Impact of the C-terminal domain of topoisomerase IIalpha on the DNA cleavage activity of the human enzyme.
The enzymatic function of the C-terminal domain of eukaryotic topoisomerase II is not well defined. This region of the enzyme is highly variable and hydrophilic and contains nuclear localization signals and phosphorylation sites. In contrast to eukaryotic topoisomerase II, type II enzymes from chlorella virus completely lack the C-terminal domain. These viral enzymes are characterized by a robust DNA cleavage activity, high coordination between their two active site tyrosyl residues, and reduced sensitivity to anticancer drugs. As a first step toward characterizing the contribution of the C-terminal domain of human topoisomerase IIalpha to enzyme function, the protein was truncated at amino acid 1175, which corresponds to the C-terminal residue of Paramecium bursaria chlorella virus-1 topoisomerase II as determined by BLAST sequence alignment. Although the overall catalytic activity of the resulting enzyme, hTop2alphaDelta1175, was lower than that of full-length topoisomerase IIalpha, the mutant protein displayed a double-stranded DNA cleavage activity that was approximately 2-3-fold higher. While the DNA breaks created by hTop2alphaDelta1175 were primarily double stranded, cuts generated by topoisomerase IIalpha were primarily single stranded. Thus, the enhanced cleavage observed for hTop2alphaDelta1175 appears to be due, at least in part, to an increase in active site coordination. Finally, hTop2alphaDelta1175 displayed a distinctly lower susceptibility to anticancer agents than did topoisomerase IIalpha, despite the fact that it showed a similar binding affinity for etoposide. Therefore, the C-terminal domain of human topoisomerase IIalpha appears to play significant roles in modulating the DNA cleavage/ligation reaction of the enzyme and its response to anticancer agents.
Dickey
Jennifer S
JS
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Osheroff
Neil
N
D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D016831:Crithidia fasciculata; D004247:DNA / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D005047:Etoposide / Q000633:toxicity; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D005047:Etoposide; D004247:DNA; D004250:DNA Topoisomerases, Type II