Enzymes and Proteins

1. Helicase

• Unwinds double helix
• Breaks hydrogen bonds between bases
• Creates replication fork

2. Single-Strand Binding Proteins (SSB)

• Bind to separated strands
• Prevent strands from reannealing
• Protect single-stranded DNA

3. Topoisomerase

• Relieves tension from unwinding
• Prevents supercoiling ahead of replication fork

4. Primase

• RNA polymerase enzyme
• Synthesizes short RNA primers (5-10 nucleotides)
• DNA polymerase needs primer to start

5. DNA Polymerase III (prokaryotes) / DNA Polymerase δ (eukaryotes)

• Main replication enzyme
• Adds nucleotides to 3' end only (5'→3' direction)
• Proofreads as it goes (3'→5' exonuclease activity)

6. DNA Polymerase I (prokaryotes)

• Removes RNA primers
• Replaces with DNA nucleotides
• 5'→3' exonuclease activity

7. DNA Ligase

• Seals gaps between Okazaki fragments
• Forms phosphodiester bonds
• Creates continuous strand

Leading vs. Lagging Strand

Leading Strand

• Synthesized continuously in 5'→3' direction
• Same direction as replication fork movement
• Only one primer needed

Lagging Strand

• Synthesized discontinuously in 5'→3' direction
• Opposite direction to fork movement
• Forms Okazaki fragments (1000-2000 nucleotides)
• Multiple primers needed
• Fragments joined by DNA ligase

Replication Steps

1. Initiation:

   • Helicase unwinds DNA at origin
   • SSB proteins stabilize
   • Primase adds RNA primers

2. Elongation:

   • DNA polymerase adds nucleotides (5'→3')
   • Leading strand: continuous synthesis
   • Lagging strand: Okazaki fragments formed

3. Termination:

   • DNA polymerase I removes RNA primers
   • Replaces with DNA
   • DNA ligase seals gaps
   • Two identical DNA molecules

Proofreading and Repair

Proofreading:

• DNA polymerase checks each nucleotide
• 3'→5' exonuclease removes errors
• Error rate: ~1 in 10 billion

Mismatch repair:

• Enzymes scan for mismatched bases
• Remove and replace incorrect nucleotides
• Occurs after replication

DNA repair mechanisms:

• Base excision repair
• Nucleotide excision repair (UV damage)
• Direct repair

Telomeres and Telomerase

Problem: DNA polymerase can't replicate ends of linear chromosomes

Telomeres:

• Repetitive sequences at chromosome ends (TTAGGG in humans)
• Protect genes from being lost
• Shorten with each division

Telomerase:

• Enzyme that extends telomeres
• Active in germ cells, stem cells
• Inactive in most somatic cells
• Overactive in cancer cells (immortality)

Key Concepts

1. Semiconservative: each new DNA has one old + one new strand
2. 5'→3' direction: DNA polymerase adds to 3' end only
3. Leading strand: continuous synthesis
4. Lagging strand: discontinuous, forms Okazaki fragments
5. Proofreading: ensures high fidelity (~1 error in 10¹⁰)
6. Telomeres: protect chromosome ends, shorten with age