BIOB11 - LECTURE 4

Learning Objectives

  • Describe major steps (and associated challenges/enzymes) involved in DNA replication.

    • DNA replication involves several key steps:

    1. Initiation: Begins at the origin of replication (ori), requires protein binding for sequence recognition and DNA unwinding. Helicases are essential for unwinding the DNA double helix. Challenge: DNA stability. Enzyme: Helicase.

    2. Unwinding and Stabilization: Single-strand DNA-binding (SSB) proteins bind to stabilize single-stranded DNA, preventing it from reannealing or forming hairpins. Challenge: Single-stranded DNA tendency to fold. Enzyme: SSB proteins.

    3. Primer Synthesis: DNA primase synthesizes a short RNA primer to provide a 3’OH group for DNA polymerase to initiate synthesis. Challenge: DNA polymerase's inability to start from scratch. Enzyme: DNA Primase.

    4. Elongation: DNA polymerase adds nucleotides complementary to the template strand, synthesizing the new DNA strand in the 5’ to 3’ direction. Challenge: Maintaining accuracy and speed. Enzyme: DNA Polymerase III.

    5. Topological Stress Relief: Topoisomerases relieve torsional stress caused by unwinding DNA, preventing overwinding. Challenge: Torsional stress. Enzyme: Topoisomerase.

    6. Lagging Strand Synthesis: The lagging strand is synthesized discontinuously in short fragments (Okazaki fragments), each requiring a new RNA primer. Challenge: Directionality of DNA polymerase. Enzyme: DNA Polymerase III and DNA Primase.

    7. Primer Removal and Gap Filling: DNA polymerase I removes RNA primers and replaces them with DNA nucleotides. Challenge: Removing RNA primers. Enzyme: DNA Polymerase I.

    8. Ligation: DNA ligase covalently joins Okazaki fragments to create a continuous strand. Challenge: Connecting DNA fragments. Enzyme: DNA Ligase.

  • Explain the mechanisms that ensure DNA is replicated and maintained with a low error rate.

    • Several mechanisms ensure high fidelity during DNA replication:

    1. Accurate Nucleotide Selection: DNA polymerase selects correct nucleotides based on base-pairing rules.

    2. Proofreading: DNA polymerase has 3’ to 5’ exonuclease activity, allowing it to remove incorrectly incorporated nucleotides immediately.

    3. Post-Replicative Repair: Systems like mismatch repair (MMR) correct errors after replication.

    4. DNA Damage Response: Cells sense and respond to DNA damage by slowing down cell division to allow repair or inducing apoptosis if the damage is too severe.

  • Explain why lagging strand synthesis requires Okazaki fragments and what the replisome is.

    • Okazaki Fragments:

    • DNA polymerase can only synthesize DNA in the 5’ to 3’ direction.

    • One template strand (leading strand) allows continuous synthesis towards the replication fork.

    • The other template strand (lagging strand) requires discontinuous synthesis in short fragments (Okazaki fragments) because it runs in the opposite direction.

    • Replisome:

    • A multiprotein complex coordinates leading and lagging strand synthesis.

    • Contains DNA polymerase, helicase, primase, and other proteins.

    • Looping of the lagging strand allows the polymerases to travel together, enhancing efficiency.

  • Draw and label a replication fork (including all 5’ and 3’ ends, RNA primers, and direction of progress).

    • Not able to draw an image. A replication fork includes:

    • A Y-shaped structure where DNA is being replicated.

    • Leading strand: Synthesized continuously towards the replication fork. Has a 5’ end furthest away from the fork.

    • Lagging strand: Synthesized discontinuously in Okazaki fragments away from the replication fork. Has a 5’ end closest to the fork.

    • RNA primers: Short RNA sequences providing a 3’OH for DNA polymerase to start synthesis.

    • Helicase: Unwinds the DNA double helix ahead of the replication fork.

    • Single-strand binding proteins (SSB): Stabilize single-stranded DNA.

    • DNA polymerase: Synthesizes new DNA strands.

    • Topoisomerase: Relieves torsional stress ahead of the replication fork.

  • Identify key differences between eukaryotic and prokaryotic DNA replication and explain what features necessitate these differences.

    • Eukaryotic vs. Prokaryotic DNA Replication

    • Origins of Replication:

      • Prokaryotes: Single origin of replication.

      • Eukaryotes: Multiple origins of replication.

    • Genome Size:

      • Prokaryotes: Smaller, circular DNA.

      • Eukaryotes: Larger, linear chromosomes.

    • Complexity:

      • Eukaryotes have more complex regulation and coordination with the cell cycle.

    • Enzymes:

      • Eukaryotes: Use multiple DNA polymerases.

    • Nucleosomes:

      • Absent in Prokaryotes.

      • Present in Eukaryotes.

    • Timing:

      • Prokaryotes: DNA replication is almost continuous.

      • Eukaryotes: DNA replication is tightly controlled and coordinated with the cell cycle (S phase).

  • Explain how nucleosomes impact eukaryotic DNA replication and how epigenetic modifications are inherited/maintained during cell division.

    • Impact of Nucleosomes

    • Nucleosomes must be disassembled and reassembled during DNA replication.

    • Chromatin remodeling complexes destabilize nucleosomes to allow DNA replication.

    • Histone chaperones reestablish nucleosomes behind the replication fork.

    • Epigenetic Inheritance

    • Reestablished nucleosomes contain approximately half “old” histones and half new histones.

    • Epigenetic modifications (e.g., methylation) on old histones serve as a template for modifying new histones, maintaining epigenetic information through cell divisions.

  • Describe the purpose and mechanism of action of telomerase and how it leads to tandem repeats in DNA.

    • Purpose of Telomerase

    • Telomerase maintains chromosome ends (telomeres).

    • Prevents shortening of chromosomes during replication.

    • Mechanism of Action

    • Telomerase is a reverse transcriptase composed of RNA and protein subunits.

    • Contains an RNA template that it uses to synthesize DNA.

    • Extends the template strand by adding tandem repeats (GGGTTA in humans).

    • Primase and DNA polymerase then fill in the end of the lagging strand.

  • Relate expression of telomerase to potential impacts on health and disease.

    • Health and Disease Impacts

    • Aging: Telomere shortening is associated with aging.

    • Cancer: Many cancer cells have high telomerase activity, allowing them to bypass normal cellular senescence and continue dividing.

    • Genetic Conditions: Dyskeratosis congenita is caused by mutations in telomerase RNA, leading to shortened telomeres and premature aging.

  • Identify types of DNA damage caused by different DNA damaging agents, and what type of repair systems are likely to act on each for repair.

    • Types of DNA Damage and Repair Systems

    • Reactive Molecules/Chemicals: Cause structural changes; repaired by nucleotide excision repair (NER) or base excision repair (BER).

    • Thermal Energy: Causes depurination (loss of a purine base); repaired by base excision repair (BER).

    • Ionizing Radiation: Causes breaks in the DNA backbone; repaired by homologous recombination or nonhomologous end joining (NHEJ).

    • UV Radiation: Causes pyrimidine dimers; repaired by nucleotide excision repair (NER).

  • Describe the general steps and key proteins involved in different methods of DNA repair and why some mechanisms are more active at particular DNA sequences or times in the cell cycle.

    • DNA Repair Mechanisms

    • Nucleotide Excision Repair (NER):

      • Removes bulky lesions.

      • Excision nucleases cut the damaged strand.

      • DNA helicase separates the strands.

      • DNA polymerase fills the gap.

      • DNA ligase seals the strand.

    • Base Excision Repair (BER):

      • Repairs altered bases.

      • DNA glycosylase removes the altered base.

      • AP endonuclease removes the remaining sugar-phosphate.

      • DNA polymerase fills the gap.

      • DNA ligase seals the strand.

    • Mismatch Repair (MMR):

      • Corrects mismatched base pairs.

      • MutS recognizes the mismatch.

      • MutL and MutH are involved in excising the incorrect nucleotide.

      • DNA polymerase fills the gap.

      • DNA ligase seals the strand.

    • Homologous Recombination:

      • Repairs double-strand breaks using a homologous template.

      • Nuclease digests 5’ ends.

      • RecA/Rad51 catalyzes strand invasion.

      • DNA polymerase synthesizes new DNA.

      • DNA ligase seals the strand.

    • Nonhomologous End Joining (NHEJ):

      • Repairs double-strand breaks without a template.

      • Ku protein detects DSB and binds the ends.

      • DNA ends are processed and joined by DNA ligase IV.

  • Explain how lack of DNA repair or the process of repair itself could introduce particular types of genetic changes/mutations.

    • Consequences of Impaired DNA Repair

    • Unrepaired Damage: Can lead to mutations if DNA replication occurs before repair.

    • Error-Prone Repair: Some repair mechanisms (e.g., NHEJ) can introduce deletions or insertions, leading to mutations.

    • Homologous Recombination: Can cause loss of heterozygosity (LOH) if the homologous sequence used as a template contains a different allele.

  • Explain why inheriting a defect in a DNA repair gene could result in sensitivity to UV light and cancer.

    • Inherited Defects in DNA Repair

    • Xeroderma Pigmentosum (XP): Mutations in NER genes result in an inability to repair UV-induced DNA damage.

    • UV Sensitivity: XP patients are highly sensitive to UV light and must avoid sunlight.

    • Increased Cancer Risk: Due to the inability to repair DNA damage, XP patients have a high risk of developing skin cancer.