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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
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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
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________ is large (very) multi-subunit complex
DNA Pol III
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Core Enzyme
3 subunits (α,ε,θ)active polymerase Non-processive160,000 m.w.
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Holo Enzyme
Core dimer7 addnl subunits (β,τ,γ,δ,δ')Highly processive 925,000 m.w.
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β clamp:
dimer of β-subunits forms a ring (clamp)
-
Processivity clamp is ______
universal mechanism
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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)
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Is replication unidirectional or bidirectional
- bidirectional
- unidirectional in some prokaryotes
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Enzymological & Topological Mechanism (E. coli)
Steps:
1)Initiation:
- assembly of replication apparatus at origin
- Initiation occurs at Ori C
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Enzymological & Topological Mechanism (E. coli)
Steps:
2.) Elongation
coordinate synthesis of leading & lagging strands
-
Enzymological & Topological Mechanism (E. coli)
Steps:
Termination:
completion of synthesis; separation of daughter chromosomes
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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
-
Unwinding by DNA _______
helicases
-
SSB
Single-stranded DNA binding proteins
-
DNA winds around the _______________
bacterial SSB homotetramer
-
Complete E. coli replication initiation cycle
-
Elongation
- a)Schematic of replication forks at origin
- b) Lagging strand synthesis
- c) Trombone model
-
Trombone model
- i.Coordination of leading & lagging strand synthesis
- ii.Clamp loading
- iii.Incorporating Okazaki fragments
-
Termination
- a)Elongation blocked by tus binding to ter sites
- b)separate catenated daughter chromosomes
-
Replication of E. coli chromsome
- 1.Coordination of replication forks - the replisome
- 2.Coordination of replication & division
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