title: Essential and distinct roles of the F-box and helicase domains of Fbh1 in DNA damage repair.
abstract: BACKGROUND
  DNA double-strand breaks (DSBs) are induced by exogenous insults such as ionizing radiation and chemical exposure, and they can also arise as a consequence of stalled or collapsed DNA replication forks. Failure to repair DSBs can lead to genomic instability or cell death and cancer in higher eukaryotes. The Schizosaccharomyces pombe fbh1 gene encodes an F-box DNA helicase previously described to play a role in the Rhp51 (an orthologue of S. cerevisiae RAD51)-dependent recombinational repair of DSBs. Fbh1 fused to GFP localizes to discrete nuclear foci following DNA damage.
  
  
  RESULTS
  To determine the functional roles of the highly conserved F-box and helicase domains, we have characterized fbh1 mutants carrying specific mutations in these domains. We show that the F-box mutation fbh1-fb disturbs the nuclear localization of Fbh1, conferring an fbh1 null-like phenotype. Moreover, nuclear foci do not form in fbh1-fb cells with DNA damage even if Fbh1-fb is targeted to the nucleus by fusion to a nuclear localization signal sequence. In contrast, the helicase mutation fbh1-hl causes the accumulation of Fbh1 foci irrespective of the presence of DNA damage and confers damage sensitivity greater than that conferred by the null allele. Additional mutation of the F-box alleviates the hypermorphic phenotype of the fbh1-hl mutant.
  
  
  CONCLUSIONS
  These results suggest that the F-box and DNA helicase domains play indispensable but distinct roles in Fbh1 function. Assembly of the SCFFbh1 complex is required for both the nuclear localization and DNA damage-induced focus formation of Fbh1 and is therefore prerequisite for the Fbh1 recombination function.
authors.affiliation: Laboratory of Genome Dynamics, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan. sakaguchi@biken.osaka-u.ac.jp
authors.forename: Chikako
authors.initials: C
authors.lastname: Sakaguchi
authors.forename: Takashi
authors.initials: T
authors.lastname: Morishita
authors.forename: Hideo
authors.initials: H
authors.lastname: Shinagawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Hishida
mesh_terms: D021581:Active Transport, Cell Nucleus; D002467:Cell Nucleus / Q000737:chemistry; D004249:DNA Damage; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D004260:DNA Repair / Q000502:physiology*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D044783:F-Box Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D005813:Genes, Synthetic; D008741:Methyl Methanesulfonate / Q000633:toxicity; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000502:physiology; D044843:SKP Cullin F-Box Protein Ligases / Q000235:genetics / Q000502:physiology; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D013329:Structure-Activity Relationship
chemical_list: D004271:DNA, Fungal; D044783:F-Box Proteins; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; D008741:Methyl Methanesulfonate; D044843:SKP Cullin F-Box Protein Ligases; C504781:SKP1 protein, S pombe; D004265:DNA Helicases; C504739:Fbh1 protein, S pombe