replisome structure and clamp loader.txt

  1. replisome structure components
    • DNA helicase: unwinds DNA
    • Primase: makes short RNA primers to initiate DNA synthesis
    • DNA Polymerase: involved in leading and lagging strand synthesis
    • ssDNA binding proteins: lagging strand protects ssDNA from nucleases and inhibits 2ndary structure
    • Circular sliding clamps and clamp loading complex: proteins encircle duplex and tether polymerase to DNA for high processivity and replication rate
  2. Clamp loader
    • Need ATP binding to trigger the arrangement of AAA+ ATPase modules
    • This ATP binding to AAA+ triggers opening of clamp
  3. Clamp loading mechanism
    • Once clamp is opened (via ATP binding to AAA+)…
    • 1. the Primer Template DNA threads through the gaps
    • ssDNA passes through domain 2 and 5 of clamp. duplex DNA (dsDNA) located inside clamp loader
    • 2. ATP hydrolysis activated through switch residue. Switch (Lys 80 of gp44) releases backbone of Walker B glutamate & NOW BINDS to DNA phosphate backbone
    • 3. ATP hydrolysis at B subunit, breaks interface at AAA+ of B & C: allows closure of clamp around primer-template DNA
    • 4. ATP Hydrolysis of C & D, dissolve AAA+ modules: clamp loader ejected & clamp left loaded onto DNA
  4. Lagging strand trombone cycle
    • Mediated by CLAMP loader and sliding clamps
    • 1. lagging strand DNA loop made via Pol III core-B as it extends Okazaki fragment and through fork progression producing ssDNA.
    • 2. DnaG primase synthesizes new RNA primer
    • 3. clamp loader loads new Beta clamp on RNA primer
    • 4. when Lagging strand synth. complete, lagging Pol III core disengages from its clamp
    • 5. this Pol III now associates w/NEW beta clamp (via clamp loader) at upstream primer to start new Okazaki synthesis
Card Set
replisome structure and clamp loader.txt