Chapter 25 - DNA Replication Flashcards
What are the requirements for DNA polymerase?
(1) Template DNA
(2) Primer with free 3’-OH
(3) dNTPs
(4) Mg+
What is the DNA polymerase reaction?
dNTP + DNA –> DNA(n+1) + PPi
PPi –> 2Pi
(uses two ATP equivalents!)
5’–>3’ polymerase function
Polymerizes dNTP monomers into polymer
5’–>3’ exonuclease
Removes primer from DNA, removes damaged DNA
3’–>5’ exonuclease
Proofreading. Km increases for incorporation of the next base when there is a mismatch. It can be overcome by increasing [dNTP], but not possible in vivo. Random movement –> moves mismatch to exonuclease site –> mismatch is cut out.
Which DNA polymerase is the primary duplicating enzyme?
DNA pol III
DNA Polymerase I
(1) 5’ –> 3’ polymerase (polymerization)
(2) 5’–>3’ exonuclease (remove primer, remove damaged DNA)
(3) 3’–>5’ exonuclease (proofreading)
- Low processivity
- Slow, 800nucleotide/s
- Abundant, too many
DNA Polymerase III
(1) α2 (5’–>3’ polymerase)
(2) ε2 (3’–>5’ exonuclease)
(3) θ2 (increase efficiency)
(4) τ2 (dimerization, holds together)
(5) X (RNA primer –> DNA switch)
(6) β2 (ring clamp, wraps around DNA duplex and increases processivity)
Clamp loader (γ complex) wraps the β2 ring clamp around the DNA. (3γ, δ, δ’, (Ψ, X ) )
How is the DNA polymerase III β2 ring clamp clamped around the DNA?
Clamp loader + ATP –> conformational change that leads the clamp loader (γ complex) to bind to the β clamp and open it. It then binds to DNA, and once the DNA has been encircled, the bound ATP is hydrolyzed and the β ring closes.
What are enzymes/proteins involved in DNA replication?
(1) RNA primase – initiator at ORI of leading strands and at each Okazaki fragments for primer
(2) Single stranded binding proteins (SSBs)
(3) DNA helicase
(4) Topoisomerase
Compare RNA polymerases and DNA polymerases.
RNA polymerases don’t need a primer, whereas DNA polymerases do.
Single Stranded Binding Proteins (SSBs)
- “helix-destabilizing protein” (gp32)
- binds specifically to single-strand DNA on the backbone
- Stabilizes single-stranded DNA in order to keep template in an extended, single-strand conformation with bases exposed and ready for base-pairing with incoming nucleotides.
- Not only facilitates DNA denaturation, but also DNA renaturation.
- binding interactions are electrostatic
- cooperative binding
- protects from nucleases and unwanted interactions
RNA primase
RNA polymerase that creates the RNA primer for DNA replication. Required for initiator at ORI of leading strand and at each Okazaki fragments.
DNA helicase
Couple ATP hydrolysis to the disruption of π-π, hydrogen-bonding interactions. Helps “unwind” the helix.
DNA Polymerase I
(1) 5’ –> 3’ polymerase (polymerization)
(2) 5’–>3’ exonuclease (remove primer, remove damaged DNA)
(3) 3’–>5’ exonuclease (proofreading)
- Single polypeptide chain
- Low processivity
- Slow, 800nucleotide/s
- Abundant, too many
DNA Polymerase III
(1) α2 (5’–>3’ polymerase)
(2) ε2 (3’–>5’ exonuclease)
(3) θ2 (increase efficiency)
(4) τ2 (dimerization, holds together)
(5) X (RNA primer –> DNA switch)
(6) β2 (ring clamp, wraps around DNA duplex and increases processivity)
Clamp loader (γ complex) wraps the β2 ring clamp around the DNA. (3γ, δ, δ’, (Ψ, X ) )
How is the DNA polymerase III β2 ring clamp clamped around the DNA?
Clamp loader + ATP –> conformational change that leads the clamp loader (γ complex) to bind to the β clamp and open it. It then binds to DNA, and once the DNA has been encircled, the bound ATP is hydrolyzed and the β ring closes.
DNA helicase
Couple ATP hydrolysis to the disruption of π-π, hydrogen-bonding interactions. Helps “unwind” the helix.
