Essential and distinct roles of the F-box and helicase domains of Fbh1 in DNA damage repair

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.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.Homologous recombination (HR) is a major error-free pathway of DSB repair found in all organisms thus far examined (for reviews, see [1-3]). Extensive studies of HR repair mechanisms in the budding yeast Saccharomyces cerevisiae have shown that HR requires members of the RAD52 epistasis group, including RAD50, MRE11, XRS2, RAD51, RAD54, RAD55, RAD57, and RAD59 [4-6]. More recent studies of HR mechanisms in the fission yeast Schizosaccharomyces pombe have revealed many similarities with HR in S. cerevisiae and have led to many insights into the mechanisms of HR-dependent DSB repair and the identification of novel genes with homologues in higher eukaryotes [7,8]. For example, Rhp51-interacting proteins such as the Swi5-Sfr1 mediator complex function in a separate pathway from Rhp55-Rhp57 to promote an Rhp51 strand exchange reaction [9,10]. Furthermore, the fbh1 gene encodes a protein consisting of a unique domain architecture, wit