SCFhFBH1 can act as helicase and E3 ubiquitin ligase.
In our previous study, we found that a human F-box DNA helicase, named hFBH1, interacted with SKP1 to form an SCF (SKP1-Cul1-F-box protein) complex together with CUL1 and ROC1 in an F-box-dependent manner. The complex immunoprecipitated from crude cell extracts catalyzed polyubiquitin formation in the presence of the ubiquitin-activating and ubiquitin-conjugating enzymes, E1 and E2, respectively. In this report, we characterized the enzymatic properties of the recombinant SCF(hFBH1) complex purified from insect cells expressing hFBH1, SKP1, CUL1 and ROC1. The SCF(hFBH1) complex was isolated as a single tight complex that retained DNA helicase, DNA-dependent ATPase and E3 ubiquitin ligase activities. The helicase and ATPase activities residing in the SCF(hFBH1) complex were indistinguishable from those of the hFBH1 protein alone. Moreover, the ubiquitin ligase activity of the SCF(hFBH1) complex was hardly affected by single-stranded or double-stranded DNA. The multiple activities present in this complex act independently of each other, suggesting that the SCF(hFBH1) complex can catalyze a ubiquitination reaction while acting as a DNA helicase or translocating along DNA. The potential roles of the SCF(hFBH1) complex in DNA metabolism based upon the enzymatic activities associated with this complex are discussed.
Kim
Jeong-Hoon
JH
National Creative Research Initiative Center for Cell Cycle Control, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejoen, 305-701, Korea.
Kim
Jaehoon
J
Kim
Do-Hyung
DH
Ryu
Gi-Hyuck
GH
Bae
Sung-Ho
SH
Seo
Yeon-Soo
YS
D000251:Adenosine Triphosphatases / Q000378:metabolism; D004247:DNA / Q000494:pharmacology; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D011994:Recombinant Proteins / Q000378:metabolism; D044843:SKP Cullin F-Box Protein Ligases / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014452:Ubiquitins; D004247:DNA; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C464443:FBH1 protein, human