The Novel Human DNA Helicase hFBH1 Is an F-box Protein

Jaehoon Kim,Jeong-Hoon Kim,Sung-Hak Lee,Do-Hyung Kim,Ho-Young Kang,Sung-Ho Bae,Zhen-Qiang Pan,Yeon-Soo Seo

doi:10.1074/jbc.m201612200

Abstract

We have identified a novel DNA helicase in humans that belongs to members of the superfamily I helicase and found that it contains a well conserved F-box motif at its N terminus. We have named the enzyme hFBH1 (human F-box DNA helicase 1). Recombinant hFBH1, containing glutathione S-transferase at the N terminus, was expressed in Sf9 cells and purified. In this report, we show that hFBH1 exhibited DNA-dependent ATPase and DNA unwinding activities that displace 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 and ROC1. In addition, the SCF complex containing hFBH1 as an F-box protein displayed ubiquitin ligase activity. We demonstrate that hFBH1 is the first F-box protein that possesses intrinsic enzyme activity. The potential role of the F-box motif and the helicase activity of the enzyme are discussed with regard to regulation of DNA metabolism.

Highlights

DNA helicases are ubiquitous enzymes that play essential roles in various DNA transactions involved in replication, repair, and recombination [1, 2] and have been implicated in a number of human genetic disorders [3, 4]
It was noted that all three homologs included a well conserved F-box motif (Fig. 1B) that is required to form a complex with SKP1, a key component of the SCF complexes involved in ubiquitin-dependent proteolysis [16]
The SCF Complex Containing hFBH1 Has Ubiquitin Ligase Activity—Because we confirmed that hFBH1 formed a complex with all three other subunits (SKP1, CUL1, and ROC1) for an SCF complex, we examined whether the SCF complex containing hFBH1 as an F-box protein (SCFhFBH1) possesses ubiquitin ligase activity as is expected for an SCF complex

Summary

Introduction

DNA helicases are ubiquitous enzymes that play essential roles in various DNA transactions involved in replication, repair, and recombination [1, 2] and have been implicated in a number of human genetic disorders [3, 4]. Analysis of the genome of Saccharomyces cerevisiae indicates that there are 134 open reading frames containing with helicase-like features that represent ϳ2% of its genome [7] The presence of such a large number of helicases or helicase-like proteins in cells, many of unknown function in vivo, most likely reflects a complexity of nucleic acid metabolic reactions and the distinct structural template requirements for a given helicase. The chromatin-remodeling factor SWI2/SNF2 containing these motifs lacks helicase activity, its ability to hydrolyze ATP is stimulated by DNA [10, 11], suggesting that ATP hydrolysis provides the energy required to alter protein-DNA structure rather than duplex DNA or RNA structure This suggests that proteins with helicase motifs may have functions that do not involve unwinding of nucleic acids. The potential biological role of this interesting human DNA helicase will be discussed

Methods

Results

Conclusion