HBG 21 ( DNA Replication and Cell Cycle)

• Cell cycle phases: interphase, DNA synthesis, and mitosis/meiosis
• Functions of proteins responsible for DNA replication:

  • Helicase
  • SSB protein
  • Primase
  • DNA polymerases
  • DNA ligase
• Relation of oogenesis and spermatogenesis to meiosis
• Cell cycle involves:

  • Cell growth
  • Replicating genetic material
  • Cell dividing
• Mitosis phases:

  • Interphase:
  • G1: increase cellular contents, grow
  • S: increase genetic contents, chromosomal replication
  • G2: checking errors on chromosome
  • Mitosis
  • Cell cycle arrest (G0):
  • Quiescent or senescent
  • Post-mitotic cells
  • e.g., post-mitotic neurons
• DNA Replication:

  • Important step in the cell cycle
  • Semi-conservative replication
  • Replication origin starts DNA replication
  • Eukaryotic chromosomes have multiple origins of replication
  • Enzymes involved: DNA helicase, Topoisomerases, DNA primase, DNA polymerase III, DNA ligase
• Leading vs Lagging strands:

  • Leading strand: replicated in a continuous manner
  • Lagging strand (Okazaki fragments): small discontinuous fragments
• Limitations of DNA polymerase:

  • Synthesizes new DNA strands in the 5' to 3' direction
  • Requires a primer to continue adding nucleotides
  • Correcting errors during replication
  • Correcting damage due to environmental effects
• Xeroderma Pigmentosum:

  • Linked to defects in DNA repair
  • Inherited as an autosomal recessive trait
  • Extremely sensitive to ultraviolet light
  • Symptoms include skin atrophy, splotchy pigmentation, spidery blood vessels, and skin cancers
• Cell Cycle Checkpoints:

  • G1 Checkpoint: main decision point for a cell to divide
  • G2 Checkpoint: ensures DNA integrity and replication before M phase
  • Metaphase Checkpoint: checks alignment and tension of chromosomes before separation
• Cell division includes:

  • Nuclear components – karyokinesis
  • Mitosis:
  • Mother and daughter cells have the same genetic complement
  • Reductional
  • Results in products that have half the genetic material as the parent cell
  • Ex: gametes in animals, spores in higher plants
  • Cytoplasmic components - cytokinesis
• Mitosis:

  • Separation of sister chromatids
  • Involves an apparatus called the spindle composed of microtubules
  • Function of microtubule is to provide shape and structure to eukaryotic cells, allow cells to move, and allow cells to move their internal components
  • Microtubules are made up of protein subunits: α-tubulin, β-tubulin, Kinesin, and dynein
  • Microtubules are formed from microtubule organizing centre – centrioles (basal bodies) or centrosome
  • Centriole replicates during S and G2 phases
  • Interpolar microtubules
  • Microtubules spread out and form aster
• Meiosis:

  • Producing sex cells or gametes in animals and spores in plants
  • Aim: to reduce genetic materials into half (2n to n)
  • Two-stage process: meiosis I and II
  • Cells entering meiosis have replicated their chromosomes (4n)
  • Involves pairing of homologous chromosomes during prophase, then separate into two daughter cells
  • Involves recombination of homologous chromosomes - crossing over occurs during prophase
  • End up with four cells, each with the haploid cell (n)
• Gametes production:

  • Spermatogenesis:
  • After mitosis, spermatogonia become primary spermatocytes (2n)
  • After meiosis I, primary spermatocyte divides into two secondary spermatocytes
  • After meiosis II, each secondary spermatocyte divides into two spermatids (1n)
  • Spermatids develop into mature spermatozoa or sperm cells
  • Oogenesis:
  • Starts in the germinal epithelium, giving rise to ovarian follicles
  • Involves three oogenetic subprocesses: Folliculogenesis, Oocytogenesis, Ootidogenesis
  • Ootidogenesis results in ovum and secondary polar body
• To apply DNA replication knowledge for clinical scenarios (bench to bedside):

  • Topoisomerases play important roles in basic cellular biology
  • Some drugs target topoisomerases, including anticancer drugs
  • Nucleoside analogues are also called nucleoside reverse transcriptase inhibitors
  • Nucleotide analog: synthetic or natural chemical compound resembling nucleotides in structure and/or function
• Cell cycle phases:

  • Interphase:
  • G1: increase cellular contents, grow
  • S: increase genetic contents, chromosomal replication
  • G2: checking errors on chromosome
  • Mitosis
  • Meiosis
• Proteins responsible for DNA replication:

  • Helicase: unwinds DNA molecules using energy derived from ATP
  • SSB protein
  • Primase: catalyzes the synthesis of short RNA strands that are complementary to the template strands
  • DNA polymerases
  • DNA ligase
• Oogenesis and spermatogenesis processes can be related to meiosis
• DNA replication:

  • Important step in the cell cycle
  • By DNA polymerase
  • Semi-conservative replication
  • Replication origin starts DNA replication
  • Eukaryotic chromosomes have multiple origins of replication
  • Enzymes needed:
  • DNA helicase unwinds DNA molecules using energy derived from ATP
  • Topoisomerases catalyze the breaking and reannealing of the original DNA phosphodiester linkages
  • DNA primase catalyzes the synthesis of short RNA strands that are complementary to the template strands
  • DNA polymerase III uses the free 3’-hydroxyls of the RNA primers to extend the chains
• • Leading strand is replicated in a continuous manner
  • Lagging strand (Okazaki fragments) is replicated in small discontinuous fragments
• Limitations of DNA polymerase:

  • Synthesizes new DNA strands in the 5' to 3' direction
  • Cannot add nucleotides in a 3' to 5' direction
  • Requires a primer to continue adding the rest of the pairs of the segment until a new double strand of DNA is completed
  • Prone to making errors
  • Correcting errors during replication (proofreading)
  • Correcting damage due to environmental effects
• Cell division includes:

  • Nuclear components – karyokinesis
  • Mitosis:
  • Mother and daughter cells have the same genetic complement
  • Reductional
  • Results in products that have half the genetic material as the parent cell
  • Ex: gametes in animals, spores in higher plants
• Cytoplasmic components - cytokinesis

  • Able to identify the mitotic stages
• Mitosis:

  • Separation of sister chromatids
  • Involves an apparatus called the spindle which is composed of microtubules
  • Function of microtubule is to provide shape and structure to eukaryotic cells, allow cells to move, and allow cell to move its internal components
  • Microtubules are made up of protein subunits: α-tubulin, β-tubulin, Kinesin, and dynein
  • Microtubules are formed from microtubule organizing centre – centrioles (basal bodies) or centrosome
  • Centriole replicates during S and G2 phases
  • Interpolar microtubules
  • Microtubules spread out and form aster
• Meiosis:

  • Producing sex cells or gametes in animals and spores in plants
  • Aim: to reduce genetic materials into half (2n to n)
  • Two-stage process: meiosis I and II
  • Cells entering meiosis have replicated their chromosomes (4n)
  • Starts with pairing of homologous chromosomes during prophase, then separate into two daughter
  • Involves recombination of homologous chromosome - crossing over occurs during prophase
  • End up with four cells, each with the haploid cell (n)
• Gametes production:

  • Spermatogenesis:
  • After mitosis – spermatogonia become primary spermatocytes (2n)
  • After meiosis I - primary spermatocyte divides two secondary spermatocytes
  • After meiosis II – each secondary spermatocyte divides into two spermatids (1n)
  • Spermatids develop into mature spermatozoa or sperm cells
  • Oogenesis:
  • Starts in the germinal epithelium, giving rise to ovarian follicles
  • Involves three oogenetic subprocesses: Folliculogenesis, Oocytogenesis, Ootidogenesis
  • Oogenesis occurs in the ovary
• Take home messages:

  • A continuously dividing cell goes through G1, S, G2 phases and mitosis
  • A cell may get out of the cycle (enter G0 phase) and stop dividing
  • A cell can be prompted to get out of G0 and start dividing again by external factors such as growth factors
  • Cyclins control cell division
  • Some cells can divide indefinitely in nondifferentiated state and are called stem cells
  • Cancer is the disease of uncontrollable cell division
  • In mitosis, one cell divides into two cells where both daughter cells have identical sets of chromosomes to the parent cell
• • All cells in our body except for sperms and oocytes are produced as a result of mitosis
  • Meiosis is used ONLY to produce sperms and oocytes
• Topoisomerases as Drug Targets:

  • DNA topoisomerases play important roles in basic cellular biology
  • Some drugs that target the topoisomerases are anticancer drugs
  • Anticancer drugs inhibit the topoisomerase molecule from re-ligating DNA strands after cleavage
• Nucleoside Analogs and HIV Therapy:

  • Nucleoside analogues are also called nucleoside reverse transcriptase inhibitors
  • Their target is the HIV enzyme reverse transcriptase
  • Incorporation of nucleoside analogues aborts DNA synthesis
• At the end of this lecture, you should:

  • Know the cell cycle phases: interphase, DNA synthesis, and mitosis/meiosis
  • Understand the functions of the proteins responsible for DNA replication, including helicase, SSB protein, primase, DNA polymerases, and DNA ligase
  • Be able to identify the mitotic stages
  • Relate the process in oogenesis and spermatogenesis to meiosis
  • Apply DNA replication knowledge for clinical scenarios (bench to bedside)