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RNA Polymerase
ssDNA Template
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DNA Polymerase
- dsDNA Template
- Can Only extend
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Primase
- E.coli enzyme
- Builds RNA primer from scratch
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Simple overview of DNA polymerase mechanism
- DNA polymerases uses Mg2+ to attach a dNTP to the 3' end of the nascent strand
- A pyrophosphate is released
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Phosphodiester bond
- Links the 5' phosphate to the 3' O
- Formed by a condensation reaction
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Who isolated RNA Pol I
Arthur Kornberg
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Who isolated RNA Pol III
Thomas Kornberg - Also discovered Pol II
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RNA Pol I
- Most Abundant
- Relatively minor role
- Primer removal, DNA repair
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RNA Pol III
- Regarded as the most active
- Chromosome replication
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DNA Pol IV, V uses
SOS Repair
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All three DNA polymerases can elongate DNA? (T/F)
T - But they can't all initiate it
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Exonuclease Activity
- Proofreading
- Direction, 3'->5'
- DNA Pol I, II and III
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5' -> 3' Exonuclease activity
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Fidelity
- How accurate a polymerase is
- Includes low misincorporation (errors) and proofreading
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Processivity
The number of nucleotides a polymerase can churn out before dropping off
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Highest fidelity polymerase
DNA Pol III
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Highest and lowest processivity polymerase
- Pol III - 500k
- Pol I - 200
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Effect of proofreading on fidelity
Can increase fidelity by two orders of magnitude
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Names of the DNA Pol III subunits
- Primer Breakdown
- Replace gap from the primer with DNA
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Rate of DNA Pol III
1000 nt/sec
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Subunits of RNA Pol III found in triplet
α, ε, θ
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Subunits of the DNA Pol III γ complex
γ, δ, δ', χ, ψ
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Other subunits of DNA Pol III
β, τ
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DNA Pol III holoenzyme
- Composed of two enzymes
- DNA Pol III
- Sliding clamp loader
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Role of the τ proteins in DNA Pol III
Hold the DNA Pol III core proteins together
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Replisome
DNA Pol III Holoenzyme + Helicase + Primase
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Lagging strand synthesis uses...
The second arm of DNA Pol III
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Eukaryotic version of the sliding clamp
Proliferating cell nuclear antigen (PCNA)
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Primosome
Heliacase + Primase at replication fork
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How long are okazaki fragments?
1000nt - 2000nt
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DNA ligase
Fixes the gap between okazaki fragments
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DNA ligase mechanism
- A lys (NH2) on the catalytic site binds AMP (ATP -> AMP, PPi released)
- The ligase formas a phosphoanhydride bond with the 5' phosphate adjacent to the nick
- The 3' OH then attacks the phosphoanhydride
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The DNA moves, but the replisome is stationary. (T/F)
True
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Tus proteins
- Terminator utillization substance
- Stop the replication fork moving
- Only act in one direction
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