molecular mechanisms in cell division

Cell DivisionDr Sufyan Akram

Overview of Cell DivisionPhases of Cell DivisionMolecular mechanisms in Cell Division

Important structures and key components in DNA synthesis

DNA polymerases and the process of DNA replicationProof reading and repair

Regulation of Cell Cycle

Overview

The Eukaryotic Cell Cycle

Most eukaryotic cells will pass through an ordered series of events in which the cell duplicates its contents and then divides into two cells

This process of cell division in multicellular organisms must be highly ordered and tightly regulated

Mitosis

Mitosis is the process by which a eukaryotic cell duplicates its DNA and then divides into two daughter cells, each of which contains the exact genetic material as the mother cell and gets roughly an equal share of other cellular components

If the DNA of a human cell were uncoiled and stretched, it would extend approximately 2 meters!

Meiosis

Meio: to reduceA form of nuclear division in which the

chromosome number is halved from the diploid number (2n) to haploid number (n)

It is preceded by DNA replication during interphase in the parent cell. This is followed by 2 cycles of nuclear division and cell divisions- Meiosis I and Meiosis II

Mitosis vs Meiosis

Mitosis generates two genetically identical diploid

daughter cells

Meiosis generates four haploid daughter cells, none of which are genetically identical

Introduction

Starting from a single-celled zygote… An adult human being has approximately 100,000 billion cells

Cell division does not stop with formation of mature organism, but continues throughout its life

Tens of millions of cells undergo division at any given moment in an adult human. This amount of division is needed to replace cells that have aged or died

Introduction

Two major cell cycle phases - based on cell activities readily visible under light microscope:

Interphase - occupies bulk of cycle; divided into G1 (first gap), S (synthesis) & G2 (second gap)

M phase – M for "mitotic"; this stage includes mitosis (duplicated chromosomes are separated into 2 nuclei) & cytokinesis (entire cell & its cytoplasm divide into 2 daughter cells)

Phases of Cell Cycle

G1 - growth phase 1S - DNA synthesisG2 - further growth

M - cell division Mitosis:

– prophase, prometaphase, metaphase, anaphaseand telophase

Cytokinesis

Inte

rpha

seM

itotic

pha

se

G1 (G=gap) is the interval between the completion of mitosis and the beginning of DNA synthesis

During G1, the cell monitors its own environment and size before it commits itself to DNA replication. Cells in G1 (if not committed to DNA replication) can pause their progress and enter a specialized resting state G0

S phase - replication of nuclear DNA

Interphase Mitotic phaseG1 S G2 Pro Prometa Meta Ana Telo

G2 is the second “Gap” phase:Nucleus well defined and bound by nuclear

envelope

Outside nucleus are two pairs of centrioles formed during early interphase

Microtubules extend from centrioles in a radial array called asters



ProphaseChanges occurs in both nucleus and cytoplasmNucleus: Chromatin fibres become more tightly

coiled and condense into discrete chromosomes. The duplicated chromosome appears as 2 identical sister chromatids joined by centromere

Cytoplasm: formation of mitotic spindle begins


PrometaphaseNuclear envelope develop fragments.

Microtubules can now invade the nucleus and interact with the chromosomes

Microtubule attach to kinetochore on each chromosomes centromere

Asters, radiate from centrioles and anchor themselves to membrane plasma


MetaphaseCentrioles at opposite poles of the cell

Chromosome convene on the metaphase plate (imaginary plane of equal distant between spindles of two poles)


AnaphaseBegins when paired centromeres of each

chromosome separate, liberating each sister chromosome from one another (each chromatid is considered one full fledged chromosome)

Chromosomes begin moving along microtubule toward opposite poles of the cell


TelophaseNucleolus begins to form at the two poles of the

cells. Nuclear envelopes are formedChromatin fibre of each chromosome become

less tightly coiled

Mitosis ends and cytokinesis begins


CytokinesisOccurs by a process called cleavage: begins with

a cleavage furrow, a shallow grove near the metaphase plate

In cytoplasmic side of the furrow, are contractile actin proteins. As the actin microfilament contract, its diameter shrinks, cleavage furrow deepens until cell pinched into two

Molecular basis

DNA Replication

DNA replication begins at specific locations in the genome, called "origins“

Unwinding of DNA at the origin, and synthesis of new strands, forms a replication fork. In addition to DNA polymerase, the enzyme that synthesizes the new DNA by adding nucleotides matched to the template strand, a number of other proteins are associated with the fork and assist in the initiation and continuation of DNA synthesis

Replication Fork

The replication fork is a structure that forms within the nucleus during DNA replication. It is created by helicases, which break the hydrogen bonds holding the two DNA strands together

The resulting structure has two branching "prongs", each one made up of a single strand of DNA

DNA Polymerase

DNA polymerases are a family of enzymes that carry out all forms of DNA replication

To begin synthesis, a short fragment of DNA or RNA, called a primer, must be created and paired with the template DNA strand

DNA polymerase then synthesizes a new strand of DNA by extending the 3' end of an existing nucleotide chain, adding new nucleotides matched to the template strand one at a time via the creation of phosphodiester bonds

Polymerase Chain Reaction

The PCR does in the test tube what every bacterium does in its tube of media or on an agar-plate and each of us do every day: we all produce billions of exact copies of our own DNA; AMPLIFYING our DNA millions of time

The enzyme DNA polymerase was discovered in the 1950s and our knowledge of the process has been increasing ever since

PCR “Reaction Mix” TARGET DNA to be copied. In theory only a single

molecule is needed A set of short (15 to 40 bases) single stranded PRIMERS

of DNA, that will bind to complementary regions of the opposing stands of the target DNA molecule

An excess of the 4 nucleotide triphosphates, ATP, GTP, CTP, TTP

The enzyme, DNA polymerase

Various buffers and cofactors like magnesium ions required by DNA polymerase

PCR

Double helix target DNA strands are separated so the primers could bind and the DNA polymerase could function

Heat separates DNA strands and that complementary strands then rejoin through base pairing when the temperature is subsequently lowered

PCR

Lowering the temperature enough to allow the primers, which were small and in vast excess, to bind (ANNEAL) to their respective complementary target DNA sequence

DNA polymerase allows polymerization reaction with the triphosphate nucleotides to occur

5′

3′

3′

5′

5′

3′

3′

5′

5′

3′

3′

5′

5′

5′3′

3′

1

2

3

DNA

5′

3′

3′

5′

5′

5′3′

3′

5′

3′

3′

5′

5′

5′3′

3′

4

5

The DNA polymerase fills in the missing portion of each strand making two new double stranded regions of DNA

The whole process is repeated several times thus yielding exponential amount of DNA strands

2 4 8 16 32 After 12 cycles… 8192

After 20 cycles… 2097152

Beginning with a single piece of DNA, PCR can generate 100 billion identical copies of a specific DNA sequence !!!

PCR takes place in a tube which is kept in a machine called “thermal cycler”

Regulation

Cell Cycle Regulation

For all living eukaryotic organisms it is essential that the different phases of the cell cycle are precisely coordinated

Errors in this coordination may lead to chromosomal alterations. Chromosomes or parts of chromosomes may be lost, rearranged or distributed unequally between the two daughter cells

Cell Cycle Regulation

Nutrients

Growth factors

Cell size Regulatory proteins &

Protein kinases

Cell-cell contact

CheckpointsMuch of the control of the progression through the

phases of a cell cycle are exerted at checkpoints

There are many such checkpoints but the three most critical are those that occur near the end of G1 prior to S-phase entry, near the end of G2 prior to mitosis, and at metaphase…

M

S

G 2

G 1G0

G2 Checkpoint

G1 Checkpoint

Metaphase Checkpoint

Is cell big enough?Is environment

favourable?

Is all DNA replicated?

Is cell big enough?Is environment

favourable?

Are all chromosomes

aligned on spindle?

molecular mechanisms in cell division

Technology

s synthesis g2

human cell

mother cell

parent cell

s dna synthesisg2

repairregulation of

cell divisionsmeiosis

growthm cell divisionmitosis