DNA Replication and Repair

DNA Replication and Repair

Semiconservative vs. Conservative Replication

• Semiconservative Replication: Separates parent strands, builds new complementary strands.
• Conservative Replication: Copies DNA molecule "as is," leaving original strands together.
• Meselson and Stahl Experiment (1958): Demonstrated semiconservative replication using isotopes (15N and 14N).
  • E. coli grown in 15N medium, then transferred to 14N medium.
  • Analysis showed new DNA strands contained both 14N and 15N.

DNA Structure and Antiparallel Strands

• DNA strands run antiparallel (opposite directions).
• 3' end: Hydroxyl attached to the 3’ carbon of the sugar.
• 5' end: Phosphate attached to the 5’ carbon of the sugar.

Step 1: Strand Separation

• Replication begins at replication origins.
• Helicase: Unwinds DNA, breaking hydrogen bonds between base pairs.
• Replication Fork: Y-shaped structure formed as strands separate.
• Single-Strand Binding Proteins (SSB): Prevent rejoining of parent DNA strands.
• Replication forks proceed in opposite directions, forming a replication bubble.

Step 2: Building Complementary Strands

• DNA Polymerases: Enzymes that join new nucleotides.
  • Add nucleotides to the 3’ end of a new strand, reading the template 3’ to 5’.
  • New strand assembled in the 5’ to 3’ direction.
• RNA Primase: Builds small complementary RNA segments (RNA primers) to start replication.
• DNA Polymerase III: Adds DNA nucleotides to the RNA primer.
• Leading strand: Continuously built toward the replication fork.
• Lagging strand: Built in segments (Okazaki fragments) away from the fork.
• DNA Polymerase I: Removes RNA primers and replaces them with DNA nucleotides.
• DNA Ligase: Catalyzes the formation of phosphodiester bonds to link Okazaki fragments.

Step 3: Dealing with Errors During DNA Replication

• DNA polymerase enzymes proofread and correct base-pair mismatches.
• DNA repair mechanisms (DNA polymerase I and II) correct missed errors.

Important Enzymes

• Helicase: Unwinds DNA helix.
• Single-strand binding proteins: Prevents parent DNA strands from rejoining.
• RNA primase: Places RNA primers on template strands.
• RNA primers: Act as starting strands for DNA polymerase.
• DNA polymerases: Assembles nucleotides into new DNA strands, removes RNA primers, proofreads and repairs.
• DNA ligase: Forms phosphodiester bonds to join Okazaki fragments.

Replication Bubble Overview

• Replication bubbles form in DNA with replication forks on each side.