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Polymerase structure
- all polymerases exhibit the following:
- 1. 2 ALPHA helices acting as THUMB and FINGERS
- 2. Beta sheet acts as PALM (holds DNA)
- 3. PALM domain has 3 Asp residues that coordinate Mg ions and Catalyze polymerase reaction
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Mechanism of POL I
- 1. DNA rearrangement, DNA template strand moves to PALM and
- 2. DNA held in place by conformational change in THUMB and movement of finger
- 3. NeW dNTP moves in by binding to Mg ion (1st Mg) and FINGER region
- 4. CLosing of fINGERS and BINDING of Aspartate
- 5. Aspartate binds to 2nd Mg ion
- 6. Catalysis of reaction removing pyrophosphate and ATTACHMENT to the growing DNA
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Role of Aspartate 882
- Serves as ANCHOR during FINGER-closing transition
- does this by bridging the ACTIVE site - linking 3'-OH and dNTP-Mg. Specifically interacts with Mg (metal B)
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Role of Asp705
- 1. Enhances Catalysis, AFTER cLOSED complex forms
- In this closed ternary complex structure, Asp rotates and binds to 1st Mg (metal B)
- 2. Binding of ligand to 2nd Mg (metal A) after FINGER closed conformation, in prep. for Phosphoryl transfer
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Role of Mg ions
- First Mg necessary for binding to dNTPs, needed in order to bind to Pol-DNA comlpex
- 2nd Mg required for COMPLETION of catalysis. Appears to not be needed until AFTER FInger closing step
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Role of dNTP transfers
dNTP binds to Polymerase DNA complex as: dNTP-Mg (Mg#1)
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Role of Eukaryotic polymerase Alpha, delta and epsilon
- Alpha: Involved in initiation, by making RNA primer and adding dNTPs to primer
- Delta: can sense RNA primer and create flap for removal
- Epsilon: pol epsilon tail may act as a built-in DNA clamp, as it mediates DNA interactions. Also can play a role in BLOCKING cells from entering cell cycle
- Delta and Epsilon: Needed for elongation, they replace pol alpha binding with clamps
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Role of FEN1
- exonuclease which REMOVES primer
- Removes primer by creating BEND in DNA allowing 5' flap to insert into active site
- 3' overhang remains in hydrophobic pocket, which later bonds with 5' phosphate with help from DNA ligase
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