DC Field | Value | Language |
---|---|---|
dc.contributor.advisor | Seo, Yeon-Soo | - |
dc.contributor.advisor | 서연수 | - |
dc.contributor.author | Kim, Jeong-Hoon | - |
dc.contributor.author | 김정훈 | - |
dc.date.accessioned | 2011-12-12T07:53:57Z | - |
dc.date.available | 2011-12-12T07:53:57Z | - |
dc.date.issued | 2004 | - |
dc.identifier.uri | http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=237522&flag=dissertation | - |
dc.identifier.uri | http://hdl.handle.net/10203/27556 | - |
dc.description | 학위논문(박사) - 한국과학기술원 : 생명과학과, 2004.2, [ xii, 119 p. ] | - |
dc.description.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... | eng |
dc.language | eng | - |
dc.publisher | 한국과학기술원 | - |
dc.subject | helicase | - |
dc.subject | F-box | - |
dc.subject | SCF | - |
dc.subject | ubiquitin | - |
dc.subject | ubiquitin | - |
dc.subject | helicase | - |
dc.subject | F-box | - |
dc.subject | SCF | - |
dc.title | (The) novel DNA helicase hFBH1 is an F-box protein that forms an SCF complex with E3 ligase and helicase activities | - |
dc.title.alternative | F-box DNA helicase, hFBH1과 $SCF^{hFBH1}$ E3 ligase의 생화학적 특성연구 | - |
dc.type | Thesis(Ph.D) | - |
dc.identifier.CNRN | 237522/325007 | - |
dc.description.department | 한국과학기술원 : 생명과학과, | - |
dc.identifier.uid | 020005069 | - |
dc.contributor.localauthor | Seo, Yeon-Soo | - |
dc.contributor.localauthor | 서연수 | - |
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