Assembly of a polymeric chain of SUMO1 on human topoisomerase I in vitro.
Human (h) DNA topoisomerase I has been identified as a major SUMO1 target in camptothecin-treated cells. In response to TOP1-mediated DNA damage induced by camptothecin, multiple SUMO1 molecules are conjugated to the N-terminal domain of a single TOP1 molecule. To investigate the molecular mechanism of SUMO1 conjugation to TOP1, an in vitro system using purified SAE1/2, Ubc9, SUMO1, and TOP1 peptides was developed. Consistent with results from in vivo studies, multiple SUMO1 molecules were found to be conjugated to the N-terminal domain of a single TOP1 molecule. Systematic analysis has identified a single major SUMO1 conjugation site located between amino acid residues 110 and 125 that contains a single lysine residue at 117 (Lys-117). Using a short peptide spanning this region, we showed that a poly-SUMO1 chain was assembled in this peptide at Lys-117. Interestingly, a Ubc9-poly-SUMO1 intermediate had accumulated to a high level when the sumoylation assay was performed in the absence of hTOP1 substrate, suggesting a possibility that the poly-SUMO1 chain is formed on Ubc9 first and then transferred en bloc onto hTOP1. This is the first definitive demonstration of the assembly of a poly-SUMO1 chain on protein substrate. These results offer new insight into hTOP1 polysumoylation in response to TOP1-mediated DNA damage and may have general implications in protein polysumoylation.
Yang
Meiluen
M
Institute of Biochemistry and Molecular Biology, School of Life Science, National Yang Ming University, Taipei 112, Taiwan.
Hsu
Chia-Tse
CT
Ting
Chun-Yuan
CY
Liu
Leroy F
LF
Hwang
Jaulang
J
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D004264:DNA Topoisomerases, Type I / Q000737:chemistry*; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D008239:Lysine / Q000737:chemistry; D008954:Models, Biological; D008957:Models, Genetic; D010455:Peptides / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D025842:SUMO-1 Protein; D017422:Sequence Analysis, DNA; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry*; D013917:Thrombin / Q000737:chemistry; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D010455:Peptides; D011108:Polymers; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D004247:DNA; D044763:Ubiquitin-Conjugating Enzymes; D005982:Glutathione Transferase; D013917:Thrombin; D004264:DNA Topoisomerases, Type I; D000098843:Ubiquitin-Conjugating Enzyme UBC9; D008239:Lysine