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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
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Clamp loader
- Need ATP binding to trigger the arrangement of AAA+ ATPase modules
- This ATP binding to AAA+ triggers opening of clamp
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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
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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
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