BIOL2104: Cell Division and DNA Replication Study Notes

Cell Division and Replication

Introduction to the Course

• Course: BIOL2104 - Week 2

• Date: Thursday, January 16, 2025

Agenda Overview

1. Finish discussing mitosis

2. DNA replication in prokaryotic versus eukaryotic cells

3. Applications of DNA replication processes

4. Mutations related to DNA replication

Identifying Mitosis Phases

• Question: Identify which circled image shows a cell in telophase.

• Related Questions:

  1. How many chromosomes are in the cell pictured?

  • a) 6

  • b) 3

  • c) 2

  • d) 0

  1. How many chromatids are in the cell pictured?

  • e) 6

  • f) 3

  • g) 2

  • h) 0

  1. How many chromosomes are in the cell pictured?

  • a) 8

  • b) 6

  • c) 4

  • d) 2

  1. How many DNA molecules are in the cell pictured?

  • e) 16

  • f) 8

  • g) 4

  • h) 2

  1. How many homologous pairs of chromosomes are in the cell pictured?

  • a) 16

  • b) 8

  • c) 4

  • d) 2

• Note: For detailed answers, refer to the student announcements.

Learning Objectives

By the end of the week, students will be able to:

1. Describe key enzymes and processes involved in DNA replication.

2. Compare and contrast DNA replication in prokaryotic and eukaryotic cells.

3. Predict effects of changes in replication machinery on molecules, cells, and organisms.

4. Describe lab techniques based on DNA replication.

5. Compare and contrast insertion versus deletion mutations.

6. Describe consequences of mutations in somatic cells versus germ cells.

DNA Replication

• Importance: Accurate DNA replication is essential for successful cell division.

• Concept: Watston & Crick proposed the mechanism of DNA replication as semi-conservative replication.

  • Definition: Each strand of the original DNA molecule serves as a template for the production of the complementary strand.

Building Materials for DNA Replication

1. Template: Single-stranded DNA

2. Raw materials (substrates): Deoxynucleotide triphosphates (dNTPs)

3. Energy sources: Powering the reactions

4. Enzymes and proteins: Read the template and assemble the new strand

Prokaryotic DNA Replication Process

1. Initiation

2. Unwinding

3. Elongation

4. Termination

Circular DNA Replication (Theta Replication)

• Process:

  1. Replication forks assemble at the origin of replication.

  2. Bidirectional progression of replication forks.

  3. Convergence of bidirectional replication forks.

  4. Separation of almost fully replicated daughter genomes.

Rolling Circle Replication

• Initiation: Begins with a break in one of the nucleotide strands, leading to the exposure of a 3’ hydroxyl (OH) and a 5’ phosphate.

• Nucleotide Addition: Nucleotides are added to the 3’ end of the broken strand using the inner strand as a template.

Steps of Prokaryotic DNA Replication

1. Initiation

• Replicator: Origin of replication is called oriC and consists of approximately 245 base pairs.

• Initiator protein: Binds to oriC causing it to unwind, known as DnaA in E. coli.

2. Unwinding

• DNA gyrase: An enzyme that controls supercoiling of DNA, reducing torsional strain in front of the replication fork.

• Helicase: Breaks hydrogen bonds between base pairs of the two strands, moving 5’ to 3’.

• Single-strand binding proteins: Attach to DNA to protect and prevent secondary structures from forming.

3. Elongation

• DNA polymerases: Add new nucleotides in a 5’ to 3’ direction and require a 3’ OH group to which a new nucleotide can be added.

• Primase: Synthesizes short stretches (10-12 nucleotides) of RNA nucleotides, known as primers, providing a starting point for DNA polymerase.

• Ligase: Joins 3’ OH and 5’ phosphate between lagging strand fragments.

Prokaryotic DNA Polymerases

1. Pol I:

   • First polymerase discovered (1956).

   • Functions: 5’ to 3’ polymerase activity and both 5’ to 3’ and 3’ to 5’ exonuclease activity.

   • Main role: Removes RNA primer and replaces it with DNA; important for repair; low efficiency.

2. Pol II:

   • 5’ to 3’ polymerase activity and 3’ to 5’ exonuclease activity.

3. Pol III:

   • The primary enzyme responsible for DNA synthesis; high efficiency.

4. Pol IV and Pol V:

   • Involved in cell response to DNA damage, with 5’ to 3’ polymerase activity.

Schematic of DNA Polymerase III Holoenzyme

• Contains 10 different subunits.

• Functional components include:

  • Clamp (β)

  • Core polymerase (α, ε, θ)

  • γ complex (γ, δ, δ', χ, ψ)

Detailed Elongation Process

• Leading Strand vs. Lagging Strand:

  • Leading strand: Synthesized continuously.

  • Lagging strand: Synthesized in fragments known as Okazaki fragments.

  • Helicase and Primase roles in strand synthesis.

Prokaryotic Replisome Components

• Key Components:

  • Exonuclease

  • Polymerase

  • Clamp

  • Gyrase

  • Ligase

  • Helicase

  • Leading strand clamp loader

  • Primase

Termination of DNA Replication

• Tus-Ter Complex:

  • Binding of the protein Tus to the ter sites blocks helicase movement in one direction.

  • Reasons for multiple ter sites include management of replication forks.

Differences Between Prokaryotic and Eukaryotic DNA Replication

• Eukaryotic DNA Replication:

  • Locations are within the nucleus compared to cytosol in prokaryotes.

  • Multiple origins of replication (10,000 to 100,000 in humans).

  • Cyclins trigger initiation of the replication process.

  • Involves complex DNA polymerases (α, β, γ, δ, ε).

Eukaryote Polymerases

• 
  

• Polymerases and their functions:
  

  Polymerase	Synthesis	Exonuclease	Functions
  α	Yes	No	Initiation & repair (primase function)
  δ	Yes	Yes	Lagging-strand synthesis, repair
  ε	Yes	Yes	Leading-strand synthesis
  γ	Yes	Yes	Mitochondrial DNA replication & repair
  ζ	Yes	No	Translesion DNA synthesis
  Midterm Style Questions

  1. Impact of Mutation in DNA Polymerase III:

     • Discuss potential effects of reduced 3' to 5' exonuclease activity on fidelity during DNA replication.

  2. Topoisomerase II as Anticancer Drug Target:

     • Explain the rationale behind targeting topoisomerase II with anticancer drugs.

  Recap: Key Differences in DNA Replication

  • Prokaryotic vs. Eukaryotic Replication: Must differentiate based on location, origins, forks, number of replicons, involved polymerases, distortion management, size of Okazaki fragments, speed, and final products.