Unit 2: Replication of DNA

DNA Replication Overview

• DNA replication produces two identical DNA replicas from one original molecule, ensuring each daughter cell has complete DNA after cell division.
• Critical for genetic continuity across generations.

Modes of DNA Replication

• Conservative Replication: Original strands re-associate, yielding one all-old and one all-new DNA double helix.
• Semi-Conservative Replication: Each parental strand serves as a template for new strands; results in two DNA molecules with one strand old and one strand new.
• Dispersive Replication: Daughter DNA comprises interspersed segments of both parental and newly synthesized DNA.

Meselson-Stahl Experiment

• Conducted in 1958, demonstrating semi-conservative replication using E. coli with heavy nitrogen isotopes.
• After transition from 15N to 14N medium, DNA density showed intermediate results, confirming semi-conservative replication with hybrid DNA bands.

Key Features of DNA Replication

• Semi-Conservative Process: Each new DNA molecule contains one original and one newly synthesized strand.
• Bi-Directional: Origin of replication (ori) allows replication to proceed in both directions, forming a replication bubble.
• Requires a Primer: Short nucleic acid sequence is needed for DNA polymerases, which cannot initiate synthesis de novo.
• Directionality: DNA strands are synthesized from 5' to 3', adding nucleotides at the 3' end.
• Semi-Discontinuous Process: Leading strand is synthesized continuously, while the lagging strand is synthesized in short segments called Okazaki fragments.

Role of DNA Polymerases

• Enzymes that catalyze DNA synthesis by forming phosphodiester bonds among nucleotides.
• E. coli DNA Polymerases:
  • DNA Polymerase I: Main role in DNA replication, recombination, and repair; has 5' to 3' polymerase and exonuclease activity.
  • DNA Polymerase II: Primarily involved in DNA repair.
  • DNA Polymerase III: Major enzyme for replication; involved in leading and lagging strand synthesis with high processivity.

Process of DNA Replication

• Initiation:

  • Begins at the origin of replication (OriC), which includes specific 9-mer and 13-mer sequences.
  • Initiator protein DnaA binds to OriC, facilitating strand separation.
  • DnaB (helicase) unwinds DNA using ATP, while single-stranded binding proteins prevent reannealing.
  • DnaG (primase) synthesizes short RNA primers for DNA polymerases to start synthesis.

• Elongation:

  • DNA Polymerase III adds nucleotides in 5' to 3' direction on both leading and lagging strands.
  • Leading strands synthesized continuously; lagging strands consist of Okazaki fragments.
  • DNA Polymerase I replaces RNA primers with DNA, sealing gaps with DNA ligase.

• Termination:

  • Occurs when replication forks reach the designated Ter region on DNA.
  • Parent DNA is copied semi-conservatively, producing two identical daughter DNA molecules.

Eukaryotic DNA Polymerases

• Eukaryotic cells have multiple types of DNA polymerases, primarily functioning in nuclear replication.
• Key Eukaryotic DNA Polymerases:
  • DNA Polymerase δ: Major enzyme replicating eukaryotic DNA with proofreading capability.
  • DNA Polymerase α: Synthesizes primers and has primase activity.
  • DNA Polymerase γ: Responsible for mitochondrial DNA replication.
  • DNA Polymerase ε: Involved in leading strand synthesis.

Comparison of Prokaryotic and Eukaryotic DNA Replication

• Prokaryotic:

  • Occurs in the cytoplasm.
  • Fast replication speed.
  • Single origin of replication.

• Eukaryotic:

  • Takes place in the nucleus.
  • Slower replication process.
  • Multiple origins of replication.