(The) novel DNA helicase hFBH1 is an F-box protein that forms an SCF complex with E3 ligase and helicase activities

Abstract

We identified a novel helicase in humans that belongs to members of superfamily I helicase, and found that it contains a well-conserved F-box motif at its N-terminus, and named the enzyme hFBH1 (human F-box DNA Helicase 1). A recombinant hFBH1 containing glutathione-S transferase at the N-terminus was expressed in Sf9 cells and purified. We show that the hFBH1 exhibited DNA-dependent ATPase and DNA unwinding activities that unwinds duplex DNA in the 3’ to 5’ direction. The hFBH1 enzyme interacted with human SKP1 and formed an SCF (SKP1/Cullin/F-box) complex together with human Cullin. The $SCF^{hFBH1}$ complex, immunoprecipitated from cell extracts, catalyzed polyubiquitin formation in the presence of the ubiquitin-activating and ubiquitin-conjugating enzymes, E1 and E2, respectively. We also describe the purification and enzymatic properties of the recombinant $SCF^{hFBH1}$ complex from insect cells expressing hFBH1, SKP1, CUL1, and ROC1. The $SCF^{hFBH1}$ complex was purified as a single tight complex that retained DNA helicase, DNA-dependent ATPase, and ubiquitin ligase activities. The helicase and ATPase activities residing in the $SCF^{hFBH1}$ complex were indistinguishable from those of the protein hFBH1 alone. Moreover, the ubiquitin ligase activity of the $SCF^{hFBH1}$ complex was hardly affected by single-stranded or double-stranded DNA. In addition taking advantage of biochemical fractionation, we found out UbcH5a and UbcH5c as stimulating factors of ubiquitin ligase activity of $SCF^{hFBH1}$ complex. This stimulation was dependent on cdc34 and required ubiquitin available 48 lysine residue. We demonstrate that hFBH1 is the first F-box protein that has its own intrinsic enzyme activity. The multiple activities present in this complex act independently of each other, suggesting that the $SCF^{hFBH1}$ complex can catalyze a ubiquitination reaction while it acting as a DNA helicase or translocating along DNA. The potential role of the $SCF^{hFBH1}$ compl...