molecular mechanisms in cell division

1. Cell Division Dr Sufyan Akram

2. Overview of Cell Division Phases of Cell Division Molecular mechanisms in Cell Division Important structures and key components in DNA synthesis DNA polymerases and the process of DNA replication Proof reading and repair Regulation of Cell Cycle

3. Overview

4. 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

5. 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!

6. Meiosis Meio : to reduce A 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

7. Mitosis vs Meiosis Mitosis generates two genetically identical diploid daughter cells Meiosis generates four haploid daughter cells, none of which are genetically identical

8. 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

9. 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)

10. Phases of Cell Cycle G1 - growth phase 1 S - DNA synthesis G2 - further growth M - cell division Mitosis: prophase, prometaphase , metaphase, anaphase and telophase Cytokinesis Interphase Mitotic phase

11. G 1 (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 G S phase - replication of nuclear DNA Interphase Mitotic phase G1 S G2 Pro Prometa Meta Ana Telo

12. G 2 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 Interphase Mitotic phase G1 S G2 Pro Prometa Meta Ana Telo

13. Interphase Mitotic phase G1 S G2 Pro Prometa Meta Ana Telo Prophase Changes occurs in both nucleus and cytoplasm Nucleus: 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

14. Interphase Mitotic phase G1 S G2 Pro Prometa Meta Ana Telo Prometaphase Nuclear 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

15. Interphase Mitotic phase G1 S G2 Pro Prometa Meta Ana Telo

16. Interphase Mitotic phase G1 S G2 Pro Prometa Meta Ana Telo Metaphase Centrioles at opposite poles of the cell Chromosome convene on the metaphase plate (imaginary plane of equal distant between spindles of two poles)

17. Interphase Mitotic phase G1 S G2 Pro Prometa Meta Ana Telo

18. Interphase Mitotic phase G1 S G2 Pro Prometa Meta Ana Telo Anaphase Begins 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

19. Interphase Mitotic phase G1 S G2 Pro Prometa Meta Ana Telo Telophase Nucleolus begins to form at the two poles of the cells. Nuclear envelopes are formed Chromatin fibre of each chromosome become less tightly coiled Mitosis ends and cytokinesis begins

20. Interphase Mitotic phase G1 S G2 Pro Prometa Meta Ana Telo Cytokinesis Occurs 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

21. Interphase Mitotic phase G1 S G2 Pro Prometa Meta Ana Telo

22. Molecular basis

23. 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

24. 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

25. 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

26. 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

27. 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

28. 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

29. 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

30. 5 ′ 3 ′ 3 ′ 5 ′ 5 ′ 3 ′ 3 ′ 5 ′ 5 ′ 3 ′ 3 ′ 5 ′ 5 ′ 5 ′ 3 ′ 3 ′ 1 2 3 DNA

31. 5 ′ 3 ′ 3 ′ 5 ′ 5 ′ 5 ′ 3 ′ 3 ′ 5 ′ 3 ′ 3 ′ 5 ′ 5 ′ 5 ′ 3 ′ 3 ′ 4 5

32. 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

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

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

35. Regulation

36. 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

37. Cell Cycle Regulation Nutrients Growth factors Cell size Regulatory proteins & Protein kinases Cell-cell contact

38. Checkpoints Much 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…

39. M S G 2 G 1 G G 2 Checkpoint G 1 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?