Topoisomerase

Topoisomerase

Immunodepletion and antibody blocking showed topo II activity required for chr. assembly and condensation
1. once condensation completed, topo II no longer required - blocking had no effect
  1. Topo II molecules easily extracted from chr. under MILD conditions
    1. Morphology of chr. well preserved
    2. mAb, MPM-2 (recognizes mitosis-specific phosphoproteins) resistant to the same treatment
  2. Localization: Immunofluorescence showed topo II uniformly distributed throughout condensed chr. NOT just axis
2. Chr. organization and axial structures are maintained after topo II extraction
  1. => topo II doesn't play a scaffolding role in the structural maintenance of mitotic chromosomes
    1. chr. scaffold = framework structure of nonhistone proteins that organize chromatin loops in mitotic chromosomes
Topo II = enzyme that catalyzes strand passing of double stranded DNA
1. 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.
  1. Topo II required for the individualization of chromosomes in vertebrates, and for disentangling replicated sister chromosomes in all oganisms
2. All eukaryotic cells have at least one type II topoisomerase
  1. Vertebrates have 2 isoforms: topo IIa and topo IIb, with distinct localization patterns in vivo
    1. During mitosis, topo IIa is enriched on chromosomes, whereas topo IIb is mostly cytoplasmic
Topo I
1. 13S condensin (I) is able to introduce +ve supercoils into a closed circular DNA with the help of bacterial/eukaryotic topo I
  1. reaction is ATP dependent
supercoiling
1. Topo II
  1. Right-handed knots into circular nicked plasmids

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