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Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom
Received 6 April 2005/ Returned for modification 22 April 2005/ Accepted 17 June 2005
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ABSTRACT |
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INTRODUCTION |
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Despite its beneficial functions, inappropriate recombination can result in genome instability. The evolution of an efficient "recombinosome" has therefore had to go hand in hand with enzymes that temper its action. In humans the tumor suppressor protein BRCA2 may help to control when and where Rad51 loads onto DNA (44). In yeast inappropriate loading of Rad51 can be reversed by the Srs2 DNA helicase, which dissociates Rad51 nucleofilaments (17, 43). Members of the widely conserved RecQ DNA helicase family, which includes Sgs1 and Rqh1 in budding and fission yeasts, respectively, and BLM and WRN in humans, can direct the way in which recombination junctions are resolved (7, 12, 13, 45). Defects in BLM and WRN result in the cancer-prone diseases Blooms syndrome and Werners syndrome, respectively, illustrating the importance of this activity.
Our studies of homologous recombination and its control have made use of the fission yeast Schizosaccharomyces pombe as a model system. In S. pombe there are two Rad52-like proteins, Rad22 and Rti1 (also known as Rad22B) (37, 41, 42). These were thought to be functionally redundant (38). However, we showed recently that Rad22 alone is essential for Rhp51-dependent recombination (Rhp51 is the homologue of Rad51 in S. pombe) (8). Rad22 is therefore just as important for recombination in S. pombe as Rad52 is in Saccharomyces cerevisiae. Previous studies had failed to detect the importance of Rad22 because of suppressor mutations in the rad22 mutant strains that were used. Intriguingly, suppressors of rad52 have not been identified in S. cerevisiae (38). Here we reveal the identity of the suppressor as the F-box DNA helicase Fbh1 and show that it plays an important role in controlling the action of Rhp51. Fbh1 is conserved in humans but is absent in S. cerevisiae and several other model eukaryotic systems (15). Its presence in S. pombe therefore provides a unique opportunity for its study in a genetically tractable system.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Strains and plasmids.
The S. pombe strains used in this study are listed in Table 1. The fbh1::kanMx6 mutant contains an insertion of a kanR marker between the two SalI sites in fbh1, whereas the fbh1
::kanMx6 mutant contains a replacement of the entire fbh1+ open reading frame by a kanR marker and was constructed by PCR-based gene targeting (3). Construction of the ECFP-rhp51+-kanR strain will be described elsewhere. The fbh1+ open reading frame was cloned into pREP41-EYFP to give plasmid pMW651, expressing N-terminally tagged EYFP-fbh1+ from the thiamine-repressible nmt1 promoter. Plasmid pMW637 is a pREP41 derivative that expresses wild-type fbh1+ from an nmt1 promoter. The F-box (Fbh1L14A/P15A) mutant and helicase-defective (Fbh1D485N) versions of this plasmid were derived from pMW637 by using a QuikChange site-directed mutagenesis kit (Stratagene), and the changes were confirmed by nucleotide sequencing. In order to construct the fbh1L14A/P15A and fbh1D485N mutant strains, the fbh1L14A/P15A and fbh1D485N mutant genes were subcloned into plasmid pFA6a-KanMx6 (3) upstream of the kanR marker. The region of genomic DNA (
600 bp) that lies immediately downstream of fbh1 was then cloned into these plasmids downstream of the kanR marker to make plasmids pMW695 (containing fbh1L14A/P15A) and pMW696 (containing fbh1D485N). NdeI-plus-BamHI digestion of these plasmids results in a linear fragment, which consists of mutant fbh1 and downstream genomic DNA flanking a kanR marker. This was used to transform strain MCW1221. Geneticin-resistant transformants were screened for successful replacement of fbh1 by the fbh1 mutant-kanR cassette by PCR. The L14A/P15A and D485N mutations in these strains were confirmed by nucleotide sequencing.
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Plasmid loss assay.
Wild-type and fbh1 cells harboring vector pREP41 (containing the LEU2 marker, which complements the leu1-32 auxotrophic marker in the parental strains) were grown for 24 h in nonselective YES liquid medium. Cells were harvested, washed in water, appropriately diluted, and plated on YES medium. Colonies were formed after 4 days at 30°C, and retention or loss of the pREP41 plasmid was determined by replica plating onto Edinburgh minimal medium lacking leucine. Three independent wild-type (pREP41) and fbh1 (pREP41) transformants were assayed for plasmid loss. The percent plasmid loss per generation (
) was determined by the formula = {1–e[1/n x ln(Rn/R0]} x 100, where R0 and Rn are the percentages of cells that retain the plasmid at generations 0 and n, respectively.
Spot assays. Exponentially growing cells from liquid cultures were harvested, washed, and resuspended in water at a density of 1 x 107 to 1 x 103 cells/ml. Aliquots (10 µl) of the cell suspensions were spotted onto solid media containing various concentrations of chemical genotoxins (MMS, hydroxyurea [HU], or CPT) or irradiated with various doses of UV light using a Stratalinker (Stratagene). In each spot assay, the undiluted spot (designated 1 in each of the figures) represents 1 x 105 cells, whereas the 0.1, 0.01, and 0.001 spots represent 1 x 104, 1 x 103, and 1 x 102 cells, respectively. For each spot assay four different doses of each genotoxin was used. However, for most figures only a single dose is displayed for simplicity. Plates were incubated at 30°C for up to 6 days and were normally photographed on each day, starting from day 2, in order to monitor differences in growth rates between the strains. The lengths of incubation for the spot assays shown in the figures are indicated in the relevant figure legends. All spot assays were repeated at least once to ensure reproducibility. The spot assays reveal relative differences in survival and growth following a single (acute) exposure to UV or prolonged (chronic) exposure to MMS, HU, and CPT.
MMS, CPT, and UV each generate DNA lesions that result in replication fork blockage or breakage. Specifically, UV and MMS generate base damage that can block replication fork progression, whereas CPT inhibits the religation step during the topoisomerase I reaction cycle, which results in increased numbers of single-strand breaks in the DNA at which replication forks collapse (26). As mentioned in the introduction, homologous recombination is used to tolerate and/or repair blocked and broken replication forks (8). HU inhibits ribonucleotide reductase, which results in a decrease in deoxynucleoside triphosphate pools and subsequent replication fork stalling. Chronic exposure to HU results in DNA breakage, which is repaired by homologous recombination (29).
Doubling time and plating efficiency. Doubling times were determined by measuring the optical density at 600 nm during exponential growth in liquid YES at 30°C. Mean values are from at least four independent cultures. Plating efficiency was determined by plating a known number of cells from cultures in liquid YES during exponential growth at 30°C onto YES agar. Cells were counted using a hemocytometer. Mean values are from at least four independent cultures.
Quantitative UV survival assays. Dilutions of cells growing exponentially were plated in triplicate onto YES plates and UV irradiated using a Stratalinker (Stratagene). Plates were incubated at 30°C for 6 days before colonies were counted. All data points represent the mean value from three independent cultures.
Recombination assays. Mitotic recombination was assayed by the recovery of Ade+ recombinants from strains containing the intrachromosomal recombination substrate shown in Fig. 4D, as described previously (7). Recombinant frequencies represent the mean value from at least 15 colonies for each strain.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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strain (not shown). We also found that our de novo rad22 strain readily acquires faster-growing colonies, which are more resistant to genotoxins (not shown). These data indicate that rad22 mutant strains can acquire suppressor mutations.
To identify the suppressor in one of the rad22 mutant strains, we backcrossed it to a wild-type strain and obtained non-rad22 mutant segregants that grew more slowly than the wild type and exhibited hypersensitivity to the genotoxins MMS and CPT (for an explanation of the different genotoxins used in this study, see "Spot assays" in Materials and Methods). In preliminary experiments (data not shown), we demonstrated 1:1 segregation of these phenotypes through further crosses, which showed that they were likely to be due to a single-gene mutation. Moreover, when the non-rad22 mutant segregants were crossed with a nonsuppressed rad22 strain, half of the rad22 progeny were suppressed, consistent with these segregants carrying the suppressor mutation.
The suppressor was cloned by complementation of its mutant phenotypes by using a genomic DNA library. Two complementing clones were isolated from approximately 20,000 transformants. Each contained a different but overlapping piece of genomic DNA with a single common gene encoding the homologue of the human F-box DNA helicase 1 (Fbh1) (not shown). Nucleotide sequencing of fbh1 in the suppressed rad22 strain, and in the suppressor mutant strain derived from it, revealed a frameshift mutation at codon 568 (an A insertion), which would result in a truncated protein lacking key helicase motifs (Fig. 1A), and a T-to-G transversion resulting in a F735C change. We have named this fbh1 allele fbh1-1.
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To characterize Fbh1, we made two strains, one containing a replacement of the entire fbh1 gene with a geneticin resistance marker (fbh1kanR) and a second one containing a replacement of amino acids 497 to 808 with a geneticin resistance marker (fbh1::kanR) (Fig. 1A). A comparison of these strains with the fbh1-1 strain revealed that the fbh1kanR strain grows slower (Fig. 1B, compare growth after 2.5 and 6 days) and is more sensitive to chronic exposure to HU, MMS, and CPT than either the fbh1-1 or fbh1::kanR strain. All three fbh1 mutant strains display similar levels of survival following acute exposure to UV, and the growth and genotoxin hypersensitivities of the fbh1-1 and fbh1::kanR strains are equivalent (Fig. 1B). These data show that Fbh1 promotes the tolerance/repair of DNA damage and is required for normal growth. Furthermore, as both fbh1-1 and fbh1::kanR mutants would express truncated forms of Fbh1 missing key helicase motifs (see Fig. 1A), we suspect that Fbh1 with an intact F-box, but little or no helicase activity, retains some ability to promote growth and DNA repair.
To investigate the relative importance of Fbh1's DNA helicase and putative ubiquitin ligase activities in promoting DNA repair, we constructed strains containing mutations in the F-box (L14A/P15A) and helicase (D485N in motif II) domains of Fbh1 (Fig. 1A) and compared them to the fbh1 strain for growth and genotoxin resistance (Fig. 1C). Equivalent mutations in other proteins have disabled F-box and helicase functions, respectively (25, 28). We also confirmed by Western blot analysis that Fbh1L14A/P15A and Fbh1D485N were expressed at levels comparable to those of the wild-type protein (not shown). The fbh1D485N strain exhibits growth and sensitivity to genotoxins similar to those of the fbh1 strain, whereas fbh1L14A/P15A displays only modest hypersensitivity to CPT (Fig. 1C). These data indicate that Fbh1's DNA helicase activity is critical for promoting DNA repair. However, this contradicts data obtained with the fbh1-1 and fbh1::kanR strains, which suggest that Fbh1 can promote some DNA repair without its DNA helicase activity (see above). One possible explanation for this discrepancy is that fbh1D485N has a dominant negative effect, which masks any residual activity it might have in promoting DNA repair. In accord with this, overexpression of Fbh1D485N, from the nmt1 promoter in the plasmid pREP41, does have a dominant negative effect on the growth and genotoxin resistance of a wild-type strain (not shown). With respect to the importance of the F-box in Fbh1, our data with fbh1L14A/P15A suggest that it plays only a minor role in promoting DNA repair. However, we have no direct evidence that the F-box activity in Fbh1 is destroyed by the L14A/P15A mutation.
Fbh1 promotes proper chromosome segregation.
The doubling time of the fbh1 mutant strain growing in liquid culture is approximately twice that of the wild type (Table 2). At least part of this slow growth can be explained by a reduction in cell viability as judged by the plating efficiency of the fbh1 strain (Table 2). Cytological examination revealed that more than half of fbh1 cells growing in culture are elongated, with 17% greater than twice the normal haploid cell division length (14 µm) (Fig. 1D and F). Moreover, staining with the DNA-specific dye DAPI showed that 30% of fbh1 cells are anucleate or contain fragmented DNA and that 70% of septated cells contain DNA segregating unevenly between the two daughters (Fig. 1D and F). These phenotypes are indicative of a defect in chromosome segregation. Consistent with this, fbh1 cells exhibit a 5-fold-higher rate of plasmid loss than the wild type (Fig. 1F). DAPI staining also revealed a significant number of cells with an unusual "figure-eight" nuclear region, where the majority of DAPI-stained material is in one circular domain, which is closely associated with, or connected to, a second, larger circular domain that is less stained (Fig. 1E and F). The significance of this phenotype is currently uncertain.
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mutant phenotypes suggested that Fbh1 might function in an Rhp51-dependent pathway for DNA repair. To see if this was true, fbh1 and rhp51 single and double mutant strains were first compared for growth and cell viability (Table 2). The doubling time of an fbh1 rhp51 double mutant is similar to that of an fbh1 single mutant, which in turn is longer than that of an rhp51 single mutant. However, the plating efficiency of an fbh1 single mutant is slightly less than those of both the rhp51 and fbh1 rhp51 mutants (Table 2). These data indicate an epistatic relationship between fbh1 and rhp51 for promoting normal growth and cell viability.
We next compared the fbh1, rhp51, and fbh1 rhp51 strains for genotoxin sensitivity (Fig. 2A). The fbh1 rhp51 double mutant exhibits no increase in sensitivity to acute UV exposure compared to an rhp51 single mutant (Fig. 2A). In contrast, the double mutant appears to be slightly more sensitive to chronic MMS, HU, and CPT exposure than either of the single mutants (Fig. 2A). However, this is due to the double mutant growing slower than the rhp51 single mutant in the presence of genotoxins, rather than to an actual reduction in viability (not shown). Together these data indicate that Fbh1 functions in an Rhp51-dependent pathway for DNA repair.
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strain, indicating that it retained at least some functionality (not shown). When expressed in a wild-type strain in the absence of an exogenous genotoxin, the majority of cells exhibit EYFP-Fbh1 as faint fluorescence throughout the nucleus (Fig. 2B). However, a few cells (<1%) contain a single EYFP-Fbh1 focus within the nucleus (not shown), and following exposure to 10 µM CPT for 4 h, >30% of cells exhibit one or more foci (similar results were obtained with HU, MMS, and mitomycin C) (Fig. 2B, see Fig. 7, and data not shown). To see if these foci colocalize with Rhp51 foci, EYFP-Fbh1 was expressed in a strain in which Rhp51 is fused to ECFP. Following DNA damage, >90% of EYFP-Fbh1 and ECFP-Rhp51 foci colocalize (Fig. 2C and data not shown). These data are consistent with Fbh1 functioning together with Rhp51 at sites of DNA damage.
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mutant was crossed with both rqh1 and srs2 mutant strains. Each gene deletion was marked with a different selectable marker so that the genotypes of the viable progeny could readily be distinguished. For the fbh1 x rqh1 cross, >100,000 viable progeny from several independent crosses were analyzed for the presence of a rqh1 fbh1 double mutant (not shown). However, only wild-type, rqh1, and fbh1 progeny were recovered, indicating that the double mutant is unviable. In contrast, fbh1 srs2 double mutants were readily recovered from the fbh1 x srs2 cross. However, a comparison of the fbh1 srs2 double mutant with its single mutants reveals a more-than-additive reduction in growth (Fig. 3A and D). Both the nonviability of an fbh1 rqh1 double mutant and the poor growth of an fbh1 srs2 double mutant are rescued by deleting either rhp51 or its mediator rhp55 (Fig. 3B, C, and D). This indicates either that Fbh1 shares an overlapping role with both Srs2 and Rqh1 for processing Rhp51-dependent recombination intermediates or that it prevents Rhp51 action that would necessitate subsequent processing by Srs2 and Rqh1.
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Having characterized some of the basic phenotypes of an fbh1 mutant, we next looked at its genetic interaction with rad22. While we already knew that fbh1-1 partially suppressed the genotoxin hypersensitivity of a rad22 mutant, it was important to establish that the same was true for the fbh1 mutant. To this end, we constructed an fbh1 rad22 double mutant and compared it to its respective single mutants and to an fbh1-1 rad22 double mutant. Both the poor growth and genotoxin sensitivity of a rad22 mutant are suppressed by fbh1 (Fig. 4A and B and Table 2). Greater suppression is seen with fbh1 than with the fbh1-1 allele (Fig. 4B). This suggests that truncated Fbh1, which would probably be devoid of helicase activity, retains some ability to block repair in the absence of Rad22. Another important observation is that the fbh1 rad22 double mutant grows better and is more viable than an fbh1 single mutant and is also more resistant to UV light (Table 2 and Fig. 4A). Cosuppression in the double mutant indicates that both Rad22 and Fbh1 have a negative effect on growth and DNA repair in the absence of the other.
To see if rad22 suppression by fbh1 depends on the other Rad52-like protein in S. pombe, Rti1, the genotoxin sensitivities of rad22 rti1, fbh1 rad22, and fbh1 rti1 double mutants were compared to those of an fbh1 rad22 rti1 triple mutant (not shown). An rti1 single mutant exhibits no detectable hypersensitivity to UV, HU, MMS, or CPT, and this mutation has no effect on the hypersensitivity of a rad22 mutant (not shown). Likewise, it has no effect on the suppression of rad22 by fbh1 as judged by the similar sensitivities of fbh1 rad22 and fbh1 rad22 rti1 mutant strains.
An analysis similar to that described above was performed to see if rad22 suppression depends on Rhp51. The rad22 rhp51 double mutant exhibits the same hypersensitivity to genotoxins as a rad22 single mutant (8), but, unlike for the rad22 strain, these phenotypes are not suppressed by the deletion of fbh1 (Fig. 4C). Likewise, the poor growth and viability of the rad22 rhp51 double mutant are not suppressed by deleting fbh1 (Table 2). This absolute dependence on rhp51+ for the suppression of rad22 mutant phenotypes suggests that Fbh1 inhibits the action of Rhp51 in the absence of Rad22.
Most homologous recombination depends on Rad22 in S. pombe (8). To see if this role is also suppressed by deleting fbh1, we constructed relevant mutant strains containing a direct repeat of ade6– mutant alleles integrated at the ade6 locus on chromosome III (Fig. 4D). Inter- or intrachromatid recombination between the ade6– mutant alleles results in ade+ recombinants that retain (conversion types) or lose (deletion types) the intervening his3+ gene. In the wild-type strain the frequency of spontaneous ade+ recombinants is 3 x 10–4 (Fig. 4E). Approximately 30% of these recombinants are conversion types. Deletion of fbh1 has no effect on spontaneous direct repeat recombination as measured by this assay (Fig. 4E). It also does not affect the frequency of UV-induced recombinants or the hyper-deletion-type recombination in an rhp51 mutant (Fig. 4E and data not shown). In the rad22 strain the recombinant frequency drops to <1 x 10–4, and all of these recombinants are deletion types (Fig. 4E). Recombination is restored to wild-type levels by deleting fbh1, although the percentage of recombinants that are conversion types is less than in the wild type (Fig. 4E). As with the suppression of genotoxin hypersensitivity, the improvement in recombinant formation in a rad22 mutant, by deleting fbh1, is dependent on Rhp51. This is evident from the fact that an fbh1 rhp51 rad22 triple mutant exhibits the same low frequency of recombination as both a rad22 single mutant and rhp51 rad22 double mutant (Fig. 4E). With the data above, these results show that much of Rad22's role in promoting Rhp51-dependent recombination and DNA repair can be circumvented by the removal of Fbh1.
Both the F-box and helicase domains of Fbh1 contribute to the inhibition of Rhp51-dependent DNA repair in the absence of Rad22.
As mentioned above, the failure of fbh1-1 to suppress rad22 genotoxin hypersensitivity as much as fbh1 suggests that Fbh1 inhibits repair by means other than just its DNA helicase activity. To test this further, we compared the abilities of fbh1 and fbh1D485N to suppress the poor growth and genotoxin sensitivity of a rad22 mutant. Both mutant alleles reduce the doubling time of a rad22 mutant, growing in liquid culture, to similar levels (Table 2). However, an fbh1D485N rad22 double mutant does not grow as well as an fbh1 rad22 double mutant on solid media in the presence of genotoxins, and this effect is due to a combination of slower growth and reduced survival (Fig. 5A and B). This provides further evidence that Fbh1 does not depend solely on its DNA helicase activity to inhibit Rhp51-dependent repair in the absence of Rad22. To see whether Fbh1's F-box is important here, we tested whether fbh1L14A/P15A could suppress rad22. However, neither the poor growth nor genotoxin hypersensitivity of a rad22 mutant was suppressed by fbh1L14A/P15A (not shown). We also tested whether overexpression of fbh1L14A/P15A and fbh1D485N, from the nmt1 promoter in pREP41, could complement the improved genotoxin resistance of an fbh1 rad22 double mutant (i.e., make an fbh1 rad22 double mutant as sensitive to genotoxins as a rad22 single mutant) (Fig. 5C). Wild-type Fbh1 fully complements this mutant to the sensitivity of a rad22 single mutant, whereas both Fbh1L14A/P15A and Fbh1D485N only partially complement. To confirm that both protein domains are independently contributing to Fbh1 action, an fbh1L14A/P15A/D485N mutant was constructed and shown not to complement the fbh1 rad22 double mutant at all (Fig. 5C). Overall, these data indicate that Fbh1's DNA helicase activity is most critical for controlling Rhp51 in the absence of Rad22. However, the F-box also appears to have a role here, which suggests that Fbh1-directed ubiquitination may play some part in the control of Rhp51.
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rhp55 and fbh1 swi5 double mutant strains were constructed and compared to their respective single mutants for genotoxin sensitivity. In the case of the fbh1 rhp55 mutant, reciprocal suppression of genotoxin sensitivity was observed such that, depending on the genotoxin used, the double mutant is as resistant or almost as resistant as the wild-type strain (Fig. 6A). A similar result was seen for the fbh1 swi5 mutant with UV (Fig. 6B). Furthermore, swi5, which exhibits relatively little hypersensitivity to HU, MMS, or CPT, suppresses fbh1 sensitivity to these agents. These data show that the need for Rhp55/Swi5 and Fbh1 for DNA repair is largely negated by their pairwise removal. Importantly, the poor growth of an fbh1 strain is also suppressed by deleting Rhp55 or Swi5 (not shown). Taken together, these data indicate that the mediator proteins are important for promoting Rhp51 action in the presence of Fbh1. Furthermore, where Rhp51 action is attenuated by the removal of a mediator protein, the requirement for Fbh1 to promote growth and DNA repair is reduced.
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by fbh1.
To see if deleting fbh1 would suppress the need for mediator proteins altogether, fbh1 rhp55 rad22 and fbh1 swi5 rad22 triple mutants were constructed and compared to their respective double mutants for genotoxin sensitivity (Fig. 6C and D). In both cases the triple mutant was at least as sensitive as the most sensitive double mutant (i.e., rad22 rhp55 and rad22 swi5, respectively), which in turn was as sensitive as a rad22 single mutant. These data show that the deletion of fbh1 cannot suppress the need for more than one mediator protein at a time. In fact, an fbh1 rhp55 rad22 triple mutant is slightly more sensitive to genotoxins than a rad22 rhp55 double mutant (Fig. 6C and E). This suggests that Fbh1 may be required to process any residual loading of Rhp51 onto DNA that occurs in the absence of both Rad22 and Rhp55.
Fbh1 subnuclear localization in the absence of recombination proteins.
To see if the formation of Fbh1 nuclear foci depends on recombination proteins, we expressed EYFP-Fbh1 in rhp55, rhp51, and rad22 mutants, in the presence and absence of CPT, and determined the percentage of cells with foci. Rather than being required for focus formation, each of the recombination mutants exhibited a greater percentage of cells with spontaneous foci and CPT-induced foci, including cells with multiple foci (Fig. 7). This suggests that Fbh1 may target sites of DNA damage directly rather than the recombination proteins that load at these sites. In the absence of recombination proteins, sites of spontaneous or induced damage would persist, resulting in the increased numbers of Fbh1 foci. Furthermore, the removal of one recombination protein may result in the inefficient or aberrant loading of the other recombination proteins at sites of DNA damage, and this may also result in increased Fbh1 focus formation.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Genetic analyses show that any double combination of fbh1, srs2, and rqh1 mutations results in a dramatic reduction or total loss of viability, which can be remedied by removing Rhp51 (9, 19). This suggests that these DNA helicases share an overlapping function for processing Rhp51-dependent recombination intermediates. In accord with their function in controlling recombination, both srs2 and rqh1 mutants exhibit elevated levels of direct repeat recombination (7, 9). In contrast, direct repeat recombination at ade6 is normal in an fbh1 mutant. This may mean that Fbh1 controls recombination only at specific loci or that the recombination intermediates that are not processed by Fbh1 result in cell death.
Based on our analysis of DAPI-stained cells, an fbh1 mutant exhibits a defect in chromosome segregation, which presumably underlies its reduced growth and viability. Rqh1 mutants also have impaired chromosome segregation, especially following exposure to agents that cause replication fork arrest (7, 32). Here chromosome segregation is improved by the expression of the bacterial Holliday junction resolvase RusA (7). This suggests that an accumulation of unprocessed Holliday junctions between sister chromatids perturbs chromosome segregation. Unlike the case for rqh1 mutants, neither chromosome segregation nor viability of an fbh1 mutant is improved by RusA (not shown). This suggests that Fbh1 is not required for processing Holliday junctions. Instead, we suspect that Fbh1 controls recombination by dissociating recombinases from DNA while translocating along it. This would be analogous to the action of its close relative Srs2 (17, 43). Evidence that active translocation would be required comes from our analysis of the fbh1D485N mutant, which contains a mutation of a conserved residue within helicase motif II that is known to be required for ATP hydrolysis in other DNA helicases. This mutant exhibits essentially a null phenotype with respect to genotoxin sensitivity, and the mutation suppresses a rad22 mutant almost as well as a complete deletion of fbh1. Exactly which recombinases might be dissociated is not known, and Fbh1's attenuation of Rhp51 activity could be by direct removal of the Rhp51 nucleofilament and/or the removal of the mediator proteins.
If Fbh1 does indeed function in a manner analogous to that of Srs2, why should the cell require two similar DNA helicases? As mentioned above, genetic analyses indicate that there is an overlap between the Fbh1 and Srs2 pathways, but it also shows that they have distinct functions. In S. cerevisiae Srs2 is important for channeling DNA repair away from recombination to the postreplication repair pathway (1, 5, 27, 31), and it may fulfill a similar function in S. pombe (9). Fbh1 may also be required for channeling lesions away from recombination, but whether this is to direct them into a particular repair pathway is not clear at present.
A distinguishing feature of Fbh1 is its F-box; however, our analyses of the fbh1L14A/P15A mutant suggest that this may play a relatively minor role in Fbh1 function. Alternatively, the L14A/P15A mutation may not fully inactivate the F-box, in which case it remains a possibility that this domain is critical for Fbh1's function. It is therefore tempting to speculate that Fbh1 may promote ubiquitin-mediated degradation to curb recombination. We are currently investigating this possibility.
Another distinction between Fbh1 and either Srs2 or Rqh1 is that deletion of Fbh1 reduces significantly the need for certain mediator proteins. In fact, double mutant strains combining fbh1 with rad22, rhp55, or swi5 each show at least some improvement in growth/viability and/or genotoxin resistance compared to their respective single mutants. This reciprocal suppression, which is not restricted to one particular mediator protein, leads us to suggest the following model (summarized in Fig. 8). Combinations of Rad22, Rhp55-Rhp57, and Swi5-Sfr1 might make Rhp51 nucleofilament assembly fast and efficient but may also increase the chances of "inappropriate" filament assembly. Exactly what might constitute an "inappropriate" filament is unknown; however, possibilities include filament assembly at inappropriate sites on the DNA and/or at inappropriate times during the cell cycle. In our model we make the assumption that inappropriate filament assembly is generally less efficient than the appropriate assemblies, possibly due to a paucity of recombination proteins, accessibility of the DNA substrate, or competition with other proteins. We suspect that Rhp51 nucleofilaments that form inefficiently are targeted by Fbh1 for removal. These filaments may be more susceptible to dissociation by Fbh1 because they are less extensive/robust and/or engage in strand invasion with slower kinetics. In either a rad22 or rhp55 mutant filament, assembly is made less efficient, thereby making appropriate Rhp51 assemblages susceptible to Fbh1 action. At the same time, the inappropriate events would also be made less efficient such that they may not occur at all, removing the need for Fbh1. Thus, in either a rad22 or rhp55 mutant the removal of Fbh1 would redress the balance, enabling the appropriate events to occur without the danger of the inappropriate ones. However, if both Rad22 and Rhp55 are missing, then nucleofilament assembly might be so disabled that Fbh1 is needed to remove any Rhp51 that manages to load onto DNA. Residual amounts of loading would be insufficient for recombination and, by interfering with other processes, could be toxic. This could explain our observation that an fbh1 rad22 rhp55 triple mutant is slower growing and more sensitive to genotoxins than a rad22 rhp55 double mutant.
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ACKNOWLEDGMENTS |
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mutant, and Hideo Shinagawa for communicating results prior to publication. This work was supported by a project grant (065278/Z/01/Z) and Senior Fellowship in Basic Biomedical Research from The Wellcome Trust awarded to M.C.W.
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FOOTNOTES |
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