Proteomic analysis of complexes formed by human topoisomerase I.
Human topoisomerase I is a nuclear enzyme that catalyses DNA relaxation and phosphorylation of SR proteins. Topoisomerase I participates in several protein-protein interactions. We performed a proteomic analysis of protein partners of topoisomerase I. Two methods were applied to proteins of the nuclear extract of HeLa cells: a co-immunoprecipitation and an affinity chromatography combined with mass spectrometry. Complexes formed by topoisomerase I with its protein partners were immunoprecipitated by scleroderma anti-topoisomerase I antibodies. To identify binding sites for the protein partners, baits corresponding to fragments of topoisomerase I were constructed and used in the affinity chromatography. The N-terminal domain and the cap region of the core domain appeared to be the main regions that bound proteins. We identified 36 nuclear proteins that were associated with topoisomerase I. The proteins were mainly involved in RNA metabolism. We found 29 new and confirmed 7 previously identified protein partners of topoisomerase I. More than 40% proteins that associate with the cap region contain two closely spaced RRM domains. Docking calculations identified the RRM domains as a possible site for the interaction of these proteins with the cap region.
Czubaty
Alicja
A
Institute of Biochemistry, Warsaw University, ul. Miecznikowa 1, 02-096 Warszawa, Poland.
Girstun
Agnieszka
A
Kowalska-Loth
Barbara
B
Trzcińska
Agata M
AM
Purta
Elzbieta
E
Winczura
Alicja
A
Grajkowski
Wojciech
W
Staroń
Krzysztof
K
D004264:DNA Topoisomerases, Type I / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D009687:Nuclear Proteins / Q000378:metabolism*; D011487:Protein Conformation; D040901:Proteomics; D012313:RNA / Q000378:metabolism; D012261:Ribonucleoproteins / Q000032:analysis / Q000378:metabolism
D009687:Nuclear Proteins; D012261:Ribonucleoproteins; D012313:RNA; D004264:DNA Topoisomerases, Type I