Rogojina AT and Nitiss JL (2008) Isolation and Characterization of mAMSA-hypersensitive Mutants: CYTOTOXICITY OF Top2 COVALENT COMPLEXES CONTAINING DNA SINGLE STRAND BREAKS. J Biol Chem 283(43):29239-50
Abstract: Topoisomerase II (Top2) is the primary target for active anti-cancer agents. We developed an efficient approach for identifying hypersensitive Top2 mutants and isolated a panel of mutants in yeast Top2 conferring hypersensitivity to the intercalator mAMSA. Some mutants conferred hypersensitivity to etoposide as well as mAMSA, while other mutants exhibited hypersensitivity only to mAMSA. Two mutants in Top2, changing Pro473 to Leu and Gly737 to Val, conferred extraordinary hypersensitivity to mAMSA, and were chosen for further characterization. The mutant proteins were purified, and their biochemical activities were assessed. Both mutants encode enzymes that are hypersensitive to inhibition by mAMSA and other intercalating agents and exhibited elevated levels of mAMSA induced Top2:DNA covalent complexes. While Gly737Val Top2p generated elevated levels of Top2 mediated double strand breaks in vitro, the Pro473Leu mutant protein showed only a modest increase in Top2 mediated double strand breaks, but much higher levels of Top2 mediated single strand breaks. In addition, the Pro473Leu mutant protein also generated high levels of mAMSA-stabilized covalent complexes in the absence of ATP. We tested the role of single strand cleavage in cell killing with alleles of Top2 that could generate single strand breaks, but not double strand breaks. Expression in yeast of a Pro473Leu mutant that could only generate single strand breaks conferred hypersensitivity to mAMSA. These results indicate that generation of single strand breaks by Top2 targeting agents can be an important component of cell killing by Top2 targeting drugs.
|Status: Published||Type: Journal Article||PubMed ID: 18723844|
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