The ability of topoisomerase II to mediate a number of DNA rearrangements was examined at the molecular level. For this purpose a new type of defined donor and acceptor substrate have been developed, ...and used for studies of the intramolecular and intermolecular DNA ligation reactions of topoisomerase II. Intramolecular ligation occurred only to single-stranded acceptor molecules with the ability to base-pair to the donor substrate, while the intermolecular ligation reaction displayed a strong preference for double-stranded acceptor molecules with a short four base, single-stranded region. The efficiency of the intermolecular ligation reaction was highly dependent on base-pairing between the acceptor molecule and the DNA donor cleaved by topoisomerase II. Thus, acceptor molecules containing a blunt end or a four base 5' overhang without base-pairing ability ligated with an approximately eightfold reduced efficiency, as compared with the base-pairing control. Experiments demonstrated that the enzyme can ligate DNA molecules, where nucleotides were either removed or inserted in the employed acceptor molecules. The results indicate that topoisomerase II might be responsible for similar rearrangements in vivo, since gapped and nicked DNA structures appear as intermediates in processes such as replication and repair. The reaction is, however, likely to be constrained by the requirement of base-pairing for ligation.
The effect of the 2-nitroimidazole Ro 15-0216 upon the interaction between purified topoisomerase II and its DNA substrate was investigated. The cleavage reaction in the presence of this ...DNA-nonintercalative drug took place with the hallmarks of a regular topoisomerase II mediated cleavage reaction, including covalent linkage of the enzyme to the cleaved DNA. In the presence of Ro 15-0216, topoisomerase II mediated cleavage was extensively stimulated at major cleavage sites of which only one existed in the 4363 base pair pBR322 molecule. The sites stimulated by Ro 15-0216 shared a pronounced sequence homology, indicating that a specific nucleotide sequence is crucial for the action of this drug. The effect of Ro 15-0216 thus differs from that of the clinically important topoisomerase II targeted agents such as mAMSA, VM26, and VP16, which enhance enzyme-mediated cleavage at a multiple number of sites. In contrast to the previous described drugs, Ro 15-0216 did not exert any inhibitory effect on the enzyme's catalytic activity. This observation might be ascribed to the low stability of the cleavage complexes formed in the presence of Ro 15-0216 as compared to the stability of the ones formed in the presence of traditional topoisomerase II targeted drugs.
Anion‐exchange chromatography of partially purified human HL‐60 topoisomerase II resolves the known α (170 kDa) and β (180 kDa) isoenzymes at 150 mM NaCl and 230 mM NaCl, respectively. An additional ...topoisomerase II fraction was eluted by > 300 mM NaCl. It could be identified by Western blotting as a late‐eluting variant of topoisomerase IIα, which is functionally altered as compared to the early‐eluting form, having the following properties: a shift in the catalytic optimum to pH 9; increased stability in DNA complex formation; approximately 100‐fold resistance to orthovanadate; approximately 1000‐fold resistance to the cytostatic substances N‐4‐(9‐acridinylamino)‐3‐methoxyphenyl‐methanesulphonamide (amsacrine) and the podophyllotoxin etoposide (VP 16). 80% of the late‐eluting topoisomerase IIα could be captured by SDS on calf thymus DNA without further enhancement by drugs. In contrast, the early‐eluting topoisomerase IIα exhibits 10% complex formation with SDS alone, and an increase to 90% complex formation in the presence of drugs. A HL‐60 subline (HL‐60/R), approximately 1000‐fold resistant to etoposide and amsacrine, has equivalent proportions of topoisomerase IIα and topoisomerase IIβ and similar levels of both isoenzymes, as compared to the drug‐sensitive HL‐60/WT cells. However, determination of the cellular levels of the early‐eluting and late‐eluting forms of topoisomerase IIα revealed that the HL‐60/R cell line contains approximately 80% of the late‐eluting topoisomerase IIα, whereas the sensitive HL‐60/WT cell line contains only 15–20% of this form. The nuclear distribution of the two forms also differs. Sensitive HL‐60/WT cells show a diffuse nuclear distribution but in resistant cells the distribution is localized in the nucleoli. Apparently two functionally distinct subforms of topoisomerase IIα coexist in drug‐sensitive and drug‐resistant HL‐60 cells and changes in their relative levels affect the cellular sensitivity to topoisomerase‐II‐targeting drugs.
We investigate the efficacy of inducing reproductive death of cancer cells by ultraviolet, visible and near infrared femtosecond laser pulses. The reproductive cell death may result from (numerous) ...single photon absorption events or from multi-photon absorption processes. The high intensity of femtosecond laser pulses facilitate multi-photon processes, which potentially may arrest the reproduction of cells at wavelengths that otherwise are harmless.
We have found that deletion of a 70-amino acid domain, spanning from position 141 to 210 in the N-terminal part of human topoisomerase I, has no effect on the catalytic activity of the enzyme in ...vitro but suppresses the lethal consequence of overexpressing human topoisomerase I in a rad52 top1 Saccharomyces cerevisiae strain. By immunostaining, the 70-amino acid domain is shown to be necessary for nuclear location of topoisomerase I. We demonstrate that the nuclear localization signal from the SV40 large T antigen can substitute for the 70-amino acid domain, restoring both the lethal effect of overexpression and the correct subcellular localization of topoisomerase I. Thus, we have identified a domain in the N-terminal part of human topoisomerase I, nonessential for catalytic activity in vitro but serving an in vivo function by directing the enzyme to the nucleus. Based on sequence comparisons, we suggest that this domain is a conserved element in the apparently non-homologous N-terminal parts of yeast and human topoisomerase I.
PDF