DNA topoisomerase II is an essential enzyme in eukaryotes and is required for many aspects of DNA metabolism including DNA replication, recombination, chromosome segregation and chromosome condensation. It is also a major component of the nuclear scaffold. Topoisomerase II from lower eukaryotes has been shown to be phosphorylated in vivo and this phosphorylation leads to a modulation of activity. However, unlike these lower eukaryotes, human topoisomerase II exists as two closely related, but genetically distinct, isozymes which have markedly different expression and localization patterns. Topoisomerase IIα is a 170kDa protein and topoisomerase IIβ is 180kDa. This study set out to analyse the phosphorylation of these specific isozymes and understand how this leads to the regulation of their distinct biological functions. In order to undertake this study, two polyclonal anti-topoisomerase II antibodies were generated and a series of other polyclonal and monoclonal antibodies characterized. Furthermore, the α isozyme of human topoisomerase II was purified to near homogeneity from cultured HeLa cells. A kinase activity with the biochemical characteristics of casein kinase II co-purified with and could phosphorylate the purified topoisomerase Hot protein. The α and β isozymes of human topoisomerase II were both shown to be phosphoproteins in vivo. The α isozyme is phosphorylated predominantly on serine residues but with a minor proportion of phosphothreonine. Both the α isozyme and a stable ISOkDa fragment of the β isozyme are phosphorylated in vitro by casein kinase II and the catalytic subunit of PKA (cAMP-dependent protein kinase). The α isozyme can also be phosphorylated in vitro by Ca<sup>2+</sup>-dependent and -independent isozymes of protein kinase C and the cell cycle-regulated p34<sup>cdc2</sup> kinase. Two-dimensional tryptic phosphopeptide mapping suggested that the pattern of phosphorylation of human topoisomerase Ha protein in vivo is complex with phosphorylation occurring on multiple residues. Comparison with in vitro maps suggested that casein kinase II and PKA could account for most of the phosphorylation seen in vivo. Using a one- dimensional phosphopeptide mapping approach, a major site of phosphorylation in vivo appeared to be within the C-terminal 20kDa, and that casein kinase II, PKA and PKC may all phosphorylate this region. Phosphorylation of human topoisomerase Hoc protein by casein kinase II, PKA and PKC all led to a stimulation of activity as measured by plasmid relaxation and decatenation. In contrast, dephosphorylation led to a marked decrease in activity of the enzyme. The dephosphorylated enzyme could be reactivated by casein kinase II but not PKA phosphorylation. These data suggest that phosphorylation plays a crucial role in the control of DNA tertiary structure in human cells via regulation of the activity of topoisomerase II proteins.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:315756 |
| Date | January 1992 |
| Creators | Fry, Andrew Mark |
| Contributors | Hickson, Ian D. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:70d2dbb9-d3fe-43ed-8206-44a95202eeff |
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