Bipartite Structure of the SGS1 DNA Helicase in Saccharomyces cerevisiae (original) (raw)

Journal Article

,

Department of Molecular Biology and Biochemistry

, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08855

Search for other works by this author on:

,

Department of Molecular Biology and Biochemistry

, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08855

Search for other works by this author on:

Department of Molecular Biology and Biochemistry

, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08855

Corresponding author: Steven Brill, Department of Molecular Biology and Biochemistry, Rutgers University, 679 Hoes Ln., CABM, Piscataway, NJ 08855. E-mail: brill@mbcl.rutgers.edu

Search for other works by this author on:

Received:

09 September 1999

Accepted:

06 December 1999

• PDF
• Split View
• • Article contents
  • Figures & tables
  • Video
  • Audio
  • Supplementary Data

Navbar Search Filter Mobile Enter search term Search

Abstract

SGS1 in yeast encodes a DNA helicase with homology to the human BLM and WRN proteins. This group of proteins is characterized by a highly conserved DNA helicase domain homologous to Escherichia coli RecQ and a large N-terminal domain of unknown function. To determine the role of these domains in SGS1 function, we constructed a series of truncation and helicase-defective (-hd) alleles and examined their ability to complement several sgs1 phenotypes. Certain SGS1 alleles showed distinct phenotypes: sgs1-hd failed to complement the MMS hypersensitivity and hyper-recombination phenotypes, but partially complemented the slow-growth suppression of top3 sgs1 strains and the top1 sgs1 growth defect. Unexpectedly, an allele that encodes the amino terminus alone showed essentially complete complementation of the hyper-recombination and top1 sgs1 defects. In contrast, an allele encoding the helicase domain alone was unable to complement any sgs1 phenotype. Small truncations of the N terminus resulted in hyperrecombination and slow-growth phenotypes in excess of the null allele. These hypermorphic phenotypes could be relieved by deleting more of the N terminus, or in some cases, by a point mutation in the helicase domain. Intragenic complementation experiments demonstrate that both the amino terminus and the DNA helicase are required for full SGS1 function. We conclude that the amino terminus of Sgs1 has an essential role in SGS1 function, distinct from that of the DNA helicase, with which it genetically interacts.

THE SGS1 gene of Saccharomyces cerevisiae is a member of the RecQ family of DNA helicases that includes the human genes BLM (Ellis et al. 1995), WRN (Yu et al. 1996), and RECQL (Puranam and Blackshear 1994), as well as rqh1+ from Schizosaccharomyces pombe (Murray et al. 1997; Stewart et al. 1997). With the exception of RECQL, these genes are related by their large size (Figure 1) and by the fact that mutations in them are associated with various forms of genomic instability and sensitivity to DNA-damaging agents (German 1963; Hoehn et al. 1975; Gebhart et al. 1988; Gangloff et al. 1994; Watt et al. 1996; Murray et al. 1997; Stewart et al. 1997). Particular attention has been drawn to this class of genes because of their causal role in the human Bloom's (BLM) and Werner's (WRN) syndromes. As a model system, the study of SGS1 in yeast promises to shed light on the role of these proteins in human disease.

Mutations in SGS1 were first identified by their ability to suppress the slow-growth phenotype of top3 strains such that top3 sgs1 double mutants grow at nearly the wild-type rate (Gangloff et al. 1994). Curiously, the sgs1 mutation has the opposite effect in a top1 background; the top1 sgs1 double mutant grows more slowly than either single mutant (Lu et al. 1996). Two-hybrid screens have detected physical interactions between Sgs1 and both Top3 (Gangloff et al. 1994) and Top2 (Watt et al. 1995). Compared to wild-type cells, sgs1 strains show a slight reduction in growth rate and a 10-fold increase in chromosome missegregation (Watt et al. 1995). sgs1 mutants also display elevated recombination: a 7-fold increase in the rate of intrachromosomal recombination at the ribosomal DNA (rDNA) locus and a 3- to 12-fold elevation at MAT (Gangloff et al. 1994; Watt et al. 1996). sgs1 mutations have been shown to increase the number of extrachromosomal rDNA circles in the cell and, as a result, to cause premature aging (Sinclair and Guarente 1997; Sinclair et al. 1997).

The RecQ family proteins have a central domain that is homologous to the bacterial DNA helicase (Figure 1) and as predicted, the BLM, WRN, and Sgs1 proteins have all been shown to possess 3′ to 5′ DNA helicase activity similar to that of RecQ (Umezu et al. 1990; Lu et al. 1996; Gray et al. 1997; Karow et al. 1997; Bennett et al. 1998). In the case of Sgs1, a region between residues 400 and 1268 was sufficient for helicase activity (Bennett et al. 1998). The amino terminus of WRN contains an exonuclease domain (Mushegian et al. 1997; Huang et al. 1998) that is absent in the other homologs, at least by homology (Figure 1). Two additional conserved regions within the family have been identified by sequence comparison. The Ct domain, which has no known function, is found only in the RecQ family of DNA helicases, while the HRD domain is also found in RNaseD homologs and has been suggested to play a role in binding nucleic acids (Morozov et al. 1997).

TABLE 1

S. cerevisiae strains used in this study

TABLE 1

S. cerevisiae strains used in this study

Functional nuclear localization signals (NLSs) have been identified at the C termini of both WRN and BLM (Kaneko et al. 1997; Matsumoto et al. 1997).

We had previously created a helicase-defective allele of SGS1 (sgs1-hd) that changed a single amino acid within the ATP-binding domain of Sgs1 (K706A). Although the mutant protein lacked detectable DNA helicase activity, sgs1-hd retained noticeable complementing activity in the top3 sgs1 and top1 sgs1 backgrounds (Lu et al. 1996). To test whether this generalized to other sgs1 phenotypes and to more accurately define the functional domains of Sgs1, we performed a structure/function analysis and tested complementation of several sgs1 phenotypes. We find that certain SGS1 mutations have distinct phenotypes. For example, sgs1-hd is null in a methylmethanesulfonate (MMS) hypersensitivity assay and in a synthetic-lethal assay, but retains partial complementing activity in the top3 sgs1 background and near wild-type activity in the top1 sgs1 background. In contrast, an allele encoding the helicase domain alone is unable to complement any sgs1 phenotype. We show by intragenic complementation that Sgs1 has a bipartite structure, consisting of the DNA helicase domain and an amino terminal domain, both of which are essential for full SGS1 activity.

MATERIALS AND METHODS

Strains: The yeast strains used in this study are listed in Table 1. Strain construction, growth, and transformation followed standard methods (Rose et al. 1990). A near-complete deletion of the SGS1 gene was constructed by amplifying the loxP-KAN-loxP cassette with the following oligonucleotides (5′-ATGGTGACGAAGCCGTCACATAACTTAAGAAGGGAGCACAAATGGCGTACGCTGCAGGTCGAC-3′) and (5′-TCACTTTCTTCCTCTGTAGTGACCTCGGTAATTTCTAAAACCTCGATCGATGAATTCGAGCTCG-3′) as described (Wach et al. 1994; Guldener et al. 1996). The PCR product was transformed into strains CHY125 and K1875, its proper integration was verified, and the KAN marker was excised to create strains NJY531 and NJY540, respectively. The resulting sgs1::loxP deletion removes all but 15 codons of SGS1 at each end of the gene. Strain NJY598 was constructed by disrupting the TOP1 gene of strain NJY531 using the same approach and the following oligonucleotides (5′-GGGAGGGCAGAGCTCGAAACTTGAAACGCGTAAAAATGACTATTGCAGCTGAAGCTTCGTACGC-3′) and (5′-TGCGAACTTGATGCGTGAATGTATTTGCTTCTCCCCTATGCTGCGGCATAGGCCACTAGTGGATCT G-3′). The resulting top1::loxP-KAN-loxP disruption removes all codons from the TOP1 gene.

Recombination assay: NJY540 cells carrying the integrated versions of the Sgs1 N- and C-terminal deletion series were grown on −ade −ura −leu plates, resuspended in water, diluted appropriately, plated onto YPD + ADE plates at ~200 cells per plate, and grown at 30°. After 3 days, seven colonies from each strain were picked, resuspended in 150 μl water, diluted, and aliquoted onto four plates: −ura and YPD at 400 cells/plate, synthetic complete medium containing 5-fluoroorotic acid (5-FOA) at 4000 cells/plate, and synthetic complete medium containing canavanine in place of arginine at 40,000 cells/plate. After 3 days at 30°, colonies were counted. The frequency of viable cells (as determined from the YPD plates) that were canavanineR or 5-FOAR (ura−) was determined as described previously and the mean value was presented as a percentage (Christman et al. 1993; Gangloff et al. 1994). The experiment was performed five times and a representative experiment is presented in Table 3.

Plasmid constructions: Plasmid pJL31 (Lu et al. 1996) contains the SGS1 gene on a 4.5-kb XhoI/SacI fragment in pRS415 (Sikorski and Hieter 1989). The natural translation start site of SGS1 in this plasmid was mutated to the context of an NdeI site by ligating a 0.15-kb promoter region of SGS1 (XhoI-NdeI PCR fragment) to the first 603 codons (NdeI/HindIII PCR fragment) and to XhoI/HindIII cut pJL31 to create pSM100 (Table 2). To generate N-terminal deletion plasmids, SGS1 fragments were amplified off pJM505 (an SGS1 clone from a CEN-LEU2 library) using NdeI-containing forward (sense) oligos that were designed to create new ATG start sites after the indicated residues of SGS1, together with an appropriate downstream (antisense) oligo containing either a HindIII or SacI restriction site. PCR products were digested with NdeI and either HindIII or SacI and subcloned into the corresponding sites of pSM100. To generate C-terminal deletions, reverse (antisense) oligos were designed to place a stop codon after the indicated residues, followed by BamHI and SacI sites. These oligos were used with an appropriate upstream (sense) oligo to amplify fragments that were subcloned into pSM100 as described above. Helicase-defective derivatives were made by subcloning the K706A mutation from pJL37 (Lu et al. 1996) on NdeI/EagI or HindIII/EagI fragments into the corresponding

TABLE 2

Plasmids used in this study

TABLE 2

Plasmids used in this study

sites of the recipient plasmid. The inserts of pSM100 derivatives were moved on ApaI/SacI fragments (with promoter) into the respective sites of pRS405 and pRS414. Plasmid pJM6702 was constructed by amplifying the GAL1-10 promoter of pBM272 (Johnston and Davis 1984) with oligonucleotides that introduce NdeI, BamHI, and SacI cloning sites adjacent to the GAL10 promoter and ligating this modified promoter to pRS415. Truncated SGS1 inserts were moved from pSM100 derivatives into this vector on NdeI/BamHI or NdeI/SacI fragments.

A C-terminal triple-HA epitope was added to the SGS1 gene of pSM100 by mutating the stop codon to a unique NotI restriction site (resulting in the addition of residues GGR to the C terminus of Sgs1) and ligating a 117-bp NotI/SacI fragment encoding 34 additional residues to Sgs1. Subsequent N-terminal deletion derivatives were constructed by moving truncation alleles on NdeI/SacII cassettes. The sgs1-ΔC795-HA and sgs1-ΔC795-myc alleles were made by fusing NotI/SacI epitope cassettes to sgs1-ΔC795 in which the sequence following residue 652 was similarly mutated to a NotI restriction site. The 13x-myc epitope was amplified as a NotI/SacI fragment using plasmid pFA6a-13Myc-kanMX6 (Longtine et al. 1998) as template. Plasmid pJM555 was constructed by ligating the ADE3 (4.5-kb BamHI/SalI fragment) and TOP3 (2.5-kb SalI/SacI fragment) genes into the BamHI and SacI sites of pRS416. All PCR reactions were carried out with Vent DNA polymerase and 12 cycles of amplification and used directly without sequencing. The absence of PCR-generated mutations was confirmed in several cases by intragenic complementation. In the case of sgs1-hd, the K706A mutation was reverted by site-directed mutagenesis and found to have wild-type activity.

MMS sensitivity: Methylmethanesulfonate was added to a final concentration of 0.012% in YPD agar before pouring and the plates were used within 3 days. Cells were scraped from freshly growing plates, resuspended in water, and OD600 was determined. Cells were transferred to microtiter plates in a vertical row at OD = 3.0 and serially diluted 1:5. A replica plater was then used to transfer a drop from each well to MMS and YPD plates.

Extract preparation and immunoblotting: Yeast cells were grown in 50 ml selective media to an OD600 = 1, harvested, washed with an equal volume of water, and resuspended in 1 ml A buffer [25 mm Tris (pH 7.5), 1 mm EDTA, 0.01% NP-40, 10% glycerol, 0.1 mm PMSF, and 1 mm DTT] with 0.1 m NaCl and the following protease inhibitors: pepstatin, 10 μg/ml; leupeptin, 5 μg/ml; benzamidine, 10 mm; bacitracin, 100 μg/ml; aprotinin, 20 μg/ml; and sodium metabisulfite, 10 mm. Cells were lysed by vortexing with glass beads at 4° and the extract was collected following centrifugation for 15 min at 4°. Extract concentrations were normalized using the Bradford assay (Bio-Rad, Richmond, CA). One hundred microliters of extract was diluted with an equal volume of RIPA buffer (Harlow and Lane 1988) and incubated with 1 μl anti-HA antibody (5 mg/ml; Boehringer Mannheim, Indianapolis) for 1 hr at 4° prior to incubation with protein A beads (Pharmacia, Piscataway, NJ) for 1 hr at 4°. Immunoprecipitations with anti-myc antibody were done under identical conditions except that 1 μl anti-myc antibody (5 mg/ml; Boehringer Mannheim) was used and protein G-agarose (Pharmacia) was used as precipitant. The beads were washed with RIPA buffer (1 ml, three times), boiled in sample buffer, and the immunoprecipitated material was resolved by 12.5% PAGE. Gels were transferred as described (Towbin et al. 1979) and immunoblotted with anti-HA or anti-myc antibody (1:10,000), HRP conjugated secondary antibody (1:10,000; GIBCO BRL, Gaithersburg, MD), and treated with chemiluminescent developer as described by the manufacturer (Amersham, Arlington Heights, IL).

Figure 1.

RecQ family of DNA helicases. (A) Schematic alignment. Protein sizes are shown at right in amino acids. The DNA helicase domain is composed of an NTP-binding motif (black), which shows the greatest similarity between the eukaryotic homologs (39–47% identity) while the 350 amino acids C-terminal to the NTP-binding motif (gray) are less well conserved (22–28% identity). The Ct and HRD domains are shown as dark gray boxes. Open boxes represent domains unique to the eukaryotic homologs. An exonuclease domain (EXO) is present in the WRN protein and nuclear localization sequences are located at the C termini of WRN and BLM (cross-hatched). (B) Sgs1 derivatives used in this study. Sgs1 truncations are named according to the number of amino acids deleted from the wild-type protein. Sgs1-hd contains the K706A mutation.

RESULTS

To identify functional domains in Sgs1, a near-complete deletion of the SGS1 gene was constructed in strain K1875 to create NJY540 (sgs1::loxP; Table 1 and materials and methods). A series of truncation derivatives of the SGS1 gene was also constructed and several representative alleles are diagrammed in Figure 1B. To express these truncations, a plasmid containing the SGS1 open reading frame and 150 bp of its promoter was prepared in which the initiating methionine codon was mutated to a unique NdeI restriction site (pSM100). This parent vector then served as recipient for SGS1 truncations on NdeI/SacI cassettes that were generated by PCR amplification.

Domains of SGS1 required for growth in the presence of MMS: sgs1 mutants have been reported to be sensitive to DNA-damaging agents (Sinclair and Guarente 1997). We confirmed that strain NJY540 (sgs1::loxP) was hypersensitive to MMS and tested whether mutant SGS1

Figure 2.

Complementation of the sgs1 MMS-hypersensitive phenotype by SGS1 truncation alleles. Strain NJY540 (sgs1::loxP) was stably transformed with the indicated SGS1 truncation alleles in the LEU2 integrating vector pRS405. Transformants were resuspended at equal concentrations and spotted in fivefold serial dilutions on YPD plates with and without MMS. Cells were grown and photographed following 2 days at 30°.

alleles could complement this phenotype. SGS1 truncation alleles were made in pRS405, integrated at the LEU2 locus of NJY540, and serial dilutions of the transformants were spotted onto plates containing MMS. As expected, vector alone did not allow growth on this medium, whereas wild-type SGS1 allowed good growth (Figure 2). None of the amino-terminal truncation alleles conferred resistance to MMS, indicating an essential role of the N terminus. The sgs1-hd allele was null in this assay, showing that helicase activity is also required for MMS resistance. A C-terminal truncation of 200 amino acids (ΔC200) allowed wild-type growth, although proteins with larger C-terminal deletions were noticeably defective. These larger C-terminal truncation alleles, such as ΔC795, allowed limited growth on MMS, but not wild-type levels. We conclude that full MMS resistance requires DNA helicase activity and all but the last 200 amino acids of Sgs1. We also note that whereas sgs1-hd is null, the allele ΔC795, which completely lacks the helicase domain, retains partial activity. This suggests that the sgs1-hd mutation may alter the function of the N terminus or the stability of the protein.

To address whether these mutations affected Sgs1 expression levels, a C-terminal triple-HA epitope tag was added to several truncated Sgs1 proteins and their relative expression levels were compared by immunoblot. Signals obtained by immunoblotting crude extracts were very weak, so consecutive immunoprecipitations and immunoblots were used to enhance these signals.

Figure 3.

Expression of Sgs1 truncation derivatives. Strain NJY531 (sgs1::loxP) was transformed with the indicated HA-tagged Sgs1 derivatives (top) in the LEU2/CEN/ARS vector pRS415. Crude protein extracts were immunoprecipitated and immunoblotted with anti-HA antibody. The positions of full-length Sgs1, the amino-terminal domain (NH2), and the helicase domain (Hel) are shown along with the IgG heavy chain. The relevant physical data of the expressed proteins are shown at the bottom.

We observed that wild-type Sgs1-HA protein migrated at 220 kD by SDS-PAGE (Figure 3). This apparent molecular weight is larger than the predicted size of 165 kD and may be due to the highly acidic amino terminus. All of the mutant proteins were stable and in some cases were overexpressed relative to the wild type; sgs1-hd and ΔN158 protein levels were slightly elevated, while ΔN322, ΔN644, and ΔC795 levels were greatly elevated. The ΔN158 and ΔN322 proteins also migrated more slowly than expected, while the ΔN644 protein, which consists of the very basic helicase domain alone, migrated at its expected size. The ΔC795 protein, which consists of the very acidic amino-terminus alone, migrated much slower than expected and was associated with a variable amount of cleaved product migrating at 110 kD. We conclude that the mutant alleles are well expressed and any loss of activity we observe is likely due to the loss of functional domains. This idea is further supported by intragenic complementation experiments described below. We also note that any potential complementing activity by these alleles might depend on their elevated expression.

Complementation of the sgs1 slx4 synthetic lethality: We have identified several nonessential SLX genes that, when mutant, cause the cell to require SGS1 for viability (J. R. Mullen, V. Kaliraman and S. J. Brill, unpublished results). A strain that is null in both SGS1 and

TABLE 3

Complementation of the sgs1 hyper-recombination phenotype

Strain NJY540 (sgs1::loxP) containing the URA3 and CAN1 genes integrated at RDN1 and LYS2, respectively (Keil and McWilliams 1993), was stably transformed with the indicated SGS1 alleles. After 3 days growth in the absence of selection, the percentage of cells that were canavanineR or ura− was determined. The data are presented as the mean of seven independent colonies with the standard deviation and fold increase over wild type. ND, not determined.

TABLE 3

Complementation of the sgs1 hyper-recombination phenotype

Strain NJY540 (sgs1::loxP) containing the URA3 and CAN1 genes integrated at RDN1 and LYS2, respectively (Keil and McWilliams 1993), was stably transformed with the indicated SGS1 alleles. After 3 days growth in the absence of selection, the percentage of cells that were canavanineR or ura− was determined. The data are presented as the mean of seven independent colonies with the standard deviation and fold increase over wild type. ND, not determined.

SLX4 (YLR135W) is inviable but can be maintained by a copy of SGS1 on a URA3-based plasmid. The strain NJY561 (slx4 sgs1 pJM500::SGS1 URA3) is unable to grow on media containing 5-FOA since selection against the URA3 plasmid is lethal in this background. We tested whether SGS1 truncations could complement this phenotype by transforming them into NJY561 and streaking the transformants onto media containing 5-FOA. Apart from wild-type SGS1, only ΔC200 was able to complement the loss of SGS1 in the slx4::loxP background (summarized in Table 5). Identical results were obtained with four other SLX mutants (data not shown). As in the MMS assay, SGS1 function requires the N terminus, the DNA helicase domain, and most of the C terminus; only the last 200 amino acids of the protein is dispensible. Finally, this assay appears more stringent than complementation of MMS hypersensitivity since no intermediate complementation was observed with SGS1 C-terminal truncations larger than ΔC200.

Complementation of the sgs1 hyper-recombination phenotype: Mutations in SGS1 cause a hyper-recombination phenotype (Gangloff et al. 1994; Watt et al. 1996). To determine which domains of SGS1 are required to complement this phenotype, we used strain NJY540 (K1875 sgs1::loxP), which contains markers for the measurement of genetic exchange; CAN1 is inserted at LYS2, creating a LYS2-CAN1-lys2 duplication, and URA3 is inserted within the rDNA cluster (Keil and McWilliams 1993). SGS1 truncation alleles were stably integrated in this strain and the frequencies of URA3 and CAN1 marker loss were determined after growth on nonselective media (Table 3). Compared to vector alone, transformation with wild-type SGS1 reduced the frequency of marker loss five- to ninefold. This reduction in marker loss agrees well with the ability of SGS1 to suppress recombination in other systems (Gangloff et al. 1994; Watt et al. 1996). As in the first two assays, the sgs1-hd allele showed negligible activity.

Interestingly, a truncation of the Sgs1 amino terminus (ΔN158) increased the recombination to a level greater than the null frequency (11–16 times over wild type). Over the course of several trials, the enhancement relative to null ranged from 1.2 to 3.3 (2.2 ± 0.9 average) for URA3 and 0.9 to 3.0 (1.7 ± 1.0 average) for CAN1. To determine if this hypermorphic phenotype was dependent on the helicase activity of the ΔN158 allele, we constructed the compound allele ΔN158-hd, which includes the K706A mutation known to inactivate the Sgs1 DNA helicase (Lu et al. 1996). Eliminating the helicase activity reduced CAN1 recombination to nine times the wild-type frequency, which was still 1.7 times the null frequency. Larger N-terminal truncations, such as ΔN322, produced recombination frequencies that were equivalent to the null. The smallest C-terminal truncation (ΔC200) retained wild-type activity, while a truncation of 300 amino acids (aa; ΔC300) was null. Unexpectedly, the ΔC795 allele, which lacks the entire DNA helicase domain, retained significant complementing activity as it did in the MMS hypersensitivity assay. Taken together, we conclude that (1) the amino terminus of Sgs1 is necessary and sufficient for suppressing recombination; (2) ΔN158 is a hypermorphic allele, with some of its activity independent of DNA helicase activity; and (3) SGS1 affects recombination at RDN1 and LYS2 similarly. As in the MMS assay, the fact that sgs1-hd fails to complement as well as ΔC795 does suggests that the -hd mutation inhibits the activity of the amino terminus.

Complementation in the top3 sgs1 mutant background: Role of SGS1 helicase activity: Given the antagonistic nature of helicase-topoisomerase interactions, it is reasonable to expect that the slow growth of top3 cells might be caused by the DNA-unwinding activity of Sgs1, unchecked by Top3. However, we previously judged sgs1-hd to be active in inhibiting the growth of top3 sgs1 cells when streaked onto agar plates (Lu et al. 1996). To avoid bias in selecting colonies and suppressors that might arise during streak purification, we observed the growth of colonies immediately following transformation of SGS1 alleles in this background. When a top3 sgs1 double mutant was transformed with vector alone it formed large colonies that were heterogeneous in size. When transformed with SGS1 it formed smaller colonies characteristic of the top3 slow-growth phenotype (Figure 4A). When the double mutant was transformed with sgs1-hd, both the number and size of the transformant colonies appeared to be suppressed relative to vector alone. However, these colonies were significantly larger and more heterogeneous than wild-type

Figure 4.

Complementation of top3 sgs1 slow-growth suppression by SGS1 truncation alleles. (A) Strain AMR60 (top3 sgs1) was transformed with the indicated SGS1 truncation alleles (left), or the indicated alleles also containing the K706A helicase-defective mutation (right), in pRS415. Selective plates were photographed following 2 days growth at 30°. (B) Strain AMR60 (top3 sgs1) was transformed with pJM555 (TOP3/URA3) and the indicated SGS1 alleles in pRS415 (LEU2). Transformants were streak purified on SD-leu plates, resuspended at equal concentrations, and spotted in 10-fold serial dilutions on SD-leu plates with or without 1 mg/ml 5-FOA. Cells were grown and photographed following 2 (-leu) or 3 (5-FOA/-leu) days at 30°. The toxic effect of ΔN158 is apparent in both the absence or presence of 5-FOA.

SGS1 colonies, indicating that sgs1-hd is not wild type but retains only partial activity in this assay (Figure 4A, top four panels).

To quantitate the activity of sgs1-hd in this assay, we used three methods. First, we picked large and small colonies from the transformation plates and measured growth rates in selective liquid medium at 30°. When top3 sgs1 cells were transformed with vector alone, the large and small colonies grew with roughly similar doubling times of 135 and 140 min, respectively. Cells transformed with SGS1 also grew at rates reflecting their colony size (205 and 235 min) as did cells transformed with sgs1-hd (210 and 155 min). We conclude that the average growth rate of sgs1-hd transformants (183 min) is intermediate to that of vector- (138 min) and SGS1- (220 min) transformed cells. In particular, even fast-growing sgs1-hd transformants grew slower than vector transformants, indicating that sgs1-hd retains partial activity in this assay. As a second method we tested how a population of top3 cells, not just a few colonies, responded to sgs1-hd. To do this, we complemented a top3 sgs1 double mutant with a TOP3/URA3 plasmid (pJM555) and one of the SGS1 alleles carried on a LEU2 plasmid. Strains carrying the two plasmids were then serially diluted and spotted on plates containing 5-FOA, to select against the TOP3/URA3 plasmid, and on control plates. As expected, cells carrying pSGS1/LEU2 grew poorly on 5-FOA/-leucine, displaying the top3 slow-growth phenotype. Cells transformed with the empty LEU2 vector grew well on 5-FOA/-leucine, displaying the top3 sgs1 phenotype of fast growth (Figure 4B). Cells carrying sgs1-hd display an intermediate phenotype, requiring an ~10-fold higher concentration of cells to equal the growth obtained with vector alone.

A third method to test the activity of sgs1-hd in the top3 sgs1 background was to place the SGS1 alleles behind the inducible GAL10 promoter and observe the effect of overexpression in the double mutant. Transformants were serially diluted and spotted onto selective plates containing either galactose or glucose. Cells transformed with vector alone grew well on both galactose and glucose, while cells transformed with SGS1 grew slowly on galactose, as expected (Figure 5). If the slow growth of top3 cells is due solely to Sgs1 helicase activity unbalanced by Top3 activity, then overexpressing sgs1-hd should have no effect. However, overexpression of sgs1-hd reduced the growth of top3 sgs1 cells such that 5- to 25-fold more sgs1-hd cells were required to equal the overall amount of growth of cells transformed with vector alone (Figure 5, bottom panels). This effect is clearly not as severe as overexpressing wild-type SGS1. Furthermore, the intermediate activity of sgs1-hd is unlikely to be due to residual helicase activity as ΔC795, which encodes only the N terminus, shows the same intermediate activity in this assay (Figure 4B). Presumably, the amino-terminal domain of Sgs1 must also contribute to growth suppression in the absence of Top3.

Role of the SGS1 amino terminus: When the top3 sgs1 double mutant was transformed with the amino-terminal truncation alleles ΔN50, ΔN101, and ΔN158, we were surprised to find that the transformants grew more slowly than cells transformed with SGS1 (Figure 4A). In the case of ΔN158, growth was so slow that transformant colonies were not visible until 3 days at 30°, compared to SGS1 transformants that were clearly visible after 2 days. This unusual phenotype of ΔN158 is discussed further below. Larger truncations, such as ΔN322 reversed this slow-growth trend while ΔN644 conferred a null phenotype (Figure 4B). The null phenotype of ΔN644 again confirms that complementation of top3 sgs1 slow-growth suppression requires the N terminus of Sgs1 and not just DNA helicase activity. We tested whether overexpression of the helicase domain would overcome this limitation. Overexpression of the N-terminal deletions, up to and including ΔN644, did inhibit

Figure 5.

Overexpression of SGS1 truncations reveals sgs1-hd activity and a role for the C terminus of Sgs1. (A) Strain AMR60 (top3 sgs1) was transformed with a galactose expression vector (pJM6702) containing the indicated SGS1 N-terminal truncation alleles (top) or the indicated alleles also containing the K706A helicase-defective mutation (bottom). Transformants were streak purified on SD-leu plates, resuspended at equal concentrations, and spotted in fivefold serial dilutions on selective plates containing either galactose or glucose. (B) As described above, but with the SGS1 C-terminal truncation series. Cells were grown and photographed after 2 (glucose) or 3 (galactose) days at 30°.

growth as wild-type SGS1 did (Figure 5A, top panels and data not shown). Thus, overexpression of the helicase domain alone can suppress the lack of the amino terminus. Accordingly, ΔN1000, which removes both the N terminus and much of the DNA helicase domain, is null in this assay. We conclude that complementation of top3 sgs1 slow-growth suppression requires both the N terminus and DNA helicase activity.

The severe growth inhibition of ΔN158 described above will be referred to as the “toxic effect” to distinguish it from the normal growth inhibition of wild-type SGS1 in the top3 background. The toxic effect of ΔN158 and its reversal by larger truncations like ΔN322 correlate with the ΔN158 hypermorphic phenotype observed in the recombination assay. To test whether this toxicity was specific to the top3 background, we transformed a sgs1 single mutant with the truncation series and found that its growth was similarly inhibited by this allele (Figure 6, left panels). We conclude that the toxic effect is specific for sgs1 mutant cells and that it is recessive since it is not observed in wild-type cells (data not shown). We tested whether the helicase activity of Sgs1 was required for the toxic effect by constructing compound

Figure 6.

Truncation of the Sgs1 amino terminus uncovers a helicase-dependent toxicity. (A) Strain NJY531 (sgs1:: loxP) was transformed with the indicated SGS1 truncation alleles (left) or the indicated alleles also containing the K706A helicase-defective mutation (right), in pRS415. Cells were grown on SD-leu and photographed following 3 days at 30°.

alleles that included the -hd mutation. The addition of the -hd mutation alleviated the toxic effect of the small amino-terminal truncations in both the top3 sgs1 (Figure 4A, right-hand panels) and sgs1 (Figure 6, right panels) backgrounds. In the top3 sgs1 background, the colony sizes after transformation of the sgs1-hd, ΔN50-hd, ΔN101-hd, and ΔN158-hd alleles were similar to each other and intermediate to those obtained with vector and wild-type SGS1. These results indicate that the ΔN158 toxic phenotype requires both the specific amino terminal truncation and DNA helicase activity.

Role of the SGS1 carboxy terminus: Sgs1 C-terminal truncations were tested for complementation in the top3 sgs1 background. As previously reported, none of the C-terminal truncation alleles, including ΔC200, significantly inhibited the growth of the top3 sgs1 double mutant using a colony size assay (Lu et al. 1996; data not shown). To quantitate the defect, we picked large and small colonies from the transformation plates and measured growth rates in selective liquid medium at 30°. Cells transformed with ΔC200 grew at rates reflecting their colony size (140 and 150 min) with an average doubling time of 145 min, relative to 220 min for SGS1 and 138 min for vector. Thus, in this assay alone, ΔC200 does not behave like wild type. Using the galactose overexpression assay, ΔC200 significantly inhibited growth in top3 sgs1 cells (Figure 5B, top panels), but this is likely due to excess helicase activity since either larger deletions (ΔC300) or inactivation of the helicase domain (ΔC200-hd) reduced this effect (Figure 5B, bottom panels). The importance of the C-terminal 200 amino acids is further emphasized by the fact that ΔC200-hd fails to inhibit growth as much as sgs1-hd when overexpressed. We conclude that complementation of top3 sgs1 slow-growth suppression is an extremely sensitive assay that requires the N terminus, DNA helicase activity, and the C-terminal 200 amino acids for full SGS1 activity.

Complementation in the top1 sgs1 mutant background: sgs1 and top1 display a synergistic growth defect

Figure 7.

Complementation of the top1 sgs1 growth defect does not require the DNA helicase domain of Sgs1. (A) Strain NJY598 (top1 sgs1) was transformed with the indicated SGS1 truncation alleles (left), or the indicated alleles also containing the K706A helicase-defective mutation (right), in pRS415. Cells were grown and photographed following 3 days at 30°. (B) NJY598 transformants containing the indicated SGS1 alleles in pRS415 were streak purified on SD-leu plates, resuspended at equal concentrations, and spotted in 10-fold serial dilutions on SD-leu plates. Cells were grown and photographed following 4 days at 22°.

such that a top1 sgs1 double mutant grows more slowly than either single mutant (Lu et al. 1996). We transformed top1 sgs1 cells with our SGS1 mutant alleles to determine how well they could improve growth in this background. As expected, NJY598 (top1 sgs1) formed small colonies when transformed with vector alone and large colonies when transformed with SGS1 (Figure 7A). Large colonies were also obtained when these cells were transformed with sgs1-hd, indicating that helicase activity is not essential in this assay (Figure 7A). We also compared the growth rates of a population of cells by serially diluting transformants and spotting on selective plates. The top1 sgs1 cells transformed with ΔC795, which encodes the N terminus, grew as well as mutants transformed with wild-type SGS1, but cells transformed with ΔN644, which encodes the helicase, grew at a rate comparable to those transformed with vector alone (Figure 7B). We conclude that DNA helicase activity is completely dispensible in this assay. As in both the MMS hypersensitivity and hyper-recombination assays, complementation by ΔC795 was better than full-length sgs1-hd, suggesting that the -hd mutation inhibits the complementing activity of the N terminus.

The toxic effect of ΔN158 was apparent in the top1 sgs1 background, since it exacerbated rather than improved the growth defect (Figure 7A). When more of the N terminus was removed, as in the ΔN322 and ΔN484 alleles, the toxic effect was lost and complementing activity returned, although not at wild-type levels. Thus, the minimal complementing region is likely to lie between residues 484 and 644 of the N terminus. In contrast to other backgrounds, the toxic effect of ΔN158 was not alleviated by the ΔN158-hd mutation (Figure 7A, right panels). This, however, is consistent with the fact that complementation in this assay does not require helicase activity. Nevertheless, the complementing activity of ΔN322 and ΔN484 was adversely affected by the -hd mutation (Figure 7A, right panels). We suspect that this result is caused by the inhibitory effect of the -hd mutation on the activity of the N terminus. We conclude that, in the top1 sgs1 background, the toxic effect is independent of helicase activity.

Intragenic complementation by SGS1: Because our assays revealed that Sgs1 contained two functional domains that could be independently manipulated, we wanted to determine if the two domains could be physically separated and still function. SGS1 truncations were subcloned into the appropriate vectors for simultaneous expression and complementation testing of two mutant alleles. Neither ΔN322 nor sgs1-hd individually conferred MMS resistance (Figure 2). However, when these two alleles were expressed simultaneously in sgs1 cells, they grew on MMS plates at a rate close to wild type (Figure 8, top). We also found that ΔN322 complemented the C-terminal truncation alleles ΔC795 and ΔC997, while complementation with ΔC1247 was impaired (Figure 8, top). In the most stringent test, we found that nonoverlapping N- and C-terminal helicase domains could complement. Figure 8 (middle) shows that ΔN644 (helicase domain) can be complemented by sgs1-hd as well as ΔC795 (N-terminal domain). In fact, significant growth on MMS is obtained with alleles ΔN644 and ΔC997, indicating that residues 450–645 are dispensible for MMS resistance. As described above, ΔC1247 fails to allow optimal growth in the presence of ΔN644. Consistent with these results, ΔC795 (N-terminal domain) cannot be rescued by sgs1-hd, although it can be rescued by other N-terminal truncation alleles, including ΔN644 (helicase domain; Figure 8, bottom panel).

To confirm that SGS1 intragenic complementation is not unique to MMS hypersensitivity, we tested the more stringent assay of restoring viability to the syntheticlethal slx4 sgs1 strain. Whereas neither ΔN322 nor sgs1-hd could individually restore viability in this background (Table 5), these alleles together rescued the slx4 sgs1 strain with a growth rate equivalent to wild type (Table 4). Moreover, ΔN644 (helicase domain) and the ΔC795 (N-terminal domain) together restored viability to the double mutant, although the growth rate was somewhat reduced. We conclude that Sgs1 can be expressed as functional N-terminal and DNA helicase domains, consistent

Figure 8.

SGS1 intragenic complementation. Strain NJY-540 (sgs1::loxP) was transformed with one SGS1 truncation allele in pRS415 (top, pSM103; middle, pSM105; bottom, pBS1) and another SGS1 truncation allele in pRS414 (top and middle, pRS414, pSM108, pSM112, pSM113, and pSM114; bottom, pSM108, pSM109, pSM110, and pSM111). Transformants were streak purified on plates lacking tryptophane and leucine, resuspended at equal concentrations, and spotted in fivefold serial dilutions on YPD plates with MMS. Plates were photographed following 2 days growth at 30°. Complementation of MMS hypersensitivity by wild-type SGS1 (pSM100) is shown at the top. On an individual basis, none of the indicated mutant alleles allowed growth on MMS (Figure 2 and data not shown).

with the dual functions of the protein, as revealed by truncation analysis.

Successful intragenic complementation suggests that either the two truncated Sgs1 proteins perform their functions separately or they assemble into a stable complex, as in β-galactosidase α-complementation. To test these possibilities, we placed a myc-epitope tag on the C terminus of the N-terminal domain (ΔC795-myc) and expressed it with the Sgs1-HA-tagged proteins described in Figure 3. Crude extracts were made from cells coexpressing these proteins and the potential complexes were analyzed by consecutive immunoprecipitation (IP) and immunoblotting. In this experiment Sgs1-HA migrated as a doublet at 220 kD perhaps due to post-translational modification (Figure 9, top). When full-length Sgs1-HA was coexpressed with ΔC795-myc and immunoprecipitated with anti-myc antibody, a small amount of Sgs1-HA could be detected in the IP following blotting with anti-HA antibody (Figure 9, middle). This result suggests that Sgs1 interacts with itself in the cell, perhaps forming a multimer via the N-terminal domain. Consistent with this idea, the HA-tagged N-terminal truncation alleles failed to coprecipitate with ΔC795-myc. A control immunoblot with anti-myc antibody revealed that ΔC795-myc was expressed in these strains (Figure 9, bottom panel). These results suggest that the N terminus and helicase domain expressed during intragenic complementation do not associate in the cell and that they perform their functions separately. We cannot, however, rule out the possibility that the truncated Sgs1 proteins were complexed in the cell and were disrupted by the lysis or immunoprecipitation procedures.

DISCUSSION

As a first step toward characterizing the RecQ family of DNA helicases, we have conducted a structure/function analysis of the yeast SGS1 gene. Table 5 summarizes the results of our experiments. An unanticipated finding of these studies was that certain SGS1 alleles have distinct phenotypes depending on the assay. In assays measuring recombination or growth rates, we identified alleles with intermediate or null phenotypes in addition to hypermorphic alleles whose phenotype was more severe than the null. In contrast, in assays measuring MMS resistance or complementation of synthetic lethality, most of the intermediate and hypermorphic alleles displayed a null phenotype. Further evidence that these alleles retained partial activity was that wild-type function could be restored through intragenic complementation.

TABLE 4

Intragenic complementation of sgs1 slx4 synthetic lethality

Strain NJY561 (sgs1 slx4) carrying plasmid pJM500 (SGS1/ URA3/ADE3) was cotransformed with the indicated SGS1 alleles on LEU2 (rows) and TRP1 (columns) plasmids as in Figure 8. The transformants were then streaked onto synthetic complete medium containing 1 mg/ml 5-FOA. Wild-type growth on this medium is indicated by + + .

TABLE 4

Intragenic complementation of sgs1 slx4 synthetic lethality

Strain NJY561 (sgs1 slx4) carrying plasmid pJM500 (SGS1/ URA3/ADE3) was cotransformed with the indicated SGS1 alleles on LEU2 (rows) and TRP1 (columns) plasmids as in Figure 8. The transformants were then streaked onto synthetic complete medium containing 1 mg/ml 5-FOA. Wild-type growth on this medium is indicated by + + .

Figure 9.

Sgs1 domains fail to interact physically. Strain NJY531 (sgs1::loxP) was transformed with the indicated SGS1-HA truncation alleles and ΔC795-myc on pRS415 and pRS414, respectively. Cells were harvested and protein extracts were prepared for immunoprecipitation and immunoblotting with the indicated antibodies.

We believe that certain mutant alleles display distinct phenotypes for two reasons: first, some assays differ in their sensitivity to Sgs1 activity and second, some assays require only one of Sgs1's two functional domains.

Compared to the null allele, ΔN158 exhibited both very high rates of recombination and toxicity in sgs1 strains, yet was null in the MMS and synthetic lethality assays (Table 5). The toxicity was most apparent in top1 sgs1 cells, where we expected any SGS1 activity to improve, not inhibit growth. And while SGS1 activity is expected to inhibit growth in the top3 background, ΔN158 inhibited growth even more dramatically than wild-type SGS1. The mechanism of ΔN158 toxicity and hyper-recombination is not understood, but it is known that overexpressing wild-type SGS1 leads to severe growth inhibition in these backgrounds (Figure 5; data not shown). Therefore, a possible explanation for the ΔN158 phenotype is that truncation of the N terminus creates a “hyperactive” protein. This idea is consistent with the simultaneous reduction in recombination rate and toxicity caused by the larger truncation encoded by ΔN322 or by a mutation in the helicase domain.

To explain these data we propose that an inhibitory domain of Sgs1 is removed by ΔN158, revealing a stimulatory domain that, in turn, is removed by ΔN322 (Figure 10A). Since removal of the stimulatory domain correlates with increased ΔN322 protein levels (Figure 3), the loss of the hypermorphic phenotype must be due to loss of function and not a loss of protein stability. A prediction of this model is that overexpression of wild-type SGS1 should stimulate recombination and other sgs1 phenotypes. Indeed, overexpression of SGS1 is known to induce nucleolar fragmentation, a phenotype of prematurely aging sgs1 cells (Sinclair et al. 1997). Does the ΔN158 hypermorphic phenotype depend on DNA helicase activity? The toxicity of ΔN158 in sgs1 and top3 sgs1 cells is relieved by a point mutation in the helicase domain. However, ΔN158-hd remains toxic in top1 sgs1 cells and causes excessive hyper-recombination in sgs1 cells even in the absence of helicase activity. Thus, the hypermorphic phenotype depends on helicase activity only when the specific assay depends on helicase activity.

How might the domain between residues 1 and 158 function to inhibit the activity of Sgs1? The simplest model (Figure 10B, top) proposes that a subdomain of the N terminus, N1, inhibits the DNA helicase (pathway 1), while the alternative, which we prefer, is that N1 inhibits an activity present in N2 (pathway 2). An obvious possibility is that N2 is a nuclease as in WRN (Huang et al. 1998), although other activities are conceivable. A second model (Figure 10B, bottom) proposes that Sgs1 exists in a complex of proteins and that N1 and N2 interact with hypothetical repressors or targeting proteins, respectively.

The ΔC795 allele, which expresses the N-terminal 652 aa alone, restored good growth to a top1 sgs1 double mutant, consistent with our previous report that a full-length helicase-defective allele was active in this assay (Lu et al. 1996). The complementing region of the amino terminus may lie C-terminal to the first 484 amino acids, since ΔN484 was also able to restore good growth to top1 sgs1 cells. We conclude that the residues between 484 and 652 contain a domain that is important for good growth in the top1 background. How could this domain function to overcome a Top1 deficiency? One possibility is that it recruits an alternate topoisomerase, such as Top2, to relax excess superhelicity. Such an idea is consistent with the fact that residues 434–744 of Sgs1 were found to interact with Top2 in a two-hybrid screen (Watt et al. 1995).

When expressed under its own promoter, ΔC795, encoding the amino terminus alone, showed at least partial activity in complementing four phenotypes: MMS hypersensitivity, hyper-recombination, top3 sgs1 growth suppression, and top1 sgs1 slow growth. In contrast, ΔN644,

TABLE 5

Complementation by SGS1 truncation alleles

a

Strain NJY561 (sgs1 slx4) carrying plasmid pJM500 (SGS1/ URA3/ADE3) was transformed with the indicated SGS1 alleles in pRS415. The transformants were then streaked onto synthetic complete medium containing 1 mg/ml 5-FOA.

b

Hypermorphic phenotype.

+ , full complementation; −, no complementation; +/ −, intermediate complementation; ND, not determined.

TABLE 5

Complementation by SGS1 truncation alleles

a

Strain NJY561 (sgs1 slx4) carrying plasmid pJM500 (SGS1/ URA3/ADE3) was transformed with the indicated SGS1 alleles in pRS415. The transformants were then streaked onto synthetic complete medium containing 1 mg/ml 5-FOA.

b

Hypermorphic phenotype.

+ , full complementation; −, no complementation; +/ −, intermediate complementation; ND, not determined.

which encodes the helicase domain alone, was null in these assays. Since intragenic complementation experiments demonstrated that the ΔN644 allele was expressed, we must conclude that the N terminus is at least as important to Sgs1 function as is the DNA helicase. This is surprising given that SGS1 is epistatic to TOP3 and it is thought that Sgs1 unwinds DNA strands to create a substrate for Top3 (Gangloff et al. 1994). Indeed, in the Escherichia coli system it has been shown that RecQ generates a substrate for strand passage by DNA topoisomerase III (Harmon et al. 1999). A possible explanation for this discrepancy is that full-length Sgs1 may be required in vivo to maintain the integrity of a larger protein complex (e.g., Sgs1, Top1, and Top2). Alternatively, Sgs1 may modify DNA as well as unwind it for processing by eukaryotic Top3. We suggest that the N terminus of Sgs1 has its own activity, such as a nuclease or perhaps a unique DNA-binding activity, which is an important component of SGS1 function.

The C-terminal truncation series revealed that the last 200 aa of Sgs1, which include the HRD domain, are dispensible for all phenotypes except complementation of top3 sgs1 slow-growth suppression. However, overexpression of ΔC200 causes slow growth in this background, as does ΔN644 (Table 5), indicating that an imbalance of helicase over Top3 activity can induce slow growth even in the absence of the N terminus. This is consistent with the fact that sgs1-hd lacks full complementation in this assay and shows that helicase activity is an important, although not exclusive, determinant in this assay. The failure of ΔC200 to complement would be expected if the C-terminal 200 aa are important for protein localization as in WRN (Matsumoto et al. 1997). Since localization did not appear to be a problem in other assays, we suspect that the C-terminal domain has another activity. One possibility is that HRD stimulates the activity of SGS1 by binding to DNA or RNA as proposed originally (Morozov et al. 1997), and that the

Figure 10.

SGS1 structure/function models. (A) The Sgs1 protein is presented schematically with functional domains shown in the N and C termini. The domain between residues 484 and 652, labeled “top1 sgs1,” represents the region required for good growth in the top1 sgs1 double mutant. (B) Models of SGS1 regulation. The upper model proposes a direct mechanism for SGS1 regulation in which the inhibitory domain, N1, inhibits the DNA helicase domain (1) or the putative stimulatory region, N2 (2). The lower model proposes an indirect mechanism for Sgs1 regulation in which activity is controlled by other factors. In this case, N1 interacts with a specific repressor protein while the stimulatory domains, N2 and the C terminus, interact with proteins involved in targeting Sgs1 to a protein complex or DNA substrate. The N4 domain is proposed to mediate interaction with Top2 (Watt et al. 1995).

requirement for this activity is only observed in the very sensitive top3 sgs1 slow-growth suppression assay.

The sgs1-hd allele, which carries a point mutation in the helicase domain, was null in three assays and intermediate in complementing the top1 sgs1 growth defect and top3 sgs1 slow-growth suppression. In some of these assays (e.g., MMS hypersensitivity, hyper-recombination, and complementation of top1 sgs1 slow growth), sgs1-hd was more defective than ΔC795, which lacks the entire helicase domain. We suspect that the defective helicase domain inhibits the function of the amino terminus. This could be explained simply if the helicase domain interacts with other proteins such as Top3 (Gangloff et al. 1994) or Top2 (Watt et al. 1995). The interaction of sgs1-hd with these proteins might tether or restrict the amino terminus from performing its normal function. Alternatively, the presence of an inactive helicase in this complex may inhibit these other enzyme activities, contributing to the sgs1-hd phenotype.

The dual functions of Sgs1 may extend to other RecQ family members such as the rqh1+ gene of S. pombe. The rqh1.r12 allele contains a single missense mutation in the ATP-binding domain of Rqh1 (Murray et al. 1997). Compared to the rqh1 null allele, rqh1.r12 confers a number of weaker phenotypes, including reduced sensitivity to UV and ionizing radiation. In addition, the null allele is synthetically lethal with the S. pombe rad3 and rad26 mutations while rqh1.r12 is not (Murray et al. 1997). Therefore, it is likely that DNA helicase activity is only one of the functions carried out by the RecQ homologs. Moreover, as a multifunctional helicase, Sgs1 is not unique in yeast. The Upf1 RNA helicase is required for both nonsense suppression and mRNA turnover in yeast (Czaplinski et al. 1998). Interestingly, inactivation of the Upf1 helicase does not cause a null phenotype; only mRNA turnover is affected. In contrast, mutations in the Upf1 N terminus affect nonsense suppression without affecting mRNA turnover (Weng et al. 1996).

Intragenic complementation experiments provide convincing evidence of the bipartite nature of Sgs1. These experiments confirm that the amino-terminal domain possesses an activity separate from the DNA helicase and that the two domains function in an interdependent manner. Further analysis of the enzymology of Sgs1 and its physical interactions with other proteins should shed light on this second function.

Acknowledgement

The authors thank Sheila Mellody for expert technical assistance. This work was supported by grants from the National Institutes of Health (R01-AG16637), the American Cancer Society (RPG-98-079-01), and the Charles and Joanna Busch Memorial Fund.

Footnotes

Communicating editor: F. Winston

LITERATURE CITED

Bennett

R J

,

Sharp

J A

,

Wang

J C

,

1998

Purification and characterization of the sgs1 DNA helicase activity of Saccharomyces cerevisiae

.

J. Biol. Chem.

273

:

9644

–

9650

.

Christman

M F

,

Dietrich

F S

,

Levin

N A

,

Sadoff

B U

,

Fink

G R

,

1993

The rRNA-encoding DNA array has an altered structure in topoisomerase I mutants of Saccharomyces cerevisiae

.

Proc. Natl. Acad. Sci. USA

90

:

7637

–

7641

.

Czaplinski

K

,

Ruiz-Echevarria

M J

,

Paushkin

S V

,

Han

X

,

Weng

Y

et al. .,

1998

The surveillance complex interacts with the translation release factors to enhance termination and degrade aberrant mRNAs

.

Genes Dev.

12

:

1665

–

1677

.

Ellis

N A

,

Groden

J

,

Ye

T-Z

,

Straughen

J

,

Lennon

D J

et al. .,

1995

The Bloom's syndrome gene product is homologous to recQ helicases

.

Cell

83

:

655

–

666

.

Gangloff

S

,

McDonald

J P

,

Bendixen

C

,

Arthur

L

,

Rothstein

R

,

1994

The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase

.

Mol. Cell. Biol.

14

:

8391

–

8398

.

Gebhart

E

,

Bauer

R

,

Raub

U

,

Schinzel

M

,

Ruprecht

K W

et al. .,

1988

Spontaneous and induced chromosomal instability in Werner syndrome

.

Hum. Genet.

80

:

135

–

139

.

German

J

,

1963

Bloom syndrome: a mendelian prototype of somatic mutational disease

.

Medicine

72

:

393

–

406

.

Gray

M D

,

Shen

J C

,

Kamath-Loeb

A S

,

Blank

A

,

Sopher

B L

et al. .,

1997

The Werner syndrome protein is a DNA helicase

.

Nat. Genet.

17

:

100

–

103

.

Guldener

U

,

Heck

S

,

Fielder

T

,

Beinhauer

J

,

Hegemann

J H

,

1996

A new efficient gene disruption cassette for repeated use in budding yeast

.

Nucleic Acids Res.

24

:

2519

–

2524

.

Harlow

E

,

Lane

D

,

1988

Antibodies: A Laboratory Manual

.

Cold Spring Harbor Laboratory Press

,

Cold Spring Harbor, NY

.

Harmon

F G

,

DiGate

R J

,

Kowalczykowski

S C

,

1999

RecQ helicase and topoisomerase III comprise a novel DNA strand passage function: a conserved mechanism for control of DNA recombination

.

Mol. Cell

3

:

611

–

620

.

Hoehn

H

,

Bryant

E M

,

Au

K

,

Norwood

T H

,

Boman

H

et al. .,

1975

Variegated translocation mosaicism in human skin fibroblast cultures

.

Cytogenet. Cell Genet.

15

:

282

–

298

.

Huang

S

,

Li

B

,

Gray

M D

,

Oshima

J

,

Mian

I S

et al. .,

1998

The premature ageing syndrome protein, WRN, is a 3′→5′ exonuclease [letter]

.

Nat. Genet.

20

:

114

–

116

.

Johnston

M

,

Davis

R W

,

1984

Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae

.

Mol. Cell. Biol.

4

:

1440

–

1448

.

Kaneko

H

,

Orii

K O

,

Matsui

E

,

Shimozawa

N

,

Fukao

T

et al. .,

1997

BLM (the causative gene of Bloom syndrome) protein translocation into the nucleus by a nuclear localization signal

.

Biochem. Biophys. Res. Commun.

240

:

348

–

353

.

Karow

J K

,

Chakraverty

R K

,

Hickson

I D

,

1997

The Bloom's syndrome gene product is a 3′-5′ DNA helicase

.

J. Biol. Chem.

272

:

30611

–

30614

.

Keil

R L

,

McWilliams

A D

,

1993

A gene with specific and global effects on recombination of sequences from tandemly repeated genes in Saccharomyces cerevisiae

.

Genetics

135

:

711

–

718

.

Longtine

M S

,

McKenzie

A

III,

Demarini

D J

,

Shah

N G

,

Wach

A

et al. .,

1998

Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae

.

Yeast

14

:

953

–

961

.

Lu

J

,

Mullen

J R

,

Brill

S J

,

Kleff

S

,

Romeo

A

et al. .,

1996

Human homologs of yeast DNA helicase

.

Nature

383

:

678

–

679

.

Matsumoto

T

,

Shimamoto

A

,

Goto

M

,

Furuichi

Y

,

1997

Impaired nuclear localization of defective DNA helicases in Werner's syndrome

.

Nat. Genet.

16

:

335

–

336

.

Morozov

V

,

Mushegian

A R

,

Koonin

E V

,

Bork

P

,

1997

A putative nucleic acid-binding domain in Bloom's and Werner's syndrome helicases

.

Trends Biochem. Sci.

22

:

417

–

418

.

Murray

J M

,

Lindsay

H D

,

Munday

C A

,

Carr

A M

,

1997

Role of Schizosaccharomyces pombe RecQ homolog, recombination, and checkpoint genes in UV damage tolerance

.

Mol. Cell. Biol.

17

:

6868

–

6875

.

Mushegian

A R

,

Bassett

D E

Jr.,

Boguski

M S

,

Bork

P

,

Koonin

E V

,

1997

Positionally cloned human disease genes: patterns of evolutionary conservation and functional motifs [see comments]

.

Proc. Natl. Acad. Sci. USA

94

:

5831

–

5836

.

Puranam

K L

,

Blackshear

P J

,

1994

Cloning and characterization of RECQL, a potential human homologue of the Escherichia coli DNA helicase RecQ

.

J. Biol. Chem.

269

:

29838

–

29845

.

Rose

M D

,

Winston

F

,

Hieter

P

,

1990

Methods in Yeast Genetics

.

Cold Spring Harbor Laboratory Press

,

Cold Spring Harbor, NY

.

Sikorski

R S

,

Hieter

P

,

1989

A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae

.

Genetics

122

:

19

–

27

.

Sinclair

D A

,

Guarente

L

,

1997

Extrachromosomal rDNA circles—a cause of aging in yeast

.

Cell

91

:

1033

–

1042

.

Sinclair

D A

,

Mills

K

,

Guarente

L

,

1997

Accelerated aging and nucleolar fragmentation in yeast sgs1 mutants

.

Science

277

:

1313

–

1316

.

Stewart

E

,

Chapman

C R

,

Al-Khodairy

F

,

Carr

A M

,

Enoch

T

,

1997

rqh1+, a fission yeast gene related to the Bloom's and Werner's syndrome genes, is required for reversible S phase arrest

.

EMBO J.

16

:

2682

–

2692

.

Thomas

B J

,

Rothstein

R

,

1989

Elevated recombination rates in transcriptionally active DNA

.

Cell

56

:

619

–

630

.

Towbin

H

,

Staehelin

T

,

Gordon

J

,

1979

Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications

.

Proc. Natl. Acad. Sci. USA

76

:

4350

–

4354

.

Umezu

K

,

Nakayama

K

,

Nakayama

H

,

1990

Escherichia coli RecQ protein is a DNA helicase

.

Proc. Natl. Acad. Sci. USA

87

:

5363

–

5367

.

Wach

A

,

Brachat

A

,

Pohlmann

R

,

Philippsen

P

,

1994

New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae

.

Yeast

10

:

1793

–

1808

.

Watt

P M

,

Louis

E J

,

Borts

R H

,

Hickson

I D

,

1995

Sgs1: a eukaryotic homolog of E. coli RecQ that interacts with topoisomerase II in vivo and is required for faithful chromosome segregation

.

Cell

81

:

253

–

260

.

Watt

P M

,

Hickson

I D

,

Borts

R H

,

Louis

E J

,

1996

SGS1, a homologue of the Bloom's and Werner's syndrome genes, is required for maintenance of genome stability in Saccharomyces cerevisiae

.

Genetics

144

:

935

–

945

.

Weng

Y

,

Czaplinski

K

,

Peltz

S W

,

1996

Genetic and biochemical characterization of mutations in the ATPase and helicase regions of the Upf1 protein

.

Mol. Cell. Biol.

16

:

5477

–

5490

.

Yu

C E

,

Oshima

J

,

Fu

Y H

,

Wijsman

E M

,

Hisama

F

et al. .,

1996

Positional cloning of the Werner's syndrome gene

.

Science

272

:

258

–

262

.

© Genetics 2000

Citations

Views

Altmetric

Metrics

Total Views 408

276 Pageviews

132 PDF Downloads

Since 3/1/2021

×

Email alerts

Citing articles via

• Latest

• Most Read

• Most Cited

More from Oxford Academic