DNA Replication

• Problems faced by DNA polymerase:

  • Can't unwind double-stranded DNA
  • Single-stranded template is unstable
  • Can't start DNA chains
  • Too slow and distributive
  • No 3’-5’ polymerase
• Solution for unwinding double-stranded DNA: DNA Helicase (MCM Complex)
• DNA Helicase is composed of 6 identical monomers forming a homohexamer
• MCM (Minichromosome Maintenance) Complex is the helicase in eukaryotes, composed of 6 distinct subunits (MCM 2-7) forming a heterohexamer
• Experimental proof for DNA Helicase:

  • Negative control: all components without helicase
  • Positive control: denatured DNA using heat, contains no helicase
  • Increasing amounts of MCM complex leads to more ssDNA in gel electrophoresis
• Solution for unstable single-stranded template: ssDNA binding proteins (SSBs)
• RPA (Replication Protein A) is a eukaryotic SSB composed of RPA70, RPA32, and RPA13
• Solution for starting DNA chains: DNA primase
• DNA primase is a protein complex consisting of Pol α + Primase
• Primase synthesizes 11-14 nucleotide RNA, always beginning with A or G, while Pol α extends with 10-20 nucleotide DNA
• Solution for being too slow and distributive: Sliding clamp
• PCNA is a eukaryotic sliding clamp that promotes processivity, only for Pol δ/Pol ε – polymerases with proofreading activity
• RFC (Replication Factor C) is a eukaryotic clamp loader that binds closed PCNA
• The replisome coordinates leading and lagging strand synthesis
• What lines the central channel of DNA Helicase?

  Positively charged residues
• What is located in the CTD of the MCM complex?

  Inward-facing ATPase
• How does the MCM complex move and on what strand?

  Moves 3’-5’ on the leading strand
• What is the localization of the DNA strands in the MCM complex?

  • 5’-end of lagging strand outside; close to Pol α
  • 3’-end of leading strand through the channel; close to Pol ε
• What is the function of SSBs?

  Protect ssDNA via binding
• How many DNA binding domains does RPA have and where are they located?

  Total: 4

  3 in RPA70

  1 in RPA32
• What is the structure of the Polα-Primase complex?

  Polα = 2 subunits: P180 and P86 (B)

  Primase = 2 subunits: P58 and P48
• What is the function of Polα subunits?

  P180: interacts with P86 via CTD

  P86 (B): Unknown function but required
• What is the structure of PCNA?

  6-fold axis of symmetry, No ATPase, Homotrimer
• What is the structure of RFC?

  Heteropentamer – RFC 1-5
• What is the mechanism of RFC?

  1. RFC 1, 3, 4 interact with closed PCNA
  2. ATP binding triggers RFC 2, 5 interaction with PCNA
  3. PCNA forced open
  4. Complex binds to primer/template junction (DNA)
  5. ATP hydrolysis (by RFC)
  6. PCNA Closure; Loss of interaction with RFC 2, 5 
  7. RFC dissociates
• What is the structure of DNA polymerase – subdomains and their function?

  • Described as a hand
  • 4 different subdomains:
  • Palm
  • Thumb (DNA channel formation)
  • Fingers (Recognition and binding of dNTP)
  • Exo (proofreading)
• What is the mechanism of DNA polymerase proofreading?

  • Initiated by incorrect nucleotide fit (incorporation)

  1. Distortion of DNA 3’-OH to Exo site
  2. Exo site removes the incorrect base
  3. Restoration of DNA to polymerase domain (synthetic mode)
• What is fractionation?

  Technique to separate components based on different physical properties