Topoisomerase

Topoisomerase

1. Immunodepletion and antibody blocking showed topo II activity required for chr. assembly and condensation

   • once condensation completed, topo II no longer required - blocking had no effect
     • Topo II molecules easily extracted from chr. under MILD conditions
       • Morphology of chr. well preserved
       • mAb, MPM-2 (recognizes mitosis-specific phosphoproteins) resistant to the same treatment
     • Localization: Immunofluorescence showed topo II uniformly distributed throughout condensed chr. NOT just axis
   • Chr. organization and axial structures are maintained after topo II extraction
     • => topo II doesn't play a scaffolding role in the structural maintenance of mitotic chromosomes
       • chr. scaffold = framework structure of nonhistone proteins that organize chromatin loops in mitotic chromosomes

2. Topo II = enzyme that catalyzes strand passing of double stranded DNA

   • Type II topoisomerases cut both strands of the DNA helix simultaneously in order to manage DNA tangles and supercoils. They use the hydrolysis of ATP.
     • Topo II required for the individualization of chromosomes in vertebrates, and for disentangling replicated sister chromosomes in all oganisms
   • All eukaryotic cells have at least one type II topoisomerase
     • Vertebrates have 2 isoforms: topo IIa and topo IIb, with distinct localization patterns in vivo
       • During mitosis, topo IIa is enriched on chromosomes, whereas topo IIb is mostly cytoplasmic

3. Topo I

   • 13S condensin (I) is able to introduce +ve supercoils into a closed circular DNA with the help of bacterial/eukaryotic topo I
     • reaction is ATP dependent

4. supercoiling

   • Topo II
     • Right-handed knots into circular nicked plasmids