Cell cycle and DNA replication Lecture PDF

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1 Cell cycle and DNA replication: Readings: p. 95-100 Marieb and Hoehn, or p. 96-101 Marieb and Hoehn (9 th ed)

Two majors types of cells – somatic and germ Somatic – undergo mitosis (cell division) at some time during their life Germ (sperm and ova) – undergo meiosis

Cell cycle and DNA replication: Cell division by mitosis – results in identical daughter cells – i.e. identical copies of genome of parent cell – are diploid (2n) Cell division by meiosis – only occurs in germ cells (ova and sperm) – results in daughter cells with different genomes – are haploid (1n) We’ll focus on somatic cell division (mitosis). Somatic cells that can undergo cell division throughout life include: •

Epithelial cells

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Smooth muscle cells

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Connective tissue cells

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Peripheral nerve cells- i.e. if a nerve cell in the finger is severed then it does eventually regenerate.

(Note that brain and spinal cord cells do not undergo cell division in adults) Some somatic cells undergo cell division frequently, such as skin cells, epithelial lining of the gut and bone marrow cells: •

Epithelial cells of intestinal tract are replaced every three days.

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Bone marrow cells are constantly dividing (new generation every 8h)

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Cell division take place during the “Cell Cycle”. (Fig. 3.31) Cell cycle includes periods of – •

Cell growth (gap phases): The G phases

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DNA replication: S phase

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Cell division (mitosis): Mitotic phase

The whole area of G1 to G2 is called interphase. Anything in yellow is the separation of the DNA

Cell Cycle divided into two major phases: •

Interphase: Includes G1 + G2 growth phases, as well as the S-phase (DNA replication)

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Mitotic phase (M phase): Where we separate copies of the DNA into two daughter cells.

3 Interphase: -In G1 and G2, we have periods of cell growth where we increase the cytoplasm and number of organelles. -During S-phase we replicate each DNA molecule. So if there ar 23 pairs of chromosomes we have to replicate all of them. 46DNA x2 = 92 DNA at the end of S-phase Once replicated, they become structures we call sister chromatids (diagram on right). Each ‘X’ is considered TWO sister chromatids.

Mitotic phase (M phase- actual process of dividing the cell) is divided into 6 stages: 1. Prophase 2. Prometaphase (or late prophase) 3. Metaphase 4. Anaphase 5. Telophase and Cytokinesis

4 At the start of the Mitotic phase (end of interphase - G2): •

DNA has been replicated (to make 92 total)

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DNA in form of chromatin (it is not in the compacted form of sister chromatids and also an electron microscope cannot distinguish them)

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Cell has two centrosomes (one was replicated during the S-phase) – each with centrioles

Centrosomes and centrioles: This is where microtubules are assembled

5 Mitosis – Stage 1: Prophase Chromatin condenses into discrete chromosomes – 46 pairs of SISTER CHROMATIDS NOT 46 pairs of chromosomes. The centrosomes start to migrate to opposite ends of the cell Centrosomes produce microtubules (these are the mitotic spindle)

Mitosis – Stage 2: Pro-metaphase (or late prophase) -Nuclear membrane dissolves -Microtubules attach to each pair of sister chromatids on either side of the pair. -Kinetochore microtubules are what comes into contact with the DNA. -Microtubules not in contact with DNA are called NONkineochore

6 Mitosis – Stage 3: Metaphase Now all 46 pairs of sister chromatids are lined up along the metaphase plate. Lined up in a way so that one member of each pair is orientated in opposite directions (i.e. north and south) -CRITICAL POINT! If not properly lined up, mitosis will not continue. Each chromatid needs a kinetocore microtubule attached to it We also have non-kinetochore microtubules, which do not attach to the chromatids. Instead they form a kind of over lapping structure (they’re eventually involved in elongation of the cell)

Mitosis – Stage 4: Anaphase This is where actual splitting occurs *NOTE: The diagram got it wrong in calling it daughter chromosomes, they’re sister chromatids -Using a molecular motor, each sister chromatid moves in opposite directions along the kinetochore microtubules. Each sister chromatid moves towards their respective centrosome -To do this movement, the microtubules need to shorten, and this is achieved by the microtubule gradually breaking down / disassembling The nonkinetochore microtubules also slide apart, pushing the ends of the cell apart (so the cell goes from a sphere shape to a oblong, football shape)

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8 Mitosis – Stage 5: Telophase and Cytokinesis -Microtubules are disassembled -Nuclear membrane is reformed around the DN -Cell starts to pinch into two areas at what was once the metaphase plate -Actin microfilaments tighten and pinch off the cells

Control of cell cycle At various points in cell cycle conditions in cell must be right for cell to proceed with cell division These point are called “Checkpoints”. At critical places in the cell cycle everything has to be right or else the cell no longer goes through cell division G1 restriction point: G2 restriction point: One in mitosis:

9 First checkpoint occurs late in G1 phase - is called a restriction point: If conditions at Restriction point are not favorable, then cell enters G0 phase - cell does not divide At G0 the cell is still alive, it just doesn’t replicate.

Two other known checkpoints are known: •

G2 checkpoint - between G2 and M phase – The

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Metaphase or spindle assembly checkpoint – Final checkpoint where if the sister chromatids are not properly lined up the cell will not undergo mitosis

Checkpoint regulation is performed by proteins called “cyclins” (which must be in sufficient numbers) without cyclin, we cannot trigger the events to scan and inspect the dividing cell. There are a number of cyclins (which we don’t need to know for an exam): •

G1/S cyclin, E cyclin

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A cyclin, D cyclin

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B and M (or mitotic) cyclin

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Cyclin concentrations vary throughout the cell cycle:

Cyclins are needed to activate a protein called: cyclin-dependent protein kinase (or cdk) A kinase is a protein which phosphorylates another protein (sticks a phosphate group onto another protein) -> This activates the protein that receives the phosphate

Role of cyclin-dependent protein kinase (or cdk) Is to phosphoraylate a key protein needed to pass the checkpoint

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Example of control by cyclin and cdk: Trinity!! My feet are nicer than yours. =] Umad? =] I refuse USOMAD XD ZZZZZZZZZZZZZZZZZZZZZZZZZZLOLSD88DDDDDDDDDDDDDyou’re weird. And mad.pop

Initiation of mitosis at the G2 checkpoint occurs when mitotic cyclin activates a mitotic cyclin cdk (MCcdk). A MCcdk is simply an activated cdk The mitotic cyclin-MCcdk Complex is called mitosis promoting factor (MPF). Once the cyclin has done its job, it’s destroyed by the cell

12 Role of Growth Factors in regulating cell cycle: Must have appropriate nutrients and growth factors available for cell division to proceed. Growth factors are peptides - examples: •

Platelet-derived growth factor

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Epidermal growth factor

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Nerve growth factor

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Insulin-like growth factor

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Cytokines

To trigger the production of cyclin we need these factors.

Growth factors stimulate the production of cyclins, for example:

The phosphorylated cdk in turn phosphorylates a target protein that allows the cell to get through the checkpoint! eeeeeeeeeeehhhh sexy ladeh OP OP OP OPPA GANGNAM STYLE

13 DNA Replication: DNA replication is an event that occurs during the “S” phase of the cell cycle. Each DNA molecule is replicated: That means that a normal human somatic cell has 46 DNA molecules (23 pairs) prior to the S phase and 92 molecules following DNA Replication.

DNA structure: (see Fig. 2.22) DNA = deoxyribonucleic acid Composed of Nucleotides Nucleotides have three regions– deoxyribose sugar, PO4, and a nitrogenous base Carbon 1 attatches to the nitrogen, 5 to the phosphate . To bond the nucleotides together, we need to link up a nucleotide to the 3’ carbon at the end of our chain of nucleotides (we can ONLY add nucleotides to a growing chain at the 3’ nucleotide at the end of our chain.) All DNA has the same 5 carbon sugar molecule

14 The nucleotides are bonded one to another to form a chain. PO4 group of one nucleotide bonds with the deoxyribose sugar of the next nucleotide in line. Two phosphates are split off and the lone phosphate creates the phosphodyester bond *remember we can only add a new nucleotide to the number 3 carbon

Nucleotides are arranged in two anti-parallel strands. The two anti-parallel strands held together by hydrogen bonds between the nitrogenous bases. 3' end 5' end

While phosphate groups are common to every part of DNA, the nitrogenous bases can be either of four different types!

3' end 5' end ways pairs with Thymine ways pairs with Guanine

ands form a spiral known as the “Double helix”.

15 Each strand has a 3-prime (3’) and a 5-prime (5’) end. -------------------------------------------DNA Replication: http://www.youtube.com/watch?v=teV62zrm2P0 Replication process – http://www.youtube.com/watch?v=AGUuX4PGlCc&feature=related Begins at several places along the DNA molecule at locations called Origins of OR’s: Areas of DNA where we break the hydrogen bonds and start to make copies of the DNA. (humans often have several along their double helix)

Replication Forks (Where we start to unravel the DNA) spread in both directions away from each origin, opening the DNA and forming a replication bubble. *Almost like a double zipper! This forms a bubble-like structure called… a replication bubble.

Helicases - enzymes that break the hydrogen bonds between the bases of the antiparallel strands at replication forks (stored within the nucleus of the cell)

Single strand binding proteins (DNA cockblockers)- keep the antiparallel strands separated by attaching to exposed DNA and preventing opposing nitrogenous bases from pairing with each other.

Both strands undergo replication at same time. The strands being replicated are called “template” strands.

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B . 2

RNA primer (set of nucleotides) is paired with bases on each strand at the Origin of Replication . Nucleotides are added to the 3’ end on the RNA primer (5’ end of nucleotide is bonded with 3’ end of primer). This (synthesis) reaction is catalyzed by DNA polymerase (Which glues old nucleotides and brand new ones together).

One after another, the appropriate nucleotides (ones that compliment the template) are bonded to the growing chain of nucleotides at the 3’ end of the chain of the new DNA strand. The new strand growing in the direction of the replication fork is called the leading strand. The new strand growing in the opposite direction of the replication fork is called the lagging strand. The cell manages to make the lagging strand work by adding in a new RNA primer that goes against the direction of the replication fork.

17 Leading strand formation is straight-forward: New nucleotides are added to 3-prime and as replication fork moves ahead (remember 3’ is always the leading point of replicating DNA). But.... the lagging strand is formed in fragments - called Okazaki fragments

Each time the replication fork moves ahead, a new RNA primer must be inserted before the lagging strand Okazaki fragment is built. And likewise, to bond Okazaki fragments together we have to get rid of the RNA primer Eventually, the RNA primers are removed and replaced with DNA. Then the Okazaki fragments are tied together by DNA ligase. (Tip: Remind yourself that ligase ties them together like how ‘ligaments’ hold things together)

DNA replication is semi-conservative: Each new DNA molecule contains one template strand and one new complimentary strand (half old, half new) To summarize: At the Origin of Replication we slap down RNA primers to begin the DNA replication. The DNA replicates with leading strands going in the direction of the Replication Fork and lagging strands go in the opposite direction. Okazaki fragments occur because as we replicate along, eventually the new DNA strand hits another RNA primer and replication halts (it’s impossible to bond a RNA primer to more than one DNA strand). We have to tie the DNA segments together with DNA ligase and afterwards we kick out the RNA primer and replace it to create a full 100% DNA strand again....