Principles of Cell Biology (BIOL2060)

Department of Biology
Memorial University of Newfoundland

The Cell Cycle: DNA replication and mitosis

The Cell Cycle

Cell cycle begins with the formation of two cells from the division of a parent cell and ends when the daughter cell does so as well.
Observable under the microscope, M phase consists of two events, mitosis (division of the nucleus) and cytokinesis (division of the cytoplasm).
As replication of the DNA occurs during S-phase, when condensation of the chromatin occurs two copies of each chromosome remain attached at the centromere to form sister chromatids.
After the nuclear envelope fragments, the microtubules of the mitotic spindle separate the sister chromatids and move them to opposite ends of the cell.
Cytokinesis and reformation of the nuclear membranes occur to complete the cell division.
Most of the time, cells are in interphase, where growth occurs and cellular components are made.
DNA is manufactured during S phase.
To prepare the cell for Sphase (DNA synthesis), G1 phase occurs (the preparation of DNA synthesis machinery, production of histones).
In an analogous manner, the cell prepares for mitosis in the G2 phase by producing the machinery required for cell division.
The length of time spent in G1 is variable.
Although growing mammalian cells often spend 8-10 hours in G1 phase, a cellular decision is made that cause cells to become arrested in G1 and thus enter the G0 state.
G2 is usually shorter than G1 and is usually 4-6 hours.

DNA Synthesis

DNA Replication is a semiconservative process that results in a double-stranded molecule that synthesizes to produce two new double stranded molecules such that each original single strand is paired with one newly made single strand.
This was demonstrated by equilibrium density centrifugation (see chapter 19 for details).
The replication of DNA most often occurs in a bidirectional manner from an origin of replication from which two replication forks move in opposite direction.
In prokaryotes, bidirectional DNA synthesis of the circular genome produces a theta structure (theta replication).
In eukaryotes, DNA replication is initiated in multiple sites along a chromosomes called replicons.
Thousands of replicons, each covering 50 to 300 kilobases, form replication bubbles that fuse to, in the end, make two daughter double stranded DNA molecules.
A number of proteins and protein complexes are involved in DNA synthesis.
DNA polymerases catalyze the synthesis of DNA by adding nucleotides, in a 5-prime to 3-prime direction, utilizing a single stranded region of DNA as a template.
Four deoxynucleotide triphosphates (dATP, dCTP, dGTP,dTTP) only are incorporated into the growing chain by releasing the terminal two phosphate groups and covalently bonding the remaining phosphate to the 3-prime hydroxyl group of the previous nucleotide residue.
Since the 5-prime end does not get added to and the 3-prime  end repeatedly does, the DNA strand is said to grow in a 5-prime to 3-prime manner.

At a replicon, one strand of DNA is made in a continuous manner (the leading strand) and the other in a discontinuous manner (the lagging strand).
DNA is made in only the 5-prime to 3-prime direction and the replication bubble opens the original double stranded DNA to expose both a 3-prime to 5-prime template (Leading strand template) and it complement.
The lagging strand must be synthesized as a series of discontinuous segments of DNA.
These small fragments are called Okazaki fragments and they are joined together by an enzyme known as DNA ligase.

DNA synthesis is not perfect initially, so a proofreading mechanism is performed by a 3-prime to 5-prime exonuclease activity that is part of DNA polymerase enzyme itself.
Since DNA polyerase requires a template and a free 3-prime hydroxyl group to add nucleotides on to, RNA primers are required to initiate DNA polymerization.
An enzyme, primase which is part of a large complex of proteins called the primosome, synthesizes a small stretch of RNA (the primer) of 3-10 nucleotide in length, which will act as a starting site for the DNA polymerase.
Okazaki fragments are initally made with RNA 5-prime ends which is digested away by the 3-prime to 5-prime exonuclease activity of the adjacent DNA polymerase enzyme just prior to the ligation of the two fragments.
To make the DNA single stranded in the first place to allow DNA synthesis, the DNA must be unwound.
Unwinding double stranded DNA requires helicases, topoisomerases and single-strand binding proteins (SSBPs).
The proteins discussed above form a ribosome-sized complex referred to as the replisome.
Arrangement of the replisome such that the lagging-strand is "looped around" allows the machinery to move in one direction while synthesizing DNA from strands in opposing polarities.

As DNA synthesis requires a RNA primer that will eventually be digested away, standard DNA replication would result in linear chromosmes that would shrink with every round of replication.
This is resolved in bacteria by the circular genome which does not have an end.
In linear chromosomes, a specialized structure the telomere solves the end of DNA replication problem.
Telomeres have highly repeated DNA sequences 5'-TTAGGG-3'.
Human chromosomes have between 100 and 1500 copies of this sequence.
Telomerase, a special DNA polymerase, can add additional copies of the 5'-TTAGGG-3' to the end of a chromosome.
The telomerase enzyme is actually a complex containing protein and RNA (a "ribozyme").
The RNA portion has a 5'-CCCTAA-3' region that acts as a template for adding the DNA repeat to the chromosome ends.
The telomerase enzyme is found mostly in the germ cells of multicellular organisms.
In somatic cells, the absence of telomerase results in shorter chromosomal ends with each division and may be the limiting factor in an organism's life span.

DNA repair

Damage to DNA occurs spontaneously.
Under normal conditions, spontaneous hydrolysis of DNA leads to depurination (breaking the glycosidic bond between the deoxyribose and the purine) or deamination (the loss of the amino group from A, C or G).
This damage can result in the inclusion of an incorrect nucleotide to produce a mutation.
Damage to DNA occurs in response to mutagens (either chemical or radiation).
Mutagenic chemicals include
    1) base analogues (similar in structure to the normal bases and can become incorporated into DNA);
    2) base-modifying agents (which can change a base) and
    3) intercalating agents (cause insertions and deletions).
Ultraviolet (UV) radiation (sunlight) can cause pyrimidine dimer formation (such as covalently linked thymines) block replication and transcription.
Ionizing radiation (such as X-rays) knock electrons off of biomolecules to generate highly reactive intermediates that causes all sorts of DNA damage.

Excision repair mechanisms remove abnormal nucleotides to correct mutations.
Base excision repair mechanisms first remove a damaged base then causes excision of the remaining sugar-phosphate unit.
Pyrimidine dimers and other bulky lesions are removed through nucleotide excision repair (NER).
NER causes two nicks which leads to the removal of a stretch of damaged single-stranded DNA (12 in E. coli and 29 in humans).
Mismatch repair corrects mutations of non-complementary bases that become included in DNA during replication that are not fixed by proof-reading.
The original strand is recognized as such by the action of DNA methylases (the old DNA strand is methylated).
Mismatch repair endonucleases cut the sugar phosphate backbone (a nick) and an exonuclease removes the incorrect nucleotides from the nicked strand.

Nuclear and Cell Division

Nuclear division (mitosis) and cytoplasmic division (cytokinesis) occur during the M phase of the cell cycle.
By morphological examination, the process of mitosis (animal cell, plant cell) can subdivided into the following sub-phases:
1) prophase, 2) prometaphase, 3) metaphase, 4) anaphase and 5) telophase
based upon the changing behaviour and appearance of the chromosomes.

1) Prophase:
The chromosomes begin to condense and become visible by light microscopy.
The two sister chromatids appear to be attached at the centromere.
The two centrosomes (which were duplicated during S-phase) begin to move to opposite ends of the cell.
The centrosomes begin assemble of the microtubule-containing structure, the mitotic spindle with dense asters forming at each centrosome.
The cytoskeletal microtubules disassociate and the tubulin is added to the mitotic spindle.
In animal cells but not plant cells, centrioles are located at the core of the centrosomes.

2) Prometaphase:
The nuclear membrane breakdown.
Centrosomes stop as locations at opposite poles of the cell.
As the nuclear membrane is no longer present, mitotic spindle microtubules make contact with the chromosomes with the CEN DNA sequences of the chromosomes’ centromere to form the kinetochore (a protein-DNA complex).
Each chromosome develops two kinetochores, one for each sister chromatid.
The kinetochores bind the free ends of the mitotic spindle microtubules to attach the chromosomes to the mitotic spindle.
The chromosomes are forced toward the centre of the cell.
The mitotic spindle microtubules can attach to the kinetochores (kinetochore microtubules), to microtubules from the other pole (polar microtubules) and to the proteins of the inner plasma membrane (aster microtubules).

3) Metaphase:
The chromosomes are completely condensed.
The chromosomes are paused and aligned at the metaphase plate, a plane half-way between the poles.
A mitotic karyotype can be constructed from cells arrested at metaphase.

4) Anaphase:
Sister chromatids separate and begin to move to opposite poles.
In anaphase A, the chromosomes are pulled by the centromeres towards the poles as the kinetochore microtubules shorten.
In anaphase B, the poles push apart as the polar microtubules get longer.
Anaphase A and B may occur in this order or at once, depending upon cell type.

5) Telophase:
The daughter chromosomes arrive at the pole and begin to revert to chromatin.
The nucleoli develop, the spindle disassembles and the nuclear envelope reappears.
Cytokinesis occurs.

The assembly of the mitotic spindle microtubules and chromosome attachment is key to ability of the mitotic spindle to move the chromosomes during mitosis.
The tubulin subunits have polarity and assembly of the mitotic spindle microtubules occurs with the minus (-) end located at the centrosome and the plus (+) end growing out.
The mitotic spindle microtubules are dynamic structures that are continually growing and shrinking by the addition and loss of tubulin subunits.
As the plus end is so named because it grows far faster than the minus end, this is where most of the increase in microtubule length occurs.
During late prophase, microtubule-forming speed up.
With the dissolution of the nuclear membrane, the plus ends of the mitotic spindle microtubules make contact with the kinetochores.
The chromosomes are both pushed and pulled into alignment by the mitotic spindle microtubules.
With anaphase, the centromeres split and the chromosomes separate and move towards opposite poles due to the action of motor proteins.

Cytokinesis divides the cytoplasm after the chromosomes have been sorted to complete the process of cell division.
Cytokinesis is not necessarily closely linked to nuclear division.
There are numerous examples of the nuclear division without cytokinesis such as early Drosophila development involves several rounds of replication without cytokinesis (the cells form later).
Cytokinesis in animal cells depends upon formation of the cleavage furrow.
This is dependent upon the tightening of a contractile ring of actin microfilaments through the action of myosin (and ATP) to pinch the cell into two.
Cytokinesis in plant cells produce a cell plate between separated daughter nuclei.
The cell plate is formed from materials (polysaccharides and glycoproteins) provides by vesicles derived from the Golgi apparatus.

Regulation of the Cell Cycle

The length of the cell cycle varies depending upon the type of cell and is extremely important in many aspects of biology, especially development.
The timing differences are primarily in the G1 phase, which may become very long (as in G0 phase).
Variation in the length of the cell cycle depends upon cell cycle checkpoints which control the cell’s progression.
These controls make certain that the cell’s machinery is operating properly with the correct timing.
In addition, the cell cycle control mechanisms must make certain that each phase of the cycle is completed properly such that the next steps are prepared for.
The control system must be able to respond to certain conditions that may affect the cell cycle.
The cell cycle checkpoints determine if a cell is ready to progress to the next stage.
Late in the G1 phase, the G1 checkpoint determines if the cell will enter the following S phase.
In animals, the G1 checkpoint or restriction point, is largely controlled by growth factors.
The G2 checkpoint determines if the cell will enter the M phase and requires the proper completion of DNA synthesis.
The third cell cycle checkpoint is the spindle assembly checkpoint which occurs between metaphase and anaphase and requires the proper attachment of all the chromosomes to the spindle apparatus.

Cell cycle control molecules were first discovered through cell fusion experiments in the 1970s.
The fusion of cells in different stages of the cell cycle (to form a heterokaryon) demonstrated that latter stages possess factor trigger progression.
G1 + S -> G1 nucleus enters S phase immediately.
S + G2 -> G2 nucleus does not enter S phase before mitosis.
M+ G1, S or G2 -> non-M phase nuclei enters mitosis, whether or not the DNA is duplicated.

The work of Dr. Masui (University of Toronto) initiated the identification of the cell cycle control molecules.
Frog oocytes are arrested in G2 until a hormone stimulates the egg to continue maturation (immature egg).
With hormonal stimulation, the eggs proceeds to once again be arrested in metaphase II of meiosis, awaiting fertilization (mature egg).
Injection of cytoplasm from a mature egg into an immature egg results in the later beginning meiosis.
The Maturation-Promoting Factor (MPF) which moves cells through the G2 checkpoint is a highly conserved factor now known as the mitosis promoting factor (MPF).
Yeast mutant strains that have defective cell cycle were identified.
One carried a mutant form of a gene (cdc2) which encodes one of the two protein that make up the MPF.
The yeast cdc2 gene encodes a cyclin-dependent protein kinase (Cdk) which is only active when bound to one of the cyclin family of proteins (the second protein in MPF).
Certain cyclins are present only during certain stages of the cell cycle.
There are several cyclin-dependent kinases and several cyclins which control the G1 checkpoint (G1 Cdk’s and G1 cyclins) and the G2 checkpoint (mitotic Cdk’s and mitotic cyclins).

Once bound to the cyclin, the mitotic cyclin-dependent kinase complex (or MPF) phosphorylates proteins involved in the early stages of mitosis.
The active MPF stimulates the breakdown of the nuclear envelope, chromosome condensation, mitotic spindle formation and degradation of key proteins.
The mitotic Cdk-cyclin complex (MPF) also controls the spindle assembly checkpoint by activating the anaphase promoting complex.
The DNA damage checkpoint is regulated by p53, "the Guardian of the Genome"!

The G1 Cdk-cyclin complex controls the G1 checkpoint by the phosphorylation of a number of proteins.
One main target is the retinoblastoma (Rb) protein.
Phosphorylation of Rb prevents the binding and inactivation of the transcription factor E2F.
When E2F is then active to allow the transcription of a number of gene products that are essential to trigger S phase.
Please note that during M phase, Rb is dephosphorylated and E2F is inhibited.
In summary, the cell cycle is regulated by the control of checkpoints by cyclin-Cdk complexes.

Notes prepared from Becker's World of the Cell, 9th edition
Hardin & Bertoni, 2015
Figures copyright of Pearson Education Inc.
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