Lecture on DNA Replication

Lecture Structure

Key Proteins: DNAA, DNAB, DNAC, Primase, SSB (Single-Stranded DNA Binding Protein), DNA Polymerase III
Initiation Site: OriC on the chromosome
Steps:
1. DNAA Protein with ATP binds to a specific 9-mer sequence at OriC
2. 13-mer sequence nearby: Strand separation
3. DNAB Protein (Helicase) and DNAC (Helicase Loader) loaded with energy from ATP
4. Helicase unwinds DNA
5. SSB occupies single-stranded DNA
6. Primase forms a short RNA primer
7. DNA Polymerase III is loaded and leading and lagging strand synthesis starts

Domains:
- Palm Domain
  - Binds magnesium and zinc ions for catalysis
  - Important in phosphodiester bond formation
  - Kinetic Selectivity: Ensures only one correct dNTP is added
  - Steric Exclusion: Prevents addition of rNTPs due to small nucleotide binding pocket
- Finger Domain
  - Contains alpha helical protein with arginine, lysine, and tyrosine residues
  - Binds incoming nucleotides and ensures correct pairing
- Thumb Domain
  - Maintains correct position of RNA primer
  - Ensures strong association of DNA Polymerase with DNA substrate

Occurs after DNA Polymerase III is loaded
Leading Strand: Continuous synthesis
Lagging Strand: Discontinuous synthesis via Okazaki fragments
- Primase forms a primer
- Primer extended in 5' to 3' direction forming loop
- Loops are released, and new primers are made
- RNA primers degraded by RNase forming Okazaki fragments
Topoisomerase removes positive supercoiling

Termination Sequences: Ter sequences on DNA
Tus Protein: Binds to termination sequences to halt replication fork
Topoisomerase: Separates two linked circular DNAs to form independent chromosomes

Hemimethylated DNA: Only the parental strand is methylated
- SeqA Protein: Binds to hemimethylated regions to prevent reinitiation
- SeqA dissociates after methylation allows reinitiation
Role of DNAA: Only ATP-bound DNA A can bind to DNA
- High ATP needed for DNA replication, ensuring replication only during high nutrient conditions