1. 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
  2. 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
  3. 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)
  4. 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
  5. 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
  6. Role of dNTP transfers
    dNTP binds to Polymerase DNA complex as: dNTP-Mg (Mg#1)
  7. 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
  8. 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
Card Set