Ubiquitin-family modifications of topoisomerase I in camptothecin-treated human breast cancer cells.
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.
Kanagasabai
Ragu
R
Department of Internal Medicine, Comprehensive Cancer Center, Mass Spectrometry and Proteomics Facility, and Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio 43210, USA.
Liu
Shujun
S
Salama
Samir
S
Yamasaki
Edith F
EF
Zhang
Liwen
L
Greenchurch
Kari B
KB
Snapka
Robert M
RM
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000201:enzymology* / Q000473:pathology; D002166:Camptothecin / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D011499:Protein Processing, Post-Translational; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism
D000972:Antineoplastic Agents, Phytogenic; D004791:Enzyme Inhibitors; D025841:Small Ubiquitin-Related Modifier Proteins; D025801:Ubiquitin; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin