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Camptothecins kill mammalian cells by stabilizing topoisomerase I-DNA strand passing intermediates that are converted to lethal double strand DNA breaks in DNA replication fork collisions. Camptothecin-stabilized topoisomerase I-DNA cleavage intermediates in mammalian cells are uniquely modified by ubiquitin-family proteins. The structure, composition, and function of these ubiquitin-family modifications are poorly understood. We have used capillary liquid chromatography-nanospray tandem mass spectrometry to analyze the endogenous ubiquitin-family modifications of topoisomerase I purified from camptothecin-stabilized topoisomerase I-DNA cleavage complexes in human breast cancer cells. Peptides shared by SUMO-2 and SUMO-3 were abundant, and a peptide unique to SUMO-2 was identified. Ubiquitin was also identified in these complexes. No SUMO-1 peptide was detected in human topoisomerase I-DNA cleavage complexes. Identical experiments with purified SUMO paralogues showed that SUMO-1 was well digested by our protocol and that fragments were easily analyzed by LC-MS/MS. Spiking experiments with purified SUMO paralogues determined that we could detect as little as 0.5 SUMO-1 residue per topoisomerase I molecule. These results indicate that SUMO-1 is below this detection level and that SUMO-2 or a mixture of SUMO-2 and SUMO-3 predominates. SUMO-1 capping seems unlikely to be limiting the growth of SUMO-2/3 chains formed on camptothecin-stabilized topoisomerase I-DNA cleavage complexes.

Ubiquitin-Family Modifications of Topoisomerase I in Camptothecin-Treated Human Breast Cancer Cells