(The) novel DNA helicase hFBH1 is an F-box protein that forms an SCF complex with E3 ligase and helicase activities = F-box DNA helicase, hFBH1과 $SCF^{hFBH1}$ E3 ligase의 생화학적 특성연구

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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...
Advisors
Seo, Yeon-Sooresearcher서연수researcher
Description
한국과학기술원 : 생명과학과,
Publisher
한국과학기술원
Issue Date
2004
Identifier
237522/325007  / 020005069
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 생명과학과, 2004.2, [ xii, 119 p. ]

Keywords

helicase; F-box; SCF; ubiquitin; ubiquitin; helicase; F-box; SCF

URI
http://hdl.handle.net/10203/27556
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=237522&flag=dissertation
Appears in Collection
BS-Theses_Ph.D.(박사논문)
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