Reconstitution and Characterization of SCFHFBH1 That Can Act Helicase and E3 Ubiquitin Ligase

초록

A human F-box DNA helicase named hFBH interacts with SKP1 to form an SCF complex (SCFhFBH1) together with CUL1 and ROC1 in an F-box dependent manner (Kim, J., Kim, J. H., Lee, S. H., Kim, D. H., Kang, H. Y., Bae, S. H., Pan, Z. Q., and Seo, Y. S. (2002) J. Biol. Chem. 277, 24530-24537). THe SCFhFBH1 complex immunoprecipitated from cell extracts catalyzed polyubiquitin formation in the presence of E1 and E2, ubiquitin-activating enzyme, respectively. In this report, we assembled in vivo and in vitro and purified the SCFhFBH1 complex from insect cells expressing hFBH1, SKP1, CUL1, and ROC1. We examined helicase and ATPase activities of the complex in comparison to hFBH1. The complex was isolated as a single tight complex and retained helicase and ATPase activities nearly identical to hFBH1 alone. In addition, it contained ubiquitin ligase activity, which was hardly affected by single-stranded or double stranded DNA. This suggests that the SCFhFBH1 can catalyze ubiquitination while it acts as DNA helicase or translocates along DNA. The association of hFBH1 to preassembled SKP1/CUL1/ROC1 complex was extremely poor at 4oC, but efficient when the mixture was incubated at 37 oC. Concurrently, it enhanced ubiquitin chain formation. Therefore, the hFBH1 protein in the complex plays several roles critical for the proper function of the complex. We discuss the potential role of the SCFhFBH1 complex in DNA metabolism based upon the enzymatic activities associated with this complex.