Covalent modification of the Werner's syndrome gene product with the ubiquitin-related protein, SUMO-1

Werner's syndrome is a potential model of accelerated human aging. The gene responsible for Werner's syndrome encodes a protein that has a helicase do main homologous to Escherichia coli RecQ. To identify binding partners that regulate the function in concert with Wrn, we screened for proteins using the yeast two-hybrid system with mouse Wrn as bait and found three. One was a novel protein, and the other two were mouse Ubc9 and SUMO-1. Ubc9 also i nteracted with the mouse homologue of the Bloom's syndrome gene product, an other eukaryotic RecQ-type helicase, but not mouse DNA helicase Q1/RecQL (R ecQL1). Deletion experiments indicated that both proteins interacted with t he N-terminal segment of Wrn (amino acid 272-514). The interaction between Wrn and SUMO-1 was weaker than that between Wrn and Ubc9. Positive interact ion was observed in the heterogeneous combination of Wrn and yeast Ubc9 (yU bc9), as well as yUbc9 and SUMO-1, in the two-hybrid system. The interactio n between yUbc9 and SUMO-1 was abolished by deleting the C-terminal Gly res idue of SUMO-1, which is reportedly required for the formation of Ubc9-SUMO -1 thioester linkage. The interaction of Wrn and SUMO-1 was also abolished by deleting the Gly residue, indicating that the interaction of Wrn and SUM O-1 is mediated by yUbc9 in the two-hybrid system. Finally, we confirmed by immunoblotting with an anti-SUMO-l antibody that Wrn was covalently attach ed with SUMO-1.