.

 

 

Mutations that exert cell cycle control in yeast are:  Cdc 28 at G1-ŕ S; Cdc 28 at S phase; cdc24 at G-2 and Cdc 34 M-ŕ G1

 

Genetic analysis of single cell systems such as Saccharomyces cerevisiae (budding yeast) and Saccharaomyces pombe (fission yeast) has yielded a wealth of database information.  Combined with genetic data, Proteome search has provided information about the number of genes and gene products involved in cell cycle control.  The database search to 95% accuracy shows that Homo sapiens have 99 gene products [28-protein for G1/S, 28 proteins for -G2/M, 23 proteins for -M, 41-Sphase, 24-others], Mus musculus contain 68 gene products and   S.cerevisiae 87 to name only few.  Important factors that operate cell cycle control system are cell cycle specific kinase complexes and few cell cycle kinase inhibitors.  Among many of the kinases cyclin dependent kinases (CDK) are important; for their activity cyclins are required, hence the name cyclin dependent kinases.  Kinases are effector molecules and cyclins are required for kinase effector function.  Perhaps the first such kinase discovered was from yeast and it was called Maturation promoting factor (MPF), later it turned out to be Mitotic Promoting Factor (MPF) or mitosis Promoting Kinase (MPK).  This protein complex turned out to be a serine-threonine protein kinase but dependent on specific cyclin.  From a variety of sources many such cyclin dependent kinases and cyclins have been discovered their nomenclature has not been strictly adhered to International nomenclature rules, so there is confusion in identifying, which is which.