Abstract
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.
Highlights
Human SUMO1 is a ubiquitin-like protein [1,2,3]
The high molecular weight species of hTOP1 were confirmed to be SUMO1-conjugated hTOP1 with antihTOP1 and anti-SUMO1 antibodies. These results indicate that the in vitro system is robust for the formation of hTOP1SUMO1 conjugates
The high molecular weight hUbc9-SUMO1 conjugates were almost undetectable, whereas the polymeric SUMO1 chains containing dimer and trimer of SUMO1 were observed when samples were analyzed under reducing conditions (Fig. 5) The residual lower molecular weight conjugates observed in Fig. 5A could be due to the formation of SUMO1-hUbc9 isopeptide bonds, whereas the residual low molecular weight species observed in Fig. 5B could be the SUMO1 dimer. These results suggest that hUbc9 forms a thioester bond with a polymeric SUMO1 chain(s) and support the notion that Ubc9 may transfer a polymeric chain to hTOP1
Summary
Human SUMO1 (small ubiquitin-related modifier) ( named UBL1, PIC1, GMP1, SMT3C, and sentrin in the literature) is a ubiquitin-like protein [1,2,3]. Proteases that activate SUMO1/Smt3p precursors and cleave SUMO1/Smt3p from their protein conjugates have been identified in yeast and mammalian cells [15,16,17,18] Many proteins such as RanGAP1 [19], promyelocytic leukemia protein [20], IB␣ [21], RAD51, RAD52 [22, 23], p53 [24], and centromere proteins [25, 26], which have diverse functions, have been shown to interact with Ubc9/SUMO1 or be covalently modified by SUMO1. Systematic mutational analysis of the SUMO consensus sequences on human TOP1 has identified Lys-117 at the N-terminal domain to be the major site for sumoylation [41] It is unclear whether multiple SUMO1 molecules are conjugated to Lys-117. Our results suggest that a poly-SUMO1 chain is formed at Lys-117 of human TOP1
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