Bio 99 Midterm 1 Lec. 5

  1. Arguments why Pol I could not be the replicating polymerase
    • 1) slow rate: 20 bp/sec
    • 2) not highly PROCESSIVE
    • 3) Pol A1 mutant: DeLucia & Cairns (1969)
    • a) 1% of wildtype Pol I activity: no effect on growth
    • b)sensitive to UV & X-irradiation
  2. DNA Pol III: Thomas Kornberg & Martin Geffeter (1970)
    1) Featuresa)5’-3’ polymerase activityi.rate ca. 1,000/secii.highly processiveb) 3’-5’ exonuclease activity (proofreading)c) no 5’-3’ exonuclease activity
  3. ________ is large (very) multi-subunit complex
    DNA Pol III
  4. Core Enzyme
    3 subunits (α,ε,θ)active polymerase Non-processive160,000 m.w.
  5. Holo Enzyme
    Core dimer7 addnl subunits (β,τ,γ,δ,δ')Highly processive 925,000 m.w.
  6. β clamp:
    dimer of β-subunits forms a ring (clamp)
  7. Processivity clamp is ______
    universal mechanism
  8. Topological considerations:
    • 1.Where and how does replication start?
    • a.Two striking features of ALL polymerases:
    • i.Synthesis is 5’-3’ (ONLY)
    • ii.Requires 3’-OH as primer
    • b.Cairns or Theta replication in E.coli
    • (circular DNA: no free ends)
  9. Is replication unidirectional or bidirectional
    • bidirectional
    • unidirectional in some prokaryotes
  10. Enzymological & Topological Mechanism (E. coli)
    Steps:
    1)Initiation:
    • assembly of replication apparatus at origin
    • Initiation occurs at Ori C
  11. Enzymological & Topological Mechanism (E. coli)
    Steps:
    2.) Elongation
    coordinate synthesis of leading & lagging strands
  12. Enzymological & Topological Mechanism (E. coli)
    Steps:
    Termination:
    completion of synthesis; separation of daughter chromosomes
  13. Model for initiation of replication at the E. Coli origin
    (4 steps)
    • 1.DnaA dimers, ea with a bound ATP, bind at the four 9 bp repeats. DNA wraps around this complex.
    • 2.The three A-T rich 13 bp repeats are denatured sequentially (supercoiling and HU)
    • 3.Hexameric DnaB protein (helicase) bind to each strand, with the aid of DnaC. DnaB helicase begins to unwind the DNA in preparation for priming and DNA synthesis.
    • 4.Next: SSB and gyrase promote thousands of bp to separate
  14. Unwinding by DNA _______
    helicases
  15. SSB
    Single-stranded DNA binding proteins
  16. DNA winds around the _______________
    bacterial SSB homotetramer
  17. Complete E. coli replication initiation cycle
    Image Upload 1
  18. Elongation
    • a)Schematic of replication forks at origin
    • b) Lagging strand synthesis
    • c) Trombone model
  19. Trombone model
    • i.Coordination of leading & lagging strand synthesis
    • ii.Clamp loading
    • iii.Incorporating Okazaki fragments
  20. Termination
    • a)Elongation blocked by tus binding to ter sites
    • b)separate catenated daughter chromosomes
  21. Replication of E. coli chromsome
    • 1.Coordination of replication forks - the replisome
    • 2.Coordination of replication & division
Author
jocelyn0399
ID
346776
Card Set
Bio 99 Midterm 1 Lec. 5
Description
bio99
Updated