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Essential and distinct roles of the F-box and helicase domains of Fbh1 in DNA damage repair
© Sakaguchi et al; licensee BioMed Central Ltd. 2008
- Received: 19 October 2007
- Accepted: 03 March 2008
- Published: 03 March 2008
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.
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
- Enhance Green Fluorescent Protein
- Focus Formation
- Homologous Recombination Repair
- Helicase Domain
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, with N-terminal F-box and C-terminal DNA helicase domains [11, 12], which is conserved in mammals but not in S. cerevisiae. The Fbh1 protein was originally identified as a 3' to 5' DNA helicase that is stimulated by RPA at low ATP concentrations . The helicase domain of Fbh1 is structurally related to the Rep, UvrD, PcrA, and Srs2 family of helicases . Previous studies have shown that S. cerevisiae Srs2 regulates RAD52-dependent HR by actively disrupting the Rad51 nucleoprotein filament [15, 16]. Interestingly, in contrast to Fbh1, Srs2 is conserved in budding and fission yeasts but not in mammals. In S. pombe, the fbh1 Δ mutation is lethal when combined with the srs2 Δ mutation, and this synthetic lethality can be suppressed by a loss of HR functions [11, 12]. Recently, Chiolo et al. reported that human FBH1 (hFBH1) suppresses specific recombination defects of S. cerevisiae srs2 mutants and that the F-box domain is essential for hFBH1 functions in this respect . Thus, the Fbh1 and Srs2 helicases appear to have only partially analogous functions in controlling HR after the formation of Rhp51 nucleoprotein filaments.
F-box proteins were first characterized as components of SCF ubiquitin-ligase complexes containing Skp1, Cullin, and F-box proteins, in which they bind substrates for ubiquitin-mediated proteolysis [18–21]. The F-box motif consists of 40–50 amino acids and is required for binding to SKP1. Therefore, the F-box motif links F-box proteins to other components of the SCF complex. Indeed, hFBH1 was shown to form an SCF complex and to have ubiquitin ligase activity in vitro [14, 22]. However, the physiological substrates of SCFFbh1 are still unknown.
In this study, we characterize the in vivo function of Fbh1, focusing on the role of its F-box and helicase domains. Our results demonstrate that the F-box domain of Fbh1 is required for its recruitment to the nucleus and to DNA damage sites, whereas the helicase domain is involved in DNA processing after the Rhp51-dependent step of HR. Thus, both domains have indispensable but distinct roles in Fbh1 functions, and assembly of the SCFFbh1 complex is a prerequisite for its DNA recombination activities.
Construction of fbh1 mutants with substitutions in the F-box or helicase motif
The role of the F-box domain
The role of the DNA helicase domain
We next examined the effect of the fbh1-hl mutation on Fbh1 function. The fbh1-hl mutant shows greater sensitivity to DNA damaging agents than does the fbh1 Δ mutant (Fig. 2A). In addition, ectopic expression of the fbh1-hl allele renders wild type cells sensitive to MMS, suggesting that the fbh1-hl alteration is a dominant mutation (see Additional file 1). Previous studies suggest that Fbh1 promotes HR repair by controlling the action of Rhp51 [11, 12], which contributes to the suppression of inappropriate recombination events. Therefore, one possibility to explain the fbh1-hl-dependent toxic phenotype is that toxic recombination intermediates caused by faulty Rhp51-dependent HR accumulate to a greater extent in the fbh1-hl mutant than in the fbh1 Δ mutant. To test this possibility, we examined the MMS sensitivity of the fbh1-hl strain in an rhp51 Δ background. As expected, fbh1-hl cells were as sensitive to MMS as fbh1 Δ cells with the rhp51 Δ background (Fig. 2C). These results indicate that Fbh1-hl is not only defective in Fbh1 function but also that it interferes with Rhp51-dependent HR. In addition, the fbh1-hl mutation does not suppress the poor growth of rad22 Δ cells, but rather, it exacerbates their decreased growth rate (Fig. 2B). However, the fbh1-fb/hl mutant, which has alterations within both the F-box and Walker A motifs, is as sensitive to DNA damaging agents as the fbh1-fb or fbh1 Δ strains (Fig. 2A), and mutations affecting both domains suppress the slow growth phenotype of the rad22 Δ strain (Fig. 2B), suggesting that F-box activity is likely to be a prerequisite for helicase activity. Thus, the F-box and DNA helicase domains play indispensable but distinct roles in Fbh1 function.
Focus formation of fbh1 mutants in response to DNA damage
Fbh1 is required for the DNA damage-induced formation of Skp1 nuclear foci
The F-box domain is responsible for the nuclear localization of Fbh1
An important unresolved issue is the identification of physiological substrates of SCFFbh1 that presumably regulate the HR pathway. A recent study has shown that hFBH1 has a short half-life when it is expressed in S. cerevisiae, and its degradation depends on the presence of a functional F-box and yeast SCF components , suggesting that one candidate for a SCFhFBH1 substrate is hFBH1 itself. The rapid turnover of hFBH1 might contribute to the tight regulation of hFBH1 helicase activity. However, if SCFhFBH1 complex formation is also necessary for its nuclear transport and recruitment to DNA damage sites, the alteration in subcellular localization caused by the F-box mutation might affect hFBH1 stability in budding yeast cells. In addition, many F-box proteins identified to date target more than one substrate for degradation [28–30]. Since our present data suggest that Fbh1 functions as an SCFFbh1 ubiquitin ligase complex for HR repair, the most plausible candidates as SCFFbh1 substrates are HR proteins, including Rhp51. For example, SCFFbh1 might contribute to regulate Rhp51-dependent HR by promoting the ubiquitination of Rhp51 or other recombination proteins. Future studies will be needed to determine whether Fbh1 is a physiological SCFFbh1 substrate and to identify other SCFFbh1 substrates, which would provide a means to conduct a more detailed analysis of its function in HR repair.
In this study, we characterized the in vivo function of Fbh1, focusing on the role of its F-box and helicase domains. Our results show that the assembly of SCFFbh1 mediated by the F-box domain of Fbh1 is required for its recruitment to both the nucleus and DNA damage sites, whereas the helicase domain is involved in controlling the action of Rhp51. Thus, Fbh1 is tightly regulated by SCF components, which might prevent it from functioning inappropriately in HR.
S. pombe strains and plasmids
Strains used in this study
h + leu1-32 ura4-D18
h+ fbh1::LEU2 leu1-32 ura4-D18
h+ fbh1::KanMX-leu1-32 ura4-D18 ade6-704
h + leu1-32 ura4-D18 fbh1-fb
h + leu1-32 ura4-D18 fbh1-hl
h + leu1-32 ura4-D18 fbh1-fb/hl
h+ ars1::pREP41-EGFP N-Fbh1 leu1-32 ura4-D18 ade6-704 fbh1::KanMX
h+ars1::pREP41-EGFP N-Fbh1-fb leu1-32 ura4-D18 ade6-704 fbh1::KanMX
h+ ars1::pREP41-EGFP N-Fbh1-hl leu1-32 ura4-D18 ade6-704 fbh1::KanMX
h+ars1::pREP41-EGFP N-Fbh1-fb/hl leu1-32 ura4-D18 ade6-704 fbh1::KanMX
smt-0 rhp51::his3 + leu1-32 ura4-D18 his3-D1 arg3-1
rhp51::his3 + fbh1-hl leu1-32 ura4-D18 his3-D1 arg3-1
smt-0 rad22::arg3 + ura4-D18 leu1-32 arg3-D1
mat1PD::LEU2 rad22::arg3 + fbh1::KanMX ura4-D18 leu1-32 arg3-D1
smt-0 rad22::arg3 + fbh1-fb ura4-D18 leu1-32 arg3-D1
mat1PD::LEU2 rad22::arg3 + fbh1-hl ura4-D18 leu1-32 arg3-D1
smt-0 rad22::arg3 + fbh1-fb/hl leu1-32 ura4-D18 his3-D1 arg3-1
skp1-94 ura4-D18 leu1-32 his3-D1
smt-0 fbh1::LEU2 rhp51::his3 + ura4-D18 leu1-32 his3-D1 arg3-D1
Expression of the GFP-Fbh1 and YFP-Skp1 fusions in S. pombe
Wild type and mutant fbh1 cDNAs were cloned separately into the vector pREP41 EGFP N  to express enhanced green fluorescent protein (EGFP) fusion proteins under the control of the medium-strength nmt1 promoter. The resulting plasmids were linearized at the unique Mlu I site within the ars1 sequence of the plasmid pREP41 and then introduced into the ars1 locus of the fbh1 Δ strain. To construct pREP41/NLS-GFP-Fbh1-fb, two complementary DNA oligonucleotides encoding a NLS sequence (PKKKRKV) from the SV40 large T antigen were synthesized and inserted at the N-terminus-encoding region. The skp1 cDNA was cloned into the plasmid pREP41 YFP N to express a yellow fluorescent protein-Skp1 fusion under the control of the nmt1 promoter. Cells harboring pREP41 YFP-Skp1 were grown in EMM2 medium with appropriate supplements and containing 0.1% MMS in the absence of thiamine for 2 h. Cells were fixed with 70% ethanol and observed by fluorescence microscopy. More than 100 individual cells were scored for each strain.
Logarithmically growing cells were harvested and resuspended in water. Five-fold serial dilutions of cultures of the indicated mutants were spotted onto YES plates containing the indicated concentration of chemical genotoxins. Plates were incubated at 30°C for 3–4 days. All spot assays were repeated at least twice to ensure that the results were reproducible.
The skp1 fragment was amplified by PCR from an S. pombe cDNA library and cloned into the vector pREP42 HA N, which encodes a triple C-terminal hemagglutinin (HA) tag . The fbh1 mutant strains C100, C101, C102 and C103 were transformed with pREP42 HA N or pREP42 HA-Skp1. The transformants were grown in EMM2 medium with appropriate supplements in the absence of thiamine to express N-terminally HA-tagged skp1 from the thiamine-repressible nmt1 promoter. Mid-log-phase cells from a 50-ml culture were collected, washed with buffer A (50 mM Tris-HCl, pH 7.5, 15 mM EGTA, 100 mM NaCl, 0.1% (w/v) Triton ×-100, protease inhibitor cocktail for yeast (Sigma), 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) and resuspended in 500 μl buffer A. Cells were disrupted with the same volume of acid-washed glass beads using a Mini-BeadBeater-8 (BioSpec Products). The supernatant fraction was collected by centrifugation and used for immunoprecipitation. Fifty microliters of protein G-agarose (GE Healthcare) was added to absorb nonspecific Protein G binding protein. Twenty microliters of anti-HA antibody (12CA5, Roche) and 40 μl Protein G-agarose were used per 400 μl cell lysate, and the mixture was rotated for 2 h at 4°C. The beads were washed three times with buffer A, resuspended in 25 μl 5% SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer, and boiled for 5 min. After centrifugation, the supernatants were separated by SDS-PAGE and analyzed by western blotting with an ECL Advance Western blotting detection kit (GE Healthcare).
Preparation of yeast extracts and Western Blotting
Total protein extract was prepared from 5 × 106 cells from logarithmically growing culture as described previously . Proteins were analyzed by SDS-PAGE, transferred to PVDF membranes, and probed with anti-Myc monoclonal antibody (Roche) or anti-a-tubulin antibody (Sigma). Detection was performed with HRP-conjugated secondary antibodies followed by treatment using the ECL advance Western blot detection kit (GE Healthcare).
We thank Y. Tsutsui and T. Toda for strains. This work was supported by Grants-in-Aid for Scientific Research from The Ministry of Education, Science, Sports, and Culture of Japan, and CREST, JST.
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