SGS1 is a multicopy suppressor of srs2: functional overlap between DNA helicases (original) (raw)

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Hocine W. Mankouri, Tim J. Craig, Alan Morgan, SGS1 is a multicopy suppressor of srs2: functional overlap between DNA helicases, Nucleic Acids Research, Volume 30, Issue 5, 1 March 2002, Pages 1103–1113, https://doi.org/10.1093/nar/30.5.1103
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Abstract

Sgs1 is a member of the RecQ family of DNA helicases, which have been implicated in genomic stability, cancer and ageing. Srs2 is another DNA helicase that shares several phenotypic features with Sgs1 and double sgs1srs2 mutants have a severe synthetic growth phenotype. This suggests that there may be functional overlap between these two DNA helicases. Consistent with this idea, we found the _srs2_Δ mutant to have a similar genotoxin sensitivity profile and replicative lifespan to the _sgs1_Δ mutant. In order to directly test if Sgs1 and Srs2 are functionally interchangeable, the ability of high-copy SGS1 and SRS2 plasmids to complement the _srs2_Δ and _sgs1_Δ mutants was assessed. We report here that SGS1 is a multicopy suppressor of the methyl methanesulphonate (MMS) and hydroxyurea sensitivity of the _srs2_Δ mutant, whereas SRS2 overexpression had no complementing ability in the _sgs1_Δ mutant. Domains of Sgs1 directly required for processing MMS-induced DNA damage, most notably the helicase domain, are also required for complementation of the _srs2_Δ mutant. Although SGS1 overexpression was unable to rescue the shortened mean replicative lifespan of the _srs2_Δ mutant, maximum lifespan was significantly increased by multicopy SGS1. We conclude that Sgs1 is able to partially compensate for the loss of Srs2.

Received November 30, 2001; Revised and Accepted January 4, 2002.

INTRODUCTION

The ability of DNA helicases to unwind DNA enables replication, transcription, recombination and repair of DNA. The fundamental importance of DNA helicases is highlighted by a variety of human genetic disorders resulting from defective helicase function (1). Although the clinical features of these disorders differ, they all exhibit genomic instability and a predisposition to cancer. One class of DNA helicases, the RecQ family (named after its homology with the prototypical bacterial RecQ helicase), has attracted particular interest recently (2). Defects in three of the five known human RecQ family members manifest as Bloom’s syndrome (BLM) (3), Rothmund–Thomson syndrome (RECQ4) (4) and Werner’s syndrome (WRN) (5). All three disorders show genomic instability associated with a predisposition to various cancers, while Rothmund–Thomson syndrome and Werner’s syndrome display features of premature ageing. At the cellular level, cells from Werner’s syndrome sufferers display genomic translocations and deletions (6), and a decreased replicative lifespan in vitro (7, reviewed in 8). Cells from Bloom’s syndrome patients similarly exhibit chromosomal rearrangements and deletions, but are characterised by a uniquely high frequency of sister chromatid exchanges (reviewed in 9).

In order to shed light on how mutations in human RecQ helicases result in genomic instability, much recent attention has been directed at RecQ orthologues in lower organisms. The genomes of Escherichia coli, Saccharomyces cerevisiae and Schizosaccharomyces pombe each contain only a single predicted RecQ helicase, thus facilitating analysis of their cellular function(s). Mutations in E.coli recQ, S.cerevisiae SGS1 and S.pombe rqh1 + all result in increased recombination (10–15), suggesting a conserved role of RecQ helicases in regulating recombination. Interestingly, the hyper-recombination phenotype of budding yeast sgs1 mutants can be complemented by the human WRN and BLM genes (12). In addition to the hyper-recombination phenotype, sgs1 mutants are sensitive to a range of genotoxins and display a reduced replicative lifespan (defined as the number of buds produced by a mother cell) (12,16–24). The human BLM gene has been shown to complement the hydroxyurea sensitivity (12) and short lifespan (22) of the sgs1 mutant, further reinforcing the notion of a conserved function of RecQ helicases. Current thinking suggests that this function may be the prevention of inappropriate recombination at stalled replication forks arising during S phase (25).

The S.cerevisiae SRS2 gene was discovered as a suppressor of the UV sensitivity of rad6 and rad18 mutants (26), and also independently in a genetic screen which isolated and characterised yeast hyper-recombination mutants (27). No obvious human homologue of Srs2 has yet been found, but the sequence of the SRS2 gene does show homology to the bacterial UvrD and Rep helicases (28) and an S.pombe orthologue has been identified recently (29). One function of Srs2 is to channel the repair of DNA lesions into the RAD6 post-replication repair pathway (30). In the absence of Srs2, DNA lesions are instead channelled into the RAD52 homologous recombination repair pathway (31). Srs2 has also been implicated in other DNA repair processes such as non-homologous end joining (32) and single-strand annealing (33).

Srs2 shares a number of interesting features with Sgs1. Both Sgs1 and Srs2 have been demonstrated to be 3′→5′ DNA helicases in vitro (34,35). The SGS1 and SRS2 gene products are generally expressed at low abundance but are up-regulated during the S phase of the cell cycle, and both have been implicated in the intra-S checkpoint response (18,36,37). Furthermore, sgs1 and srs2 mutants have a hyper-recombination phenotype (13,27). Most recently, sgs1_Δ and srs2_Δ strains have both been shown to have a shortened replicative lifespan and a greater propensity to undergo terminal cell-cycle arrest as mitotic G2/M intermediates (38). Deletion of both the SGS1 and SRS2 genes causes a severe synthetic growth defect, with low cell viability and a high incidence of G2/M terminal cell-cycle arrest, which can be suppressed by deletion of genes involved in homologous recombination (20,38,39). This suggests that unrestrained recombination causes the poor growth of the sgs1srs2 double mutant and is consistent with the idea that Srs2, like Sgs1, may act to prevent inappropriate recombination at stalled replication forks.

It was initially proposed that the Sgs1 and Srs2 helicases perform redundant functions in DNA replication and RNA pol I transcription (40). However, the DNA replication and RNA pol I defects reported are in fact likely to be secondary consequences of unrestrained recombination (20). Nevertheless, the original suggestion of functional overlap between these two helicases (40) remains possible. Consistent with this idea, we found the _srs2_Δ mutant to have a similar genotoxin sensitivity profile and replicative lifespan to the _sgs1_Δ mutant. Moreover, increased gene dosage of SGS1 partially restored genotoxin resistance and increased the maximum lifespan in the _srs2_Δ mutant. This indicates that Sgs1 can partially compensate for the loss of Srs2.

MATERIALS AND METHODS

Yeast strains and plasmids

Yeast strains and plasmids used in this study are described briefly in Table 1. The BY4743 strain was obtained from ATCC (Manassas, TN), and all other strains were obtained from Research Genetics (Groningen, The Netherlands). All deletion mutants were prepared by deletion module PCR using the Kan MX4 cassette (Saccharomyces Genome Deletion Project).

The pYES2 vector was obtained from Invitrogen (Groningen, The Netherlands). The pQE30 plasmid was obtained from Qiagen (Dorking, UK). The YCplac33 and YEplac195 vectors were gifts from Dr Alan Boyd (University of Edinburgh, Edinburgh, UK). The pNF3000 plasmid, which carries the RAD3 gene controlled by its endogenous promoter in the YEp24 vector, was generously provided by Drs Paula Fischaber and Errol Friedberg (University of Texas Southwestern Medical Center, Dallas, TX).

Plasmid construction

The YCplac33-SRS2 and YEplac195-SRS2 plasmids were constructed as follows. The 3525 bp SRS2 open reading frame (ORF) plus 610 bp upstream and 132 bp downstream was amplified from yeast genomic DNA (Promega, Southampton, UK) using the following primers: CATGAACCTGGATCCATATATAGAAATCGG (sense, _Bam_HI site underlined) and CATTAAGGGTACCTAACACACCATCCACATTTC (antisense, _Kpn_I site underlined). The PCR product was digested with _Bam_HI and _Kpn_I and ligated into _Bam_HI/_Kpn_I-digested YCplac33 and YEplac195 vectors. The pYES2-SRS2 plasmid was constructed by amplifying the 3.525 kb SRS2 ORF from YEplac195-SRS2 using the primers GGGAGGATCCATGTCGTCGAACAATGATCTTTG (sense, _Bam_HI site underlined) and GAAACTCGAGCTAATCGATGACTATGATTTCAC (antisense, _Xho_I site underlined). The PCR product was digested with _Bam_HI and _Xho_I and ligated into _Bam_HI/_Xho_I-digested pYES2 vector.

The YEplac195-SGS1 and YEplac195-SGS1 K706→A constructs were made by subcloning the 4.8 kb _Sal_I fragment containing the SGS1 gene from YCplac33-SGS1 and YCplac33-SGS1 K706→A (23) into _Sal_I-digested YEplac195. The pYES2-SGS1 plasmid was constructed by amplifying the 4.344 kb SGS1 ORF from yeast genomic DNA using the following primers: GTACACACAAGGGATCCATGGTGACGAAGC (sense, _Bam_HI site underlined) and TTTCCTCGAGTCACTTTCTTCCTCTGTAGT (antisense, _Xho_I site underlined). The PCR product was digested with _Bam_HI and _Xho_I and ligated into _Bam_HI/_Xho_I-digested pYES2 vector.

The pQE30-SGS1(1-400) plasmid was constructed as follows. The 1200 bp fragment of SGS1 was amplified from YCplac33-SGS1 using the following primers: GTACACACAAGGGATCCATGGTGACGAAGC (sense, _Bam_HI site underlined) and TTCTTCTGGTACCACCTACGGATAATCGTC (antisense, _Kpn_I site underlined). The PCR product was digested with _Bam_HI and _Kpn_I and ligated into _Bam_HI/_Kpn_I-digested pQE30 plasmid.

Construction of SGS1 mutant libraries

Initially, the YEplac195-SGS1 plasmid was modified to remove a _Not_I restriction site carried over from the original cloning vector. Transposon-based mutagenesis was then performed on this modified plasmid using the E::Z™ In-frame linker insertion kit (Cambio, Cambridge, UK). This utilises the Tn5 in vitro transposition system (41). Mutagenesis was performed in vitro by incubating 0.03 pmol of EZ::TN<_Not_I/KAN-3> transposon and plasmid DNA with 1 µl of Tn5 transposase enzyme at 37°C for 2 h. The reaction was then stopped and the reaction mixture transformed into XL1 Blue E.coli (Stratagene, Amsterdam, The Netherlands). All resultant Kan+ Amp+ colonies were pooled and grown up prior to plasmid DNA isolation. The KanR gene was spliced out by digesting the midiprep with _Not_I, leaving a 57-bp sequence at the site of transposon insertion. This sequence codes for a 19-amino acid read-through insertion, which maintains the reading frame and so avoids creation of truncation mutants. The _Not_I-cut plasmid was gel purified, re-ligated in vitro and re-transformed into XL1 Blue E.coli. The resulting Amp+ colonies were used to construct both the random mutational library and the methyl methanesulphonate (MMS) loss-of-function library.

For the random mutational library, 190 Amp+ colonies were selected at random and grown up individually. Of these, PCR analysis identified 80 colonies that contained plasmids harbouring the 57-bp insertion within the SGS1 gene. These colonies were individually grown up prior to isolation of their plasmid DNA. The position of transposon insertion was determined by digesting plasmid minipreps with _Sal_I and _Not_I.

For the MMS loss-of-function library, all original Amp+ colonies harbouring transposon-mutagenised YEplac195-SGS1 were pooled and grown up. Their DNA was then isolated and transformed into the _sgs1_Δ mutant strain. Ura+ transformants (500) were then picked and patched individually onto 10 × 10 grids on SD-URA plates. Each plate also contained positive (_sgs1_Δ-YEplac195-SGS1) and negative (_sgs1_Δ-YEplac195) controls for comparison. Plates were incubated overnight at 30°C, and then replica plated onto plates containing 0.023% MMS and left for 2 days. Loss-of-function mutations (i.e. plasmids no longer able to rescue MMS sensitivity in the _sgs1_Δ strain) were identified on replica plates, and the corresponding colonies picked from the master plate. These were grown up, pooled and their DNA recovered and transformed into XL1 Blue E.coli. Forty-two Amp+ colonies were selected for further analysis of transposon insertion by restriction digest analysis. The remaining colonies were pooled, and their DNA isolated by midiprep.

Drug sensitivity plates

This was performed as described previously for _sgs1_Δ, using identical drug concentrations (23). For quantitation of strains grown on solid media supplemented with MMS, yeast strains were grown up in minimal media for 2–3 days at 30°C, diluted and then spread on solid media plates containing increasing doses of MMS. After 3 days, the percent viability of each strain was expressed as number of colonies obtained relative to that on the control (no MMS) plate. For quantitation of MMS sensitivity of strains in liquid media, yeast strains were grown to an OD600 of 0.5–0.6 before addition of 0.01–0.12% MMS for 60 min. Cells were washed in 10% sodium thiosulfate, diluted and spread onto solid media plates. After 3 days, the percent viability of each strain was expressed as number of colonies obtained relative to that obtained for the control samples.

Detection of Sgs1 and Srs2 by western blotting

Yeast extracts were prepared by bead-beating in 8 M-urea-containing 2D-gel extraction buffer (Bio-Rad, Hemel Hempstead, UK) and run on SDS–polyacrylamide gels prior to transfer onto nitrocellulose. Recombinant his-tagged Sgs1(1-400) protein was purified from E.coli transformed with the pQE30-SGS1(1-400) plasmid using Ni2+-NTA-agarose (Qiagen). The purified protein was then used for antibody production in rabbits by following a published method (42). The resulting Sgs1p 857 antiserum was then purified using protein G–Sepharose to yield the IgG fraction used for western blotting. The Srs2p (yA-19) and Sir2p (yN-19) goat polyclonal antibodies were obtained from Autogen Bioclear (Mile Elm, UK). All primary antibodies were routinely used at dilutions of 1:500. Immunoreactive bands were detected with peroxidase-conjugated secondary antisera and visualised using enhanced chemiluminescence.

Lifespan analysis

This was performed as described previously (23). Briefly, strains were grown at 30°C until they reached an OD600 of 0.6–1.0. One microlitre of culture was streaked onto plates and left at 30°C for 1–2 h. After this time, cell doublets were moved to uninhabited regions of the plate. When these budded again, (newly formed) virgin yeast cells were removed by micromanipulation to a new location. All future buds produced by these daughter cells were micromanipulated away, and catalogued. The plates were incubated at 30°C during working hours, and moved to 4°C overnight. Lifespan was defined as number of daughter cells removed from the mother cell. All lifespans were observed a minimum of twice.

Yeast transformation and DNA extraction

This was performed as described previously (23).

RESULTS

Deletion of the SGS1 gene in both haploid and diploid strains has been reported to result in a replicative lifespan of ∼40% that of isogenic wild-type strains (22–24). To determine whether deletion of SRS2 has a similar effect, lifespan analysis was performed on _srs2_Δ and BY4743 (wild-type) isogenic diploid strains transformed with the empty YCplac33 vector (Fig. 1A). Both strains were transformed with YCplac33 to allow direct comparison with previous findings (23). The mean lifespan of the BY4743-YCplac33 strain was 16.0 ± 1.0 (n = 76), compared with 7.2 ± 0.9 (n = 38) for the _srs2_Δ-YCplac33 strain. The maximum lifespans recorded were 44 and 23, respectively. The short lifespan of the _srs2_Δ mutant observed is comparable with that of the isogenic diploid _sgs1_Δ-YCplac33 strain, which has a mean lifespan of 7.4 ± 0.8 (n = 32) and maximum of 16 (23). A similar magnitude of shortened replicative lifespan has recently been reported for the haploid _sgs1_Δ and _srs2_Δ strains (38). Therefore, deletion of SGS1 or SRS2 reduces the replicative lifespan to a similar extent in both haploid and diploid backgrounds.

We have previously characterised the sensitivity of the _sgs1_Δ mutant to MMS (DNA methylating agent), 4-NQO (causes bulky-base adducts and oxidative damage), hydroxyurea (DNA synthesis inhibitor), camptothecin (Type I topoisomerase inhibitor) and mitoxantrone (Type II topoisomerase inhibitor) (23). To assess the sensitivity of the _srs2_Δ mutant to these drugs, the diploid _srs2_Δ mutant was transformed with YCplac33 (empty vector) and YCplac33-SRS2 and compared with the BY4743 isogenic wild-type strain transformed with YCplac33. Various dilutions of _srs2_Δ-YCplac33, BY4743-YCplac33 and _srs2_Δ-YCplac33-SRS2 strains were grown on plates containing the above drugs (Fig. 1B). Similar to the _sgs1_Δ mutant, the _srs2_Δ-YCplac33 strain showed sensitivity to all DNA damaging agents tested. Transformed _srs2_Δ cells containing the YCplac33-SRS2 plasmid showed comparable drug sensitivity to the BY4743 wild-type strain under these conditions. The observation that plasmid-borne SRS2 can restore genotoxin resistance to isogenic wild-type levels thus confirms that deletion of the SRS2 gene is the sole defect in the _srs2_Δ mutant.

To assess the specificity of the drug sensitive phenotype of the _sgs1_Δ and _srs2_Δ strains, the MMS sensitivity of other yeast mutants lacking DNA helicases was assessed. The _pif1_Δ, _rrm3_Δ, _yKU70_Δ and _yKU80_Δ mutants were tested. Pif1 and Rrm3 are DNA helicases involved in regulating telomeric and mitochondrial DNA (reviewed in 43), and are also involved in the replication fork progression through the rDNA (44). The y KU70 and y KU80 gene products are involved in the repair of DNA double strand breaks (reviewed in 45) and physical and genetic interactions between RecQ helicases and Ku proteins have been demonstrated (46,47). It was found that the _pif1_Δ mutant showed a slight sensitivity to MMS, as compared with the isogenic wild-type strain. The _rrm3_Δ, _yKU70_Δ and _yKU80_Δ strains were not sensitive to MMS (results not shown). Therefore, MMS sensitivity appears to be a relatively specific phenotype for loss-of-function of either the Sgs1 or Srs2 helicases.

Based on the fact that either single mutant is viable, but the double mutant has a severe growth defect, it has been proposed that the Sgs1 and Srs2 helicases may be functionally redundant (40). We therefore reasoned that overexpression of either of these DNA helicases might compensate for loss of the other. To initially confirm overexpression, Sgs1 and Srs2 levels in vivo were assessed by western blotting. Protein was extracted from _sgs1_Δ-pYES2, _sgs1_Δ-pYES2-SGS1, _srs2_Δ-pYES2 and _srs2_Δ-pYES2-SRS2 cultures induced with galactose. Extracted protein samples were separated by SDS–PAGE, western blotted and probed for either Sgs1 or Srs2. An ∼160 kDa band corresponding to Sgs1 was present in the _sgs1_Δ_-_pYES2-SGS1, but not _sgs1_Δ-pYES2, protein extract (Fig. 2A). Similarly, an ∼150 kDa band corresponding to Srs2 was present in the _srs2_Δ-pYES2-SRS2, but not _srs2_Δ-pYES2, protein extract (Fig. 2A). For a control, all samples were probed for Sir2p, which was equally present in all protein extracts.

Although expression of SGS1 or SRS2 from the galactose-inducible pYES2 vector resulted in detectable levels of Sgs1 or Srs2 protein, it was not possible to detect Sgs1 by western blotting when SGS1 was expressed under its own promoter from the YEplac195 vector. We therefore attempted to genetically verify the overexpression of Sgs1 from this plasmid. Sgs1 helicase activity has been postulated to produce a substrate that is deleterious unless resolved by Top3 (11,48). Sgs1 overexpression should therefore be more harmful in _top3_Δ mutants than in TOP3 strains. To test this, _top3_Δ mutant and isogenic wild-type control strains were transformed with both low-copy (YCplac33) and high-copy (YEplac195) SGS1 plasmids (Fig. 2B). Both strains gave viable Ura+ transformants when transformed with the YCplac33, YCplac33-SGS1, YEplac195 and YEplac195-SGS1 K706→A plasmids. However, transformation of both strains with YEplac195-SGS1 gave viable Ura+ transformants in the wild-type strain, but very few viable colonies with the _top3_Δ mutant. Therefore, expression of Sgs1 from the multicopy YEplac195 vector, but not the low-copy YCplac33 plasmid, was detrimental to the _top3_Δ mutant. This indicates that increased gene dosage of Sgs1 does indeed result in increased Sgs1 protein levels. Furthermore, the deleterious effects of higher Sgs1 levels in the _top3_Δ mutant required the helicase activity of Sgs1, consistent with the notion that the helicase activity of Sgs1 creates a substrate that Top3 resolves (11,48).

To assess if Sgs1 and Srs2 are functionally interchangeable, the ability of high-copy plasmids carrying SGS1 and SRS2 to rescue MMS sensitivity in the _sgs1_Δ and _srs2_Δ strains was assessed (Table 2). To control for non-specific effects of helicase overexpression, the high-copy pNF3000 plasmid carrying RAD3 (a 5′→3′ DNA helicase involved in excision repair) (49) was also compared. As a further specificity control, the effects of these plasmids on the (slight) MMS sensitivity of the _pif1_Δ mutant were also investigated. High-copy plasmids encoding SRS2 (both YEplac195-SRS2 and pYES2-SRS2) were able to complement the MMS sensitivity of the _srs2_Δ mutant, but had no obvious beneficial effects in either the _sgs1_Δ or _pif1_Δ mutants. Rather, Srs2 overexpression was found to have a dominant negative effect on growth of _sgs1_Δ, _pif1_Δ and wild-type strains. High-copy vectors expressing SGS1 (YEplac195-SGS1 and pYES2-SGS1) were able to complement the MMS sensitivity of both the _sgs1_Δ and _srs2_Δ mutant, but not the _pif1_Δ mutant. Sgs1 helicase activity was essential for complementation as YEplac195-SGS1 K706→A (expressing a helicase-defective allele which has some complementing activity in the _sgs1_Δ mutant) (23,50) was not able to suppress the MMS sensitivity of the _sgs1_Δ or _srs2_Δ mutant. The pNF3000 plasmid was unable to rescue the MMS sensitivity of all three mutants tested, although this plasmid has been demonstrated previously to complement the UV sensitivity of a rad3-1 strain (49).

The ability of SGS1 to rescue the sensitivity of the _srs2_Δ mutant to MMS and hydroxyurea is shown in Figure 3A. This demonstrates that the ability of SGS1 to act as a multicopy suppressor of _srs2_Δ is not uniquely specific for sensitivity to DNA damaging agents, but is also seen for inhibitors of DNA replication. To quantify the SGS1 complementation of _srs2_Δ MMS sensitivity, _srs2_Δ-YEplac195-SGS1, _srs2_Δ-YEplac195 and BY4743-YEplac195 diploid strains were grown up, diluted and then spread onto plates containing increasing doses of MMS. After 3 days, the percent viability of each strain was expressed as number of colonies obtained relative to that on the control (no MMS) plate. It can be seen that at the 0.003% dose, where complete killing of _srs2_Δ-YEplac195 cells occurred, the YEplac195-SGS1 construct restored MMS resistance to ∼60% of that seen in the isogenic wild-type (Fig. 3B). A similar effect was observed when cells were exposed to MMS for 1 h before removal and inactivation of the drug (Fig. 3C). Under these conditions, overexpression of SGS1 restored viability to 30–40% of wild-type levels at doses where complete killing of control _srs2_Δ-YEplac195 cells occurred.

Mutational analysis suggests that Sgs1 is a multifunctional protein, as mutant alleles of sgs1 have selective complementing ability in different phenotypic assays (17,21,23,51). We therefore set out to determine whether the domains of Sgs1 required for rescue of MMS sensitivity in the _sgs1_Δ mutant differ from those required for complementation of the _srs2_Δ mutant. In order to do this, we used in vitro transposon-scanning mutagenesis on the YEplac195-SGS1 plasmid to create two distinct libraries of sgs1 mutants containing in-frame 19-amino acid insertions (see Materials and Methods). The unselected mutational library was generated by individually growing up randomly chosen bacterial colonies and PCR screening for the presence of plasmids harbouring the 57-bp insertion within the SGS1 gene. Figure 4A shows the frequency and approximate location of in-frame transposon insertions in SGS1 in this library, which comprises 75 alleles. It can be seen that there is coverage of insertion events throughout the entire SGS1 gene with no obvious hotspots, suggesting that transposon insertion is basically random. To construct a library of sgs1 MMS loss-of-function plasmids (i.e. plasmids no longer able to rescue MMS sensitivity in the _sgs1_Δ strain), _sgs1_Δ cells were transformed with the entire original pool of transposon-mutagenised YEplac195-SGS1. Of the resulting Ura+ transformants, 500 were picked and grown up individually on 10 × 10 grids before replica plating onto MMS plates. One hundred colonies failed to grow on MMS plates; these were recovered from the master plate, and their pooled plasmid DNA isolated to create the MMS loss-of-function library. Restriction analysis of 42 alleles chosen at random from this library revealed a more restricted distribution of insertion events than in the unselected library (compare Fig. 4A and B). Most notably, there was a complete absence of insertions in the C-terminal 250 amino acids of Sgs1, a domain known to be dispensable for MMS resistance (17,51) (Fig. 4B).

To determine whether the same functional domains of SGS1 are also required for complementation of _srs2_Δ, the _srs2_Δ mutant was transformed with the MMS loss-of-function SGS1 library (more than 500 Ura+ transformants obtained). For a control, the _srs2_Δ mutant was also transformed with the unselected library of 75 transposon-mutagenised SGS1 plasmids characterised in Figure 4A (more than 200 Ura+ transformants obtained). Colonies were resuspended, diluted and spread on control (no MMS) and 0.0035% MMS plates (a dose at which the _srs2_Δ mutant fails to grow). For the unselected SGS1 plasmid library, 156 colonies were obtained in total, compared with 378 colonies obtained on the control (no MMS) plates. This verifies that insertion of the 57-bp linker sequence in the SGS1 gene is not detrimental per se. In contrast, for the MMS loss-of-function SGS1 plasmid library, none of the MMS loss-of-function plasmids (in the _sgs1_Δ background) could rescue MMS sensitivity in the _srs2_Δ mutant. Therefore, we conclude that the same domains of SGS1 are required to restore MMS resistance to both _sgs1_Δ and _srs2_Δ strains, suggesting that a common molecular mechanism of action underlies complementation in both mutants.

It has been reported that sensitivity to MMS is enhanced in diploid _srs2_Δ strains relative to haploids (28). This ploidy-specific phenotype has been interpreted as additional lethal recombination events occurring between homologous chromosomes as well as sister chromatids. We therefore set out to determine whether the ability of SGS1 to complement MMS sensitivity in the _srs2_Δ mutant is due to suppression of recombination between homologous chromosomes. Haploid _sgs1_Δ and _srs2_Δ strains were used for these studies and compared with the isogenic wild-type BY4741 strain. To directly compare any ploidy-specific phenotypes, both haploid and diploid BY4741, _sgs1_Δ and _srs2_Δ mutant strains were grown on plates ± MMS (Fig. 5A). As reported previously (28), the diploid _srs2_Δ strain was more MMS sensitive than the haploid _srs2_Δ strain. The observed MMS sensitivity of strains (in order of most MMS sensitive) was as follows: _srs2_Δ diploid > _sgs1_Δ haploid = _sgs1_Δ diploid > _srs2_Δ haploid > BY4741 haploid = BY4741 diploid.

To test for any ploidy-specific effects of Sgs1 and Srs2 overexpression, haploid strains transformed with YEplac195, YEplac195-SGS1, YEplac195-SGS1 K706→A or YEplac195-SRS2 were grown on plates ± MMS. Both the haploid _sgs1_Δ-YEplac195 and _srs2_Δ-YEplac195 strains were sensitive to 0.01% MMS, whereas BY4741-YEplac195 was unaffected at these doses (Fig. 5B). The ability of YEplac195-SGS1 (and not YEplac195-SGS1 K706→A) to suppress MMS sensitivity was reproducible in both the _sgs1_Δ and _srs2_Δ haploid strains. Similarly, YEplac195-SRS2 could rescue the MMS-sensitive phenotype of the haploid _srs2_Δ mutant, but again no beneficial effects in the _sgs1_Δ haploid were evident. This demonstrates that the ability of SGS1 to function as a multicopy suppressor of the _srs2_Δ mutant is not a ploidy-specific effect.

In addition to the MMS-sensitive phenotype, the ability of the YEplac195-SGS1 plasmid to rescue the shortened replicative lifespan of the _srs2_Δ mutant was investigated. Lifespan analysis was performed on the _srs2_Δ-YEplac195 and _srs2_Δ-YEplac195-SGS1 strains (Fig. 6). Average lifespans of the strains [expressed as mean number of buds removed ± standard error of the mean (SEM)] were 11.1 ± 1.2 (n = 37) for _srs2_Δ-YEplac195 and 13.3 ± 1.4 (n = 38) for _srs2_Δ-YEplac195-SGS1. The maximum lifespans recorded were 24 and 36, respectively. Although a Student’s _t_-test revealed there to be no significant difference between the mean lifespans, a χ2 test on the 10% longest-lived subpopulations (52) revealed that Sgs1 overexpression caused a statistically significant increase in _srs2_Δ lifespan (P < 0.02).

DISCUSSION

Phenotypic overlap between _sgs1_Δ and _srs2_Δ mutants

Mutant sgs1 and srs2 strains have been shown to exhibit hyper-recombination, defective intra-S checkpoint responses and shortened replicative lifespans (13,18,27,37,38). The _sgs1_Δ mutant is also known to be sensitive to a variety of genotoxic agents (12,16,17,19,23). We report here that the _srs2_Δ mutant is similarly sensitive to MMS, hydroxyurea, camptothecin, mitoxantrone and 4-NQO, reinforcing the phenotypic overlap with the _sgs1_Δ mutant. In addition to its genotoxin sensitivity, the _sgs1_Δ mutant has a shortened replicative lifespan (22–24). We found that the _srs2_Δ mutant has a mean replicative lifespan ∼45% of that of the wild-type strain. This is comparable with the average lifespan recorded for the isogenic _sgs1_Δ mutant (43%) (23), revealing a similar magnitude of shortened lifespan for the diploid _sgs1_Δ and _srs2_Δ mutants.

These findings are in agreement with a recent report documenting the phenotypes of the haploid _sgs1_Δ and _srs2_Δ mutants (38). In that work, it was revealed that the previous characterisation of premature ageing in the sgs1 mutant as a phenocopy of normal ageing (24) was not entirely accurate. Instead, it was concluded that the short lifespan exhibited by both _sgs1_Δ and _srs2_Δ mutants was due to a combination of ageing-independent mitotic checkpoint arrest and accelerated normal ageing (38). To determine whether the increased G2/M checkpoint arrest phenotype was also apparent in our diploid strains, we followed the approach of McVey et al. (38) and re‐analysed our lifespan data for incidence of terminally budded phenotypes. This analysis was performed blind as our lifespan studies were completed before publication of their report. The morphology of _sgs1_Δ and _srs2_Δ terminal phenotypes revealed a higher percentage of budded mitotic intermediates than that observed for the isogenic wild-type strain (data not shown), thus confirming the findings of McVey et al. (38). As the severe phenotype of sgs1srs2 double mutants can be suppressed by inactivation of Rad51, Rad52 or Rad57, it is likely that it results from unrestrained recombination (38), in keeping with the idea that both Sgs1 and Srs2 act to prevent such events during S phase.

Complementation of the _srs2_Δ mutant by SGS1

It has been suggested that the Sgs1 and Srs2 helicases may be functionally redundant, based on the fact that either single mutant is viable, but the double mutant has a severe growth defect (40). Although complete redundancy can be ruled out due to the abundance of phenotypes exhibited by each single mutant (see above), the similarities of these phenotypes are consistent with a degree of functional overlap. Our finding that SGS1 can function as a multicopy suppressor of the MMS and hydroxyurea sensitivity of the _srs2_Δ mutant provides direct support for this notion. In contrast, high-copy SRS2 was unable to complement genotoxin sensitivity of the _sgs1_Δ strain. One explanation of this finding is that Sgs1 performs some cellular function(s) that cannot be substituted by Srs2, despite the ability of Sgs1 to functionally replace Srs2. However, it is worth noting that, as reported previously (53), we found that higher levels of Srs2 caused detrimental effects in the wild-type strain.

Sgs1 is a large protein that, in addition to the RecQ helicase domain, has distinct domains required for interactions with Top2, Top3 and Rad51 (11,54–57). Furthermore, mutant alleles of sgs1 have selective complementing ability in different phenotypic assays (17,23,50,51). Therefore, the domains of Sgs1 required for rescue of MMS sensitivity in the _sgs1_Δ background could potentially differ from those required for complementation of the _srs2_Δ mutant. To address this issue, we constructed libraries of SGS1 mutant plasmids containing small in-frame insertions. Unsurprisingly, a large proportion of loss-of-function mutants for MMS resistance in the _sgs1_Δ background contained transposon insertions in or around the helicase domain of SGS1. This is consistent with reports that the helicase activity of Sgs1 is required for dealing with MMS-induced DNA damage (17,21,23,51,58). Some insertions were also found N-terminal to the helicase domain, mapping close to the interaction sites for Top2 and Top3. This supports the observation that the N-terminus of Sgs1 is essential for MMS resistance (17). Interestingly, no MMS loss-of-function alleles mapped to the C-terminus of Sgs1, where the Rad51 interaction occurs (57). Again, this is consistent with reports that much of the C-terminus of Sgs1 (the final 254 amino acids) is dispensable for MMS resistance (17,51). It was found that none of these SGS1 loss-of-function plasmids tested could complement the _srs2_Δ mutant. Similarly, a point mutation causing inactivation of Sgs1 helicase activity (50) was unable to restore MMS resistance to the _srs2_Δ strain. This demonstrates that complementation of the _srs2_Δ mutant by high-copy SGS1 requires domains of Sgs1 that are directly involved in processing MMS-induced DNA lesions.

When SGS1 was over expressed in the _srs2_Δ mutant, no significant increase in the mean replicative lifespan was evident. This contrasts with the ability of SGS1 overexpression to suppress MMS sensitivity of the _srs2_Δ mutant. One possible reason for this is that other important roles of Srs2 may still be lacking and contribute to the shortened mean replicative lifespan of the _srs2_Δ mutant. Alternatively, the beneficial effects of SGS1 overexpression may be outweighed by detrimental effects associated with high levels of Sgs1. Several observations support the latter theory. First, SGS1 overexpression can increase the maximum lifespan of the _srs2_Δ mutant by 50%. Secondly, SGS1 overexpression has been reported to cause detrimental effects (18,24). Thirdly, _srs2_Δ cells over expressing SGS1 actually show an increase in terminally budded morphologies as compared with control _srs2_Δ mutants containing empty vector (data not shown). It is remarkable that high-copy SGS1 can extend maximum lifespan in the face of such deleterious effects.

Cellular functions of Sgs1 and Srs2

Proteins involved in recombinational repair (e.g. Rad52) and post-replication repair (e.g. Rad6 and Rad18) play important roles in processing DNA lesions induced by MMS, in an analogous manner to that proposed for the processing of UV-induced lesions (59). Srs2 is thought to channel lesions into post-replication repair pathways, whereas Sgs1 is thought to be more intimately involved in recombinational repair (2,57,60) (Fig. 7). The ability of Sgs1 overexpression to process MMS-induced DNA damage in the _srs2_Δ mutant implies that endogenous Sgs1 levels are normally limiting in the _srs2_Δ mutant. This effect of Sgs1 overexpression may be to increase the efficiency of Sgs1’s normal physiological function(s), thereby reducing the requirement for Srs2-dependent repair pathways. Alternatively, it may be that Sgs1 is capable of directly replacing Srs2, by processing substrates that would normally be dealt with by Srs2.

A variety of data support the notion that the _sgs1_Δ and _srs2_Δ phenotypes are a consequence of unrestrained recombination (20). Consistent with this, mutations in the RAD51 or RAD52 genes can suppress the MMS sensitivity of the _srs2_Δ mutant (61), whereas overexpression of RAD51 or RAD52 enhances the MMS sensitivity of the _srs2_Δ mutant (62). Therefore, in the absence of Srs2, the Rad52-dependent recombination repair pathway is over-active (31), leading to aberrant processing of recombination intermediates and genomic instability (Fig. 7). However, it is worth noting that, in addition to its well-established anti-recombinase function, Srs2 has recently been claimed to promote recombination in some circumstances (63). The absence of Sgs1 would also be predicted to cause aberrant processing of recombination intermediates as not only are the later steps of the homologous recombination repair pathway impaired (57,64), but also aberrant recombination intermediates cannot be ‘salvaged’ by Sgs1 (14). This model is consistent with observations that the synthetic growth defect in sgs1srs2 double mutants (40) can be rescued by mutation of genes involved in homologous recombination (20,38,39).

Although we favour the idea that Sgs1 overexpression rescues MMS sensitivity in the _srs2_Δ mutant by suppressing unrestrained recombination, it remains possible that the ability of Sgs1 overexpression to compensate for lack of Srs2 is unrelated to DNA recombination. This possibility is supported by the fact that inactivation of the homologous recombination pathway does not suppress the severe synthetic growth defects observed in the _S.pombe srs2_Δ_rqh1_Δ double mutant (29). Nevertheless, the fact that genetic interactions between SGS1/rqh1 + and SRS2 are evident in two such evolutionarily divergent species further supports the notion of functional overlap between these DNA helicases. It will be of interest to assess if overexpression of rqh1 + can rescue any phenotypes of the _S.pombe srs2_Δ mutant.

Wider implications of functional overlap between DNA helicases

These findings may have potentially important ramifications for studies of the human RecQ helicases. First, the physiological effects of overexpression of Sgs1 may warrant further characterisation, as higher levels of both the BLM and WRN helicases have been reported to exist in transformed cells and tumour cells, relative to those found in normal cells (65,66). Furthermore, the finding that RecQ helicases can show partial functional redundancy with other DNA repair pathways may be significant for the understanding of the functions of RecQ family members in maintenance of genomic stability. In addition to the functional overlap between Sgs1 and Srs2 demonstrated here, an extra copy of Ku70, a DNA repair helicase, can partially rescue the phenotypes of Drosophila lacking the RecQ helicase Dmblm (47). This functional overlap of DNA repair pathways implies that defects in human RecQ helicases would have more severe effects under conditions where the redundant pathways are limiting. This may be important in understanding the role of the human RecQ helicases in genomic stability, cancer and ageing.

ACKNOWLEDGEMENTS

We gratefully acknowledge Drs Paula Fischaber and Errol Friedberg for the generous gift of the pNF3000 plasmid. This work was supported by the Wellcome Trust in the form of Prize Studentships to H.W.M. and T.J.C.

*

To whom correspondence should be addressed. Tel: +44 151 794 5333; Fax: +44 151 794 5337; Email: amorgan@liv.ac.uk

Figure 1. The diploid _srs2_Δ mutant has a shortened replicative lifespan and is sensitive to genotoxins. (A) Lifespan analysis. The _srs2_Δ and isogenic wild-type BY4743 diploid strains were transformed with YCplac33 vector and the replicative lifespan determined manually by micromanipulation. Individual cells were followed until they ceased dividing, and replicative age was defined as the number of buds removed from a mother cell. The survivorship curve shows the percentage of the population of yeast cells that is viable plotted against replicative age. Average lifespan of the strains (expressed as mean number of buds removed ± SEM) was 16 ± 1.0 (n = 76) for the BY4743-YCplac33 strain and 7.2 ± 0.9 for the _srs2_Δ-YCplac33 strain (n = 38). Maximum lifespans recorded were 44 and 23, respectively. (B) Genotoxin sensitivity. Strains were grown up, diluted and serially spotted out onto solid media plates containing various drugs. Plates were left for 3–4 days at 30°C. Cell spots (from left to right) correspond to serial 1 in 10 dilutions of cells (starting, furthest left, with OD600 = 0.33). The diploid strains compared were _srs2_Δ-YCplac33 (_srs2_Δ), _srs2_Δ-YCplac33-SRS2 (srs2_Δ_-SRS2+) and the wild-type control, BY4743-YCplac33 (BY4743). Drug doses used were as follows: 50 mM hydroxyurea, 0.33 mM camptothecin, 0.0066% MMS, 1 mM mitoxantrone and 0.5 µM 4-NQO.

Figure 2. Confirming overexpression of the Sgs1 and Srs2 helicases in vivo. (A) The ability to detect Sgs1 and Srs2 in vivo was assessed by western blotting. Protein was extracted from the _sgs1_Δ-pYES2, _sgs1_Δ-pYES2-SGS1, _srs2_Δ-pYES2 and _srs2_Δ-pYES2-SRS2 strains incubated in galactose-containing media to induce expression. Yeast proteins were extracted, separated by SDS–PAGE and probed for Sgs1 or Srs2. For control, the samples were probed for Sir2. (B) Transformation of the MATa_top3_Δ mutant and isogenic wild-type control BY4742 strains with both low-copy (YCplac33) and high-copy (YEplac195) SGS1 plasmids was compared. Both strains were transformed with equal amounts of YCplac33, YCplac33-SGS1, YEplac195, YEplac195-SGS1 and YEplac195-SGS1 K706→A plasmids. Equivalent amounts of each transformation mixture were spread on SD-URA solid media plates and incubated at 30°C. After 3 days, plates were assessed for numbers of Ura+ transformants.

Figure 3.SGS1 is a multicopy suppressor of the drug sensitivity of the _srs2_Δ mutant. (A) The diploid _srs2_Δ mutant was transformed with a range of constructs subcloned into the high-copy vector, YEplac195. Strains were grown up in minimal media, then diluted and serially spotted out onto control (no drug) plates and plates containing either 0.0062% MMS or 50 mM hydroxyurea. Plates were left at 30°C for 3 days. Cell spots (from left to right) correspond to serial 1 in 10 dilutions of cells. (B) To quantify the _srs2_Δ_-_complementing ability of SGS1, strains were grown up as above and then spread onto plates containing increasing doses of MMS. The number of colonies visible after 3 days was then counted on each plate. For each strain, percent viability was expressed as a percentage of viable colonies obtained on the control (no MMS) plates. The diploid strains compared were _srs2_Δ-YEplac195 (_srs2_Δ), _srs2_Δ-YEplac195-SGS1 (_srs2_Δ-SGS1+) and the isogenic wild-type control, BY4743-YEplac195 (Wild type). Data presented are pooled from two independent experiments and are expressed as mean ± range. (C) To quantify the _srs2_Δ_-_complementing activity of SGS1 in liquid media, strains were grown up before addition of MMS for 60 min. MMS was then inactivated, washed off and yeast spread on solid media plates. The number of colonies visible after 3 days was then counted on each plate. For each strain, percent viability was expressed as a percentage of viable colonies obtained for the control (no MMS) samples. The diploid strains compared were _srs2_Δ-YEplac195 (_srs2_Δ), _srs2_Δ-YEplac195-SGS1 (_srs2_Δ-SGS1+) and the isogenic wild-type control, BY4743-YEplac195 (Wild type). Data presented are pooled from two independent experiments and are expressed as mean ± range.

Figure 4. Transposon-scanning mutagenesis of SGS1. (A) Bar chart showing frequency and location of in-frame transposon insertions in a non-selected library. The approximate location of transposon insertion was determined for 75 randomly chosen alleles by analysis of restriction endonuclease digests of mutagenised SGS1 plasmids. (B) Bar chart showing frequency and location of in-frame transposon insertions that abolish MMS complementation. Alleles of SGS1 (100) were selected which failed to rescue the MMS sensitivity of the _sgs1_Δ mutant. Forty-two of these alleles had their location of transposon insertion determined by analytical restriction digest mapping. The approximate positions of regions required for interaction with Top2 and Top3, the helicase domain and a region dispensable for MMS resistance (MMS–) are indicated.

Figure 5. Complementation of _srs2_Δ MMS sensitivity by SGS1 is ploidy independent. (A) MMS sensitivity of haploid and diploid wild-type, _sgs1_Δ and _srs2_Δ strains. Strains transformed with the empty YEplac195 vector were grown up, diluted and spotted out onto plates ± MMS. Plates were left at 30°C for 3 days. Cell spots (from left to right) correspond to serial 1 in 10 dilutions of cells. (B) MMS sensitivity of MATa haploid wild-type, _sgs1_Δ and _srs2_Δ strains transformed with high-copy SGS1 and SRS2 plasmids. Each strain was transformed with YEplac195 (vector), YEplac195-SRS2 (SRS2+), YEplac195-SGS1 (SGS1+) and YEplac195-SGS1 K706→A (SGS1 K706→A+). Strains were grown up in minimal media, then diluted and serially spotted out onto control (no MMS) plates and plates containing 0.01% MMS. Plates were left at 30°C for 3 days. Cell spots (from left to right) correspond to serial 1 in 10 dilutions of cells.

Figure 6. Effect of multicopy SGS1 on the short lifespan of the _srs2_Δ mutant. The diploid _srs2_Δ mutant strain was transformed with YEplac195 and YEplac195-SGS1 and the replicative lifespan determined manually by micromanipulation. Individual cells were followed until they ceased dividing, and replicative age was defined as the number of buds removed from a mother cell. The survivorship curve shows the percentage of the population of yeast cells that are viable plotted against replicative age. Average lifespan of the strains (expressed as mean number of buds removed ± SEM) was 11.1 ± 1.2 (n = 37) for _srs2_Δ-YEplac195 (_srs2_Δ-vector) and 13.3 ± 1.4 (n = 38) for _srs2_Δ-YEplac195-SGS1 (_srs2_Δ-SGS1+). Maximum lifespans recorded were 24 and 36, respectively.

Figure 7. Model of Sgs1 and Srs2 function in maintaining genomic stability. A simplified diagram summarising the likely roles of Sgs1 and Srs2 is presented. It is probable that the short lifespan and genotoxin sensitivity in the _srs2_Δ mutant is due to unrestrained recombination. If levels of Sgs1 are limiting, the ability to antagonise aberrant recombination and promote conservative homologous recombination may thus be overwhelmed. Therefore, increased levels of Sgs1 may allow _srs2_Δ mutant cells to deal with this situation. Alternatively, it may be that higher levels of Sgs1 can directly replace Srs2 and process substrates that would normally be dealt with by Srs2.

Table 1.

Saccharomyces cerevisiae strains and plasmids used in this study

Yeast strains used in this study, and their genotypes, are shown. Additionally, features of plasmids used are briefly described. All plasmids contain the AmpR selectable marker for propagation in E.coli.

*ARS1 CEN4 plasmids are single-copy plasmids, whereas 2_µ_ ori plasmids are multicopy plasmids.

Table 1.

Saccharomyces cerevisiae strains and plasmids used in this study

Yeast strains used in this study, and their genotypes, are shown. Additionally, features of plasmids used are briefly described. All plasmids contain the AmpR selectable marker for propagation in E.coli.

*ARS1 CEN4 plasmids are single-copy plasmids, whereas 2_µ_ ori plasmids are multicopy plasmids.

Table 2.

Ability of various plasmids to rescue the MMS sensitivity of the diploid _sgs1_Δ, _srs2_Δ or _pif1_Δ strains

All vectors used are 2_µ_-based high-copy plasmids. The YEplac195 and YEp24 plasmids possess the genes of interest under the control of their own endogenous promoter. The pYES2-based vectors contain the gene of interest under the control of the inducible GAL1 promoter. The pNF3000 plasmid contains the RAD3 gene under control of its own endogenous promoter. Ability to rescue MMS sensitivity is defined as clearly increased colony growth relative to an empty vector control. +, rescue; –, no rescue; N.D., not determined.

*Plasmids grown in raffinose medium, which allows ‘leaky’ expression from the GAL1 promoter. Strains containing the pYES2-SRS2 plasmid failed to grow in galactose-containing medium.

Table 2.

Ability of various plasmids to rescue the MMS sensitivity of the diploid _sgs1_Δ, _srs2_Δ or _pif1_Δ strains

All vectors used are 2_µ_-based high-copy plasmids. The YEplac195 and YEp24 plasmids possess the genes of interest under the control of their own endogenous promoter. The pYES2-based vectors contain the gene of interest under the control of the inducible GAL1 promoter. The pNF3000 plasmid contains the RAD3 gene under control of its own endogenous promoter. Ability to rescue MMS sensitivity is defined as clearly increased colony growth relative to an empty vector control. +, rescue; –, no rescue; N.D., not determined.

*Plasmids grown in raffinose medium, which allows ‘leaky’ expression from the GAL1 promoter. Strains containing the pYES2-SRS2 plasmid failed to grow in galactose-containing medium.

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