BIPARTITE NUCLEAR-LOCALIZATION SIGNALS IN THE C-TERMINUS OF HUMAN TOPOISOMERASE II-ALPHA

DNA topoisomerase II alpha is the intracellular target for several imp ortant chemotherapeutic agents, and drug-resistant human tumor cell li nes have been described in which deletions in the C-proximal region of this enzyme are associated with its cytoplasmic localization. We have identified multiple potential bipartite nuclear localization signal ( NLS) sequences in this region using a modified definition of the motif , and in the present study, we have expressed five of these as fusion proteins with beta-galactosidase. Only one sequence (spanning amino ac ids 1454 to 1497) was sufficient to cause strong nuclear localization. Subsequent mutation analyses indicated that this NLS sequence was bip artite and that both domains contain more than two basic amino acids. Substitution of the lysine residue at position 1492 in the second basi c domain with glutamine resulted in a fusion protein that localized in efficiently to the nucleus, indicating that all three basic residues i n this domain are necessary. Our results confirm that a broader defini tion is required to detect all potential bipartite NLS motifs in a pol ypeptide sequence, although functional tests are still essential for i dentification of those sequences actually capable of directing nuclear localization. (C) 1997 Academic Press.