From the Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08855
Received for publication, October 24, 2000, and in revised form, December 6, 2000
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ABSTRACT |
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Several members of the RecQ family of DNA
helicases are known to interact with DNA topoisomerase III (Top3). Here
we show that the Saccharomyces cerevisiae Sgs1 and Top3
proteins physically interact in cell extracts and bind directly
in vitro. Sgs1 and Top3 proteins coimmunoprecipitate from
cell extracts under stringent conditions, indicating that Sgs1 and Top3
are present in a stable complex. The domain of Sgs1 which interacts
with Top3 was identified by expressing Sgs1 truncations in yeast. The
results indicate that the NH2-terminal 158 amino acids of
Sgs1 are sufficient for the high affinity interaction between Sgs1 and
Top3. In vitro assays using purified Top3 and
NH2-terminal Sgs1 fragments demonstrate that at least part
of the interaction is through direct protein-protein interactions with
these 158 amino acids. Consistent with these physical data, we find
that mutant phenotypes caused by a point mutation or small deletions in
the Sgs1 NH2 terminus can be suppressed by Top3
overexpression. We conclude that Sgs1 and Top3 form a tight complex
in vivo and that the first 158 amino acids of Sgs1 are
necessary and sufficient for this interaction. Thus, a primary role of
the Sgs1 amino terminus is to mediate the Top3 interaction.
The Saccharomyces cerevisiae SGS1 gene encodes a member
of the RecQ family of DNA helicases. In addition to the RecQ protein of
Escherichia coli, this family includes the human BLM, WRN, RECQL4, and RECQ5 proteins as well as Rqh1 from
Schizosaccharomyces pombe (1-7). These proteins play an
important role in DNA metabolism as mutations in the human genes give
rise to diseases characterized by genome instability and a
predisposition to cancer. Werner's syndrome cells, which result from
mutations in WRN (2), display a genomic instability termed
variegated translocation mosaicism (8). Bloom's syndrome cells, which
result from mutations in BLM (1), are characterized by
increased rates of sister chromatid exchange and sensitivity to
DNA-damaging agents (9). Mutations in RECQL4 are found in a
subset of Rothmund-Thomson syndrome cases. These cells are
characterized by elevated rates of chromosomal breaks and
rearrangements (5, 10). All members of this family contain a
COOH-terminal domain with homology to RecQ, and all those that have
been tested exhibit a 3'- to 5'-DNA helicase activity (11-15). In
addition to the helicase domain, the eukaryotic proteins contain a
large NH2-terminal domain of about 650 amino acids whose sequence is poorly conserved between members. The
NH2-terminal domain is important for activity in yeast
(16), but with the exception of the 3'- to 5'-exonuclease domain of WRN
(17, 18) the biochemical function of the NH2-terminal
domain is unknown.
A subset of the eukaryotic RecQ family members has been shown to
interact with DNA topoisomerase III
(Top3)1 (19-22). Eukaryotic
Top3 was first identified as a hyperrecombination mutant in yeast that
also displayed a slow growth phenotype (23). Top3 has since been
identified in several organisms including S. pombe (21, 24),
Caenorhabditis elegans (25), and humans (26, 27).
Like the bacterial enzyme, eukaryotic Top3 is a type I 5'-DNA
topoisomerase with weak superhelical relaxing activity and a strict
requirement for substrates containing single-stranded DNA or
strand-passing activity (28, 29). The biological function of Top3 is
unclear, but in addition to its relaxing activity E. coli
topoisomerase III is notable for its ability to decatenate gapped
single-stranded DNA circles (29). The recent demonstration that
eukaryotic Top3 and E. coli RecQ helicase functionally
interact to catenate fully duplex DNA circles (30) suggested a role for these enzymes at the termination of DNA replication to decatenate daughter chromosomes (31, 32). Although it has been suggested that RecQ
helicases might function to restart stalled replication forks (7,
33-35) a role for Top3 in this process is unclear.
The SGS1 gene of yeast was identified as a mutation that
suppressed the slow growth phenotype of top3 mutants (22).
Thus, in contrast to top3 strains, top3 sgs1
double mutants exhibit a near wild type growth rate as well as
suppression of other top3 phenotypes (22, 36). Compared with
wild type cells the sgs1 single mutant displays increased
rates of mitotic recombination, both at the ribosomal DNA locus and
throughout the genome (22, 37), as well as increased rates of
chromosome loss and missegregation (38). Like mutations in
BLM, SGS1 mutations result in a hypersensitivity to methyl methanesulfonate (MMS) (16) and hydroxyurea (HU)
(39).
SGS1 was cloned in a two-hybrid screen with TOP3,
suggesting that Top3 interacted with the first 550 amino acids of Sgs1
(22). Because two-hybrid results do not provide evidence for direct binding, we set out to confirm this result biochemically, refine the
domain of interaction, and determine whether binding was through direct
protein-protein interaction. We identified an Top3·Sgs1 complex by coimmunoprecipitating and cofractionating these proteins from yeast extracts. The results indicate that Sgs1 and Top3 are present in a stable complex and that the NH2-terminal 158 amino acids of Sgs1 are sufficient for complex formation. The proteins do not appear to form a simple heterodimer, however, because the full-length proteins cofractionate at a large native molecular weight.
We determined that only the NH2-terminal 158 amino acids of
Sgs1 were required to bind Top3 based on an enzyme-linked immunosorbent assay (ELISA) using purified proteins. These biochemical results are
consistent with our observation that phenotypes caused by mutations in
the first 158 amino acids of Sgs1 can be suppressed by overexpressing
Top3, whereas larger deletions cannot.
Yeast Strains and Plasmids--
Strain construction, growth, and
transformation followed standard protocols (40). S. cerevisiae strain NJY620 expresses epitope-tagged versions of Sgs1
and Top3. This strain was constructed by modifying the chromosomal
SGS1 gene of wild type strain CHY125 (41) by integrating
BglII-linearized plasmid pJM1526, which places three
consecutive HA epitopes (YPYDVPDYA) at the COOH terminus of Sgs1. This
gene and protein are henceforth called SGS1-HA and Sgs1-HA,
respectively. The chromosomal TOP3 gene was modified by
integrating SphI-linearized pJM2565, which places a single V5 epitope (GKPIPNPLLGLDSTRTG, Invitrogen) followed by six histidines at the COOH terminus of Top3. This gene is henceforth referred to as
TOP3-V5 and its encoded protein as Top3-V5. Strain WFY822 was created by integrating pJM2565 into strain NJY531
(sgs1::loxP) (16). Strain NJY560 was constructed
by deleting the SGS1 and SLX4 genes of CHY125
(41) with loxP-KAN-loxP cassettes (42) and maintaining the
strain with plasmid pJM500 (SGS1/URA3). SGS1 and
sgs1-34 were integrated at the LEU2 locus of
NJY560 to create strains BSY1228 and BSY1229, respectively.
SGS1 mutant phenotypes were assayed as described (16).
Plasmid pJM1526, which expresses the epitope-tagged truncation
Sgs1645-1447-HA, contains the insert from pSM105-HA (16) in the vector pRS405 (43). Plasmid pJM2565 contains a fragment of the
TOP3 gene encoding a COOH-terminal in-frame fusion to the V5-His6 epitope (Invitrogen) in pRS404. To overexpress Top3 in yeast,
TOP3 was subcloned downstream of the GAL1
promoter in pRS424 to make pJM2566. Plasmids expressing Sgs1-HA
truncations were described (16), except for pKR1554 and pKR1555, which
express epitope-tagged proteins Sgs11-158-HA and
Sgs11-322-HA, respectively. To create these plasmids the
first 474 and 966 base pairs of SGS1 were amplified by
polymerase chain reaction so as to place an NdeI site in the
context of the initiating ATG and an NotI site at the end of
the coding region. These fragments were subcloned into
NdeI/NotI- digested pSM100-HA (16). For expression of recombinant yeast proteins in E. coli, TOP3-V5
was subcloned into the T7-inducible vector pET11a (44), yielding plasmid pSAS402. Glutathione S-transferase (GST) fusion
proteins were expressed by subcloning NdeI/BamHI
fragments from pKR1554 and pKR1555 into pET11GTK-WF to create pKR1564
and pKR1565. Plasmid pET11GTK-WF was created by destroying the
NdeI site of pET11GTK (45) and placing an in-frame
NdeI downstream of the GST target coding region by
polymerase chain reaction.
Yeast Extracts, Immunoprecipitations, and
Immunoblotting--
Extract preparation and chromatography were
performed at 4 °C. To prepare large scale extracts, yeast cells were
grown in 12 liters of yeast extract-peptone-dextrose (YPD) at 30 °C
to A600 = 1.5; the medium was
supplemented with an additional 2% dextrose and growth continued to
A600 = 2.8, which yielded 90 g of cells,
wet weight. Cells were washed once with H2O and resuspended in Buffer A (25 mM Tris-HCl (pH 7.5), 1 mM
EDTA, 0.01% (v/v) Nonidet P-40, 10% (v/v) glycerol, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM DTT)
plus 200 mM NaCl and the following protease inhibitors: 10 µg/ml pepstatin, 5 µg/ml leupeptin, 10 mM benzamidine,
100 µg/ml bacitracin. The cells were broken in a bead-beater (Biospec Products) with 50% volume of glass beads in 30-s bursts (separated by
90-s pauses) for a total of 5 min of breakage. The lysate was centrifuged at 16,000 × g for 10 min and the resulting
supernatant cleared at 235,000 × g for 90 min in a
Beckman Ti45 rotor. This centrifugation was observed to pellet a
significant portion of the chromatin as reported (46). The cleared
lysate was precipitated by stirring 350 mg of
(NH4)2SO4 /ml of lysate for 60 min
followed by centrifugation at 188,000 × g for 15 min.
The pellet was resuspended in 84 ml of Buffer A and dialyzed to a
conductivity of Buffer A plus 250 mM NaCl. Small scale
extracts for immunoprecipitations were prepared as described (16).
Protein concentrations were determined by the Bio-Rad protein assay
using bovine serum albumin as a standard. Superose 6 chromatography was
performed in Buffer B (25 mM Hepes-HCl (pH 7.5), 1 mM EDTA, 0.01% (v/v) Nonidet P-40, 0.1 mM
phenylmethylsulfonyl fluoride, 1 mM DTT) containing 150 mM NaCl at 0.4 ml/min. Fractions were collected,
precipitated with trichloroacetic acid, and resolved by 10%
SDS-PAGE.
Immunoprecipitations (IPs) were performed at 4 °C essentially as
described (16). Unless otherwise indicated, all IPs were performed by
incubating extract with 1 µl of anti-HA (Roche Molecular Biochemicals, 5 µg/µl) or anti-V5 (Invitrogen, 1 µg/µl)
antibodies for 1 h in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1% (v/v) Nonidet P-40, 0.5% (w/v)
deoxycholate, 0.1% (w/v) SDS) (47). 20 µl of protein-A Sepharose
beads (Amersham Pharmacia Biotech) was added to each sample, followed
by rocking for 1 h. The immune complexes were then washed three
times with 1 ml of RIPA buffer. Following SDS-PAGE the gels were
transferred to nitrocellulose membranes (48) and treated with either
anti-V5-horseradish peroxidase or anti-V5 as the primary
antibody (1:10,000). Blots were treated with anti-mouse horseradish
peroxidase conjugate secondary antibody as required (1:10,000; Life
Technologies, Inc.) and developed with chemiluminescence reagents (Life
Technologies, Inc.) to detect Top3-V5. Blots were reprobed with anti-HA
(1:10,000) as the primary antibody and treated as above to detect
Sgs1-HA. For phosphate labeling experiments, yeast cells were grown and
labeled with [32P]PO4 as described (49).
Extract preparation and immunoprecipitations were then performed as
described above.
Purification of Recombinant Yeast Proteins--
Plasmids
pET11GTK (expressing GST alone), pKR1564
(GST-Sgs11-158-HA), pKR1565
(GST-Sgs11-322-HA), and pSAS402 (Top3-V5) were transformed
into E. coli BL21-RIL cells (Life Technologies, Inc.). Cells
were grown by shaking in LB medium containing 0.1 mg/ml ampicillin at
37 °C to an A600 of 0.4. To induce the
expression of the recombinant protein, cultures were treated with
isopropyl-1-thio-D-galactopyranoside at a final
concentration of 0.1 mM for 2 h at 37 °C, except
for cells expressing Top3-V5, which were induced for 6 h at
20 °C. Induced cells were pelleted and resuspended in Buffer A plus
protease inhibitors (above) containing 250 mM NaCl for GST
and GST fusions, and 150 mM KCl for Top3. Extractions and
chromatography were performed at 4 °C, except where noted. Cell
suspensions were incubated with 0.1 mg/ml lysozyme for 30 min and then
sonicated three times for 1 min using a Branson sonifier 450 microtip
at setting 4, 60% duty cycle. Lysed cells were clarified by
centrifugation at 32,500 × g and the supernatant
collected as extract.
GST and GST-Sgs11-158-HA proteins were purified by batch
binding the extract from 1 liter of cells to 1 ml of
glutathione-Sepharose 4B resin (Amersham Pharmacia Biotech) for 2 h. The resin was washed with 3 column volumes of Buffer A plus 250 mM NaCl, then half-column volume fractions were eluted at
room temperature with Buffer A (pH 8.0) plus 150 mM NaCl
and 10 mM glutathione. The peak fraction was determined by
Bradford assay and SDS-PAGE, then 200 µl was fractionated on a
Superdex 75 (Amersham Pharmacia Biotech) gel filtration column in
Buffer B plus 150 mM NaCl to achieve greater purity.
GST-Sgs11-322-HA extract from 2 liters of cells was
diluted in Buffer A to a conductivity of Buffer A plus 50 mM NaCl and bound to an SP-Sepharose (Amersham Pharmacia
Biotech) column at 20 mg of extract/ml of resin. SP-Sepharose was
washed with 3 column volumes of Buffer A plus 200 mM NaCl,
then GST-Sgs11-322-HA was eluted in Buffer A plus 500 mM NaCl. The resulting SP 500 mM pool was
diluted in half with Buffer A and affinity purified by
glutathione-Sepharose 4B and Superdex 75 chromatography as above.
Top3-V5 containing extract from 3 liters of cells was bound to P-11
phosphocellulose (Whatman) at a ratio of 10 mg of extract/ml of resin
in Buffer A plus 150 mM KCl. The column was washed with 3 column volumes of Buffer A plus 400 mM KCl, then
Top3-V5-containing fractions were eluted from the column in Buffer A
plus 600 mM KCl. Top3-V5-containing fractions were
precipitated with 400 mg/ml (NH4)2SO4 for 1 h and then
pelleted at 32,500 × g. The resulting Top3-V5-containing pellet was resuspended in Buffer N (25 mM Tris-HCl (pH 8.0), 0.01% (v/v) Nonidet P-40, 10% (v/v)
glycerol, 0.1 mM phenylmethylsulfonyl fluoride, 250 mM NaCl) plus 20 mM imidazole and batch bound
to 1.5 ml of Probond nickel resin (Invitrogen) for 4 h. Resin was
poured into a column and washed with 3 column volumes of Buffer N plus
20 mM imidazole and 10 column volumes of Buffer N plus 50 mM imidazole. Top3-V5 protein was then eluted in 6 half-column volume fractions of Buffer N plus 250 mM imidazole.
ELISAs--
To detect a direct interaction between Top3 protein
and the NH2 terminus of Sgs1, 15 pmol of purified GST and
GST-Sgs1-HA fragments were first immobilized in DYNEX Imulon 2 HB 0.4-ml wells. Immobilization of GST and GST-Sgs1-HA fragments was
carried out in 75 µl of PBS (10.1 mM
Na2HPO4, 2.4 mM
KH2PO4, 137 mM NaCl, 2.7 mM KCl), pH 7.2, containing 0.1% Tween 20 (PBST) and 1 mM DTT by shaking at 60 rpm for 1 h at room
temperature. After immobilization, wells were washed once with 0.4 ml
of PBST plus 1 mM DTT, then blocked with 0.4 ml of 5%
dried milk (w/v) in PBST plus 1 mM DTT for 1 h at room
temperature. After blocking, cells were washed three times with 0.4 ml
of PBST plus 1 mM DTT. A titration of 0-40 pmol of Top3
protein was added to each set of coated wells in PBST plus 1 mM DTT in a volume of 75 µl and incubated for 30 min at
room temperature. After incubation with Top3-V5 protein the wells were
washed three times with 0.4 ml of PBST. To detect the Top3-V5 protein,
100 µl of anti-V5 antibody (diluted 1:5,000 in PBST plus 0.5% dried
milk) was added to each well for 1 h at room temperature. Wells
were then washed three times with PBST, and 100 µl of anti-mouse
horseradish peroxidase conjugate secondary antibody (diluted 1:5,000 in
PBST plus 0.75% (w/v) dried milk) was added to each well and incubated
for 1 h at room temperature. After the secondary antibody
incubation, wells were washed three times with 0.4 ml of PBST, and then
200 µl of 3,3',5,5'-tetramethylbenzidine liquid substrate system for
ELISA (Sigma) was added to each well and incubated for 30 min at room
temperature. After incubation, 100 µl of 0.5 N
H2SO4 was added to each well and the
A450 of each solution read to determine the
amount of Top3-V5 protein present.
Functional Complementation of Epitope-tagged SGS1 and TOP3--
To
characterize the interaction between Top3 and Sgs1, we constructed
yeast strains whose chromosomal copies of the SGS1 and TOP3 genes were modified to express the COOH-terminally
tagged proteins Sgs1-HA and Top3-V5 (see "Experimental
Procedures"). These strains allowed us to immunoprecipitate and
immunoblot the products of stable single-copy genes expressed under
their native promoters. To verify that the epitope-tagged alleles
behaved like wild type, we tested their ability to complement various
sgs1 and top3 phenotypes. Two very sensitive
measures of SGS1 and TOP3 activity are resistance
to the DNA-damaging agent MMS and resistance to the DNA synthesis
inhibitor HU (16). The strains expressing the tagged proteins were
serially diluted and replica plated to medium containing MMS or HU. As
shown in Fig. 1, the epitope-tagged strains grew as well as wild type on YPD plates and did not show the HU
or MMS hypersensitivity characteristic of sgs1 or
top3 strains. For example, top3 mutants grow very
slowly on YPD; SGS1 TOP3-V5 cells do not display the slow
growth of SGS1 top3 cells and in fact grow at the wild type
rate (data not shown). Similarly, sgs1 strains grow somewhat
slower than wild type, and SGS1-HA TOP3 cells grow
noticeably faster than sgs1 cells. Whereas sgs1 and top3 single mutants are hypersensitive to MMS and HU
(Fig. 1), the SGS1-HA and TOP3-V5 strains do not
display either of these sensitivities; these strains grow like wild
type in the presence of these drugs as does the SGS1-HA
TOP3-V5 double-tagged strain. Based on these growth phenotypes we
conclude that the epitope-tagged alleles SGS1-HA and
TOP3-V5 function exactly like wild type.
Coimmunoprecipitation and Cofractionation of Sgs1 and Top3--
To
identify an interaction between Sgs1 and Top3, extracts were prepared
from a wild type strain and from strain NJY620 expressing Sgs1-HA and
Top3-V5. Following incubation of the extracts with anti-HA or anti-V5
antibodies, the immune complexes were precipitated with protein A beads
and analyzed by immunoblot. Using extracts from cells expressing the
tagged proteins, we observed that anti-V5 precipitated Top3-V5, as
expected, and coprecipitated Sgs1-HA (Fig.
2A, lane 6).
Similarly, anti-HA precipitated Sgs1-HA, as expected, and
coprecipitated Top3-V5 (Fig. 2A, lane 4). These signals are specific to the epitope-tagged proteins as extract from the
untagged wild type strain showed no bands of corresponding size. We
note that under optimal conditions Top3-V5 coprecipitated Sgs1-HA more
efficiently than Sgs1-HA coprecipitated Top3-V5 (Fig. 2A,
compare lanes 2 and 4 with 6 and
8). The simplest explanation for this effect is that there
is an excess of Top3 over Sgs1 protein in the extract. Such a result is
consistent with the genetics of this system; lowering the Top3:Sgs1
ratio either by mutating TOP3 (22) or by overexpressing
SGS1 (16) results in a profound growth defect.
The previous experiment indicates that Sgs1 and Top3 interact in cell
extracts but does not address the strength of the interaction or
whether these proteins require DNA to interact. We addressed these
questions by varying the conditions of the immunoprecipitation from
nonstringent (Buffer A plus 150 mM NaCl) to very stringent (RIPA buffer plus 50 µg/ml ethidium bromide). As shown in Fig. 2B, the intensity of the Sgs1-HA signal that coprecipitated
with Top3-V5 was unaffected by changing these conditions. Likewise, the
efficiency with which Top3-V5 was coprecipitated with Sgs1-HA was
unaffected by changing these conditions (Fig. 2B,
lower panel). Both proteins were found to coprecipitate even
under the harshest conditions. We conclude that Sgs1 and Top3 are
stably bound and that their interaction is not mediated by DNA.
If Sgs1 and Top3 are present in a complex then they would be expected
to cofractionate over a gel filtration column. An extract from NJY620
cells was fractionated over a Superose 6 gel filtration column, and the
fractions were immunoblotted to determine the elution volumes of
Sgs1-HA and Top3-V5 (Fig. 3). A portion
of the Sgs1-HA and Top3-V5 proteins were found to elute with a similar profile, the peak of which corresponds to a native molecular
mass of ~1.3 MDa (Fig. 3, top and middle
panels). Additional Top3-V5 signal was detected in a second peak
close to the void volume, although this signal was not associated with
Sgs1-HA (Fig. 3, middle blot). When WFY822 (sgs1
TOP3-V5) extract was fractionated on a Superose 6 column, only the
Top3-V5 signal eluting near the void volume was detected (Fig. 3,
bottom panel). We conclude that the 1.3-MDa peak of Top3 is
Sgs1-dependent, and the Top3-V5 signal near the void is
likely to represent aggregated Top3-V5 protein that is in excess of
Sgs1-HA. If a single polypeptide of Sgs1 were to interact with a single
polypeptide of Top3, the expected size would be 240 kDa. The larger
size of 1.3 MDa suggests that these proteins have a different
stoichiometry or are complexed with additional proteins.
TOP3 and SGS1 Interact through the NH2 Terminus of
SGS1--
To determine the domain(s) of Sgs1 responsible for
interaction with Top3, strain WFY822 (TOP3-V5
sgs1::loxP) was transformed with a series of plasmids
expressing Sgs1-HA truncations under the control of the native
SGS1 promoter (16). Extracts were prepared and IPs performed
under RIPA conditions. A fragment of Sgs1 consisting of amino acids
1-652 (Sgs11-652-HA) coprecipitated with Top3-V5 (Fig.
4A), consistent with a Top3
interaction domain in the NH2-terminal 550 amino acids as
determined by the two-hybrid assay (22). In contrast, no interaction
was detected between Top3-V5 and the DNA helicase domain of Sgs1
(Sgs1645-1447-HA) (Fig. 4B). These results
indicate that the interaction between Sgs1 and Top3 is mediated through
the NH2 terminus of Sgs1. To map the interaction domain
more accurately, fragments of Sgs1-HA containing all but the
NH2-terminal 158 or 322 amino acids were expressed in the
presence of Top3-V5. Neither Sgs1159-1447-HA nor
Sgs1323-1447-HA was successfully coprecipitated with Top3-V5 (Fig. 4, C and D). This result indicates
that the first 158 amino acids are necessary to detect an interaction
with Top3-V5 under these conditions. We tested whether the first 158 amino acids were sufficient for this interaction and observed that
Sgs11-158-HA was indeed coprecipitated with Top3-V5 (Fig.
4E). When Sgs11-158-HA was immunoprecipitated
with anti-HA it migrated in between a doublet of small IgG chains on
SDS-PAGE. Comparing this signal with that of a control IP from an
untagged strain confirms the identity of this band as
Sgs11-158-HA (Fig. 4F). We were unable to test
whether Sgs11-322-HA could be coprecipitated with Top3-V5 because this protein was insoluble when expressed in yeast (data not
shown). We conclude that the first 158 amino acids of Sgs1 are
necessary and sufficient to interact with Top3-V5 in
vivo.
After determining that the first 158 amino acids of Sgs1 are sufficient
for interacting with Top3 in yeast extracts, we wanted to find out if
this was due to a direct protein-protein interaction. We initially
tried to express Top3-V5 and Sgs1-HA fragments in rabbit reticulocyte
lysates and immunoprecipitate them under mild conditions (Buffer A plus
150 mM NaCl), but these assays revealed no interaction
(data not shown). We then turned to the more sensitive ELISA to
identify a direct interaction. GST fusions of Sgs11-158-HA and Sgs11-322-HA were expressed in bacteria and purified on glutathione beads. As shown in Fig.
5A, unfused GST protein and
GST-Sgs11-322-HA were highly purified, whereas
GST-Sgs11-158-HA contained several smaller bands that are
likely to be breakdown products because their abundance varied between
preparations. Recombinant full-length Top3-V5 was highly purified using
Ni-affinity chromatography (Fig. 5A). ELISA wells were
coated with 15 pmol of purified GST, GST-Sgs11-158-HA, or
GST-Sgs11-322-HA and nonspecific sites blocked with 5%
dried milk. Increasing amounts of purified Top3-V5 protein were then
incubated in a series of wells prior to washing and detecting bound
Top3-V5 with anti-V5 antibody and a chromogenic substrate. This assay
revealed weak background binding of Top3-V5 to unfused GST protein that
saturated at 30 pmol of input Top3-V5 (Fig. 5B). In
contrast, both GST-Sgs11-158-HA and
GST-Sgs11-322-HA bound increasing amounts of Top3-V5 protein. At the highest input level of Top3-V5, these Sgs1 domains bound three times more Top3-V5 than GST alone. This result demonstrates a direct protein-protein interaction between the amino terminus of Sgs1
and Top3. Little difference between GST-Sgs11-158-HA and
GST-Sgs11-322-HA was detected, confirming that the first 158 amino acids contains a significant portion of the interacting domain.
Top3 Overexpression Complements Mutations in the NH2
Terminus of Sgs1--
We previously used a synthetic lethal screen to
identify several novel "SLX" mutants that require
SGS1 for viability (41). Phenotypically, sgs1
To address the question of why NH2-terminal deletions of
Sgs1 were defective in this assay, we tested whether overexpression of
Top3 could rescue the synthetic lethal phenotype. The starting strain,
NJY560 (slx4
The sgs1-34 mutation was isolated as a
temperature-sensitive allele of SGS1 caused by the amino
acid change Q31P. At the restrictive temperature (37 °C)
sgs1-34 behaves like sgs1- Post-translational Modification of Sgs1--
We suspected that the
failure to identify a strong Sgs1-Top3 interaction by in
vitro translation might be due to the requirement for an in
vivo modification to Sgs1. We addressed this question by asking
whether Sgs1 is phosphorylated in vivo. Yeast cells expressing Sgs1-HA or a control HA-tagged protein were grown in the
presence of 32Pi. Extracts were prepared from
these strains, and proteins were immunoprecipitated and analyzed by
SDS-PAGE and autoradiography. As shown in Fig.
7, immunoprecipitation of Sgs1-HA results
in a 32P-labeled band migrating at 220 kDa, as expected for
Sgs1-HA. The 220-kDa band is specific to Sgs1-HA as only the expected
100-kDa protein is precipitated with anti-HA from a control extract. An additional control revealed that only the expected 69-kDa RPA1 protein
was immunoprecipitated from the Sgs1-HA extract with an antiserum to
RPA1. Taken together, these data indicate that phosphorylated forms of
Sgs1 and RPA1 do not interact under these conditions. We conclude that
Sgs1 is phosphorylated during exponential growth in
vivo.
Although a DNA fragment encoding the amino-terminal 550 amino
acids of Sgs1 was isolated in a two-hybrid screen using Top3 as bait
(22), there has been no biochemical confirmation of this interaction or
any evidence of a direct interaction between these two proteins. To
address the in vivo association of these proteins we created
epitope-tagged alleles of SGS1 and TOP3 which were stably integrated at their chromosomal locations and expressed under their native promoters. These alleles were active in all of the
biological assays we examined, indicating that the tagged proteins
retain wild type function. Our data show that Sgs1 and Top3 can be
coimmunoprecipitated under stringent buffer conditions including 0.1%
SDS and ethidium bromide. These results are consistent with the idea
that these proteins are present in a stable complex in
vivo.
As summarized in Fig. 8, our deletion
analysis indicates that a domain as small as the
NH2-terminal 158 amino acids of Sgs1 is able to bind to
Top3 in vivo. ELISAs using highly purified proteins provide
biochemical evidence that Top3 binds Sgs11-158 through
direct protein-protein interactions. As expected for such a physical
interaction, Top3 overexpression suppressed phenotypes associated with
mutations in the Sgs1 amino terminus. The fact that overexpressed Top3
suppressed Sgs1 with NH2-terminal deletions as large as 158 amino acids suggests that the Top3 interaction domain extends further
than residues 1-158 in vivo (Fig. 8). Because overexpressed
Top3 failed to suppress a deletion of 322 amino acids, we conclude that
the Top3 interaction domain in vivo is larger than amino
acids 1-158 and smaller than 1-322.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Epitope-tagged alleles of SGS1
and TOP3 function like wild type. S. cerevisiae strains with the indicated genotypes were scraped from
freshly grown plates, resuspended in H2O to equal
A600, and serially diluted 1:10 in a microtiter
plate. 5 µl of each dilution was then replica plated to YPD plates
containing 100 mM HU, 0.012% MMS, or no drug as shown.
Cells were grown at 30 °C for 24 h (YPD) or 48 h (HU, MMS)
and photographed. The top row contains the wild type (WT)
parent strain CHY125.
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Fig. 2.
Coimmunoprecipitation of Sgs1-HA and
Top3-V5. Panel A, extracts were prepared from strain
NJY620 (SGS1-HA TOP3-V5) expressing Sgs1 and Top3
epitope-tagged proteins (even numbered lanes) and a wild
type strain (CHY125) expressing no epitope-tagged proteins (odd
numbered lanes). 1 mg of each extract was immunoprecipitated with
anti-HA or anti-V5 antibodies under RIPA conditions and the products
resolved by SDS-PAGE. After transfer to nitrocellulose the membrane was
probed with anti-V5-horseradish peroxidase to detect Top3
(left) or anti-HA to detect Sgs1 (right).
Panel B, IPs were performed as above except that the NJY620
extract was prepared, and the immune complexes were washed under the
following conditions: Buffer A plus 150 mM NaCl, RIPA
buffer, or RIPA buffer plus 50 µg/ml ethidium bromide, as indicated.
The upper blot was probed with anti-HA to detect Sgs1, and
the bottom blot was probed with anti-V5-horseradish
peroxidase to detect Top3.
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Fig. 3.
Cofractionation of Sgs1-HA and Top3-V5 by gel
filtration chromatography. Extracts from strains NJY620 and WFY822
(sgs1 TOP3-V5) were fractionated by Superose 6 chromatography as indicated. The fractions were trichloroacetic acid
precipitated, resolved by SDS-PAGE, and immunoblotted to detect Sgs1-HA
(top panel) or Top3-V5 (middle and bottom
panels). The relative positions of the molecular weight standards
blue dextran (2,000), thyroglobulin (669,000), and -galactosidase
(460,000) are indicated along with the peak of Sgs1-HA and Top3-V5
elution (*).
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Fig. 4.
Coimmunoprecipitation of Top3 and Sgs1
deletions. Strain WFY822 (sgs1 TOP3-V5)
was transformed with plasmids expressing the indicated Sgs1-HA protein
fragments under the control of the natural SGS1 promoter.
Cells were grown under selective conditions, whole cell extracts were
prepared, and the indicated IPs were performed under RIPA conditions.
The precipitated Sgs1-HA proteins were then detected by immunoblot
using anti-HA antibody. The 50-kDa Sgs11-158-HA protein
(panel E) migrates between two small
immunoglobulin proteins present in the anti-HA antibody. A control
extract from the untransformed parent strain (panel
F) was used to indicate the positions of the immunoglobulin
bands that are detected by the secondary antibody. The following
Sgs1-HA expression plasmids were used: panel A, pJM1541;
panel B, pSM105-HA; panel C, pSM103-HA;
panel D, pSM102-HA; panel E, pKR1554.
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Fig. 5.
Purified Top3-V5 and GST-Sgs1 protein
fragments bind in vitro. Panel A,
recombinant Top3-V5, GST-Sgs11-158-HA,
GST-Sgs11-322-HA, and GST were purified from bacterial
extracts and analyzed by SDS-PAGE and Coomassie Blue staining. The low
molecular weight bands in the GST-Sgs11-158-HA and Top3
preparations are most likely breakdown products. Proteins loaded:
marker proteins, 2 µg/band; GST-Sgs11-322-HA, 1.15 µg
total; GST-Sgs11-158-HA, 1.25 µg total; GST, 1.3 µg
total; Top3-V5, 1.0 µg total. Panel B, 15 pmol of purified
GST-Sgs11-158-HA, GST-Sgs11-322-HA, or GST
was immobilized in ELISA wells, blocked with 5% milk protein, and
challenged with the increasing amounts of Top3-V5. The wells were
washed, and the bound Top3-V5 was detected by treatment with anti-V5,
horseradish peroxidase-conjugated secondary antibodies, and a
chromogenic reagent. The reaction was stopped and the absorbance
measured at 450 nm.
and slx4
single mutants are viable, but the
sgs1
slx4
double mutant is dead. Because
Sgs1 activity is essential for viability in this background,
slx4
mutants provide a genetic system to identify
functional domains of SGS1. Structure-function analysis
previously revealed that small NH2-terminal deletions or
mutations in the DNA helicase domain of Sgs1 were lethal (16).
sgs1
pJM500,
SGS1/URA3), is nonviable on medium containing the drug
5-FOA because it selects against the SGS1/URA3
plasmid, which is essential for viability in this background. NJY560
was first transformed with a plasmid expressing the TOP3 gene under control of the inducible GAL1 promoter (pJM2566,
GAL1p-TOP3) and then with a series of SGS1
deletions in a LEU2 vector. In contrast to the
LEU2 vector alone, wild type SGS1 allowed these cells to grow on 5-FOA (Fig.
6A). Complementation of the
synthetic lethal phenotype by SGS1 is independent of Top3
overexpression because growth is observed under both repressed
(glucose) and induced (galactose) conditions. As expected, a
helicase-defective allele of SGS1 (sgs1-hd) and
all NH2-terminal truncations of Sgs1 were lethal when
streaked onto 5-FOA plates containing glucose (Fig. 6A). In
contrast, when these strains were streaked on 5-FOA galactose, the
sgs1-
N50 and sgs1-
N158 alleles displayed complementing activity. Neither
sgs1-hd nor sgs1-
N322
complemented, even when Top3 was overexpressed. These results indicate
that although DNA helicase activity is required under all conditions,
the first 158 amino acids of Sgs1 are not required if Top3 is
overexpressed. The suppression of the sgs1-
N158 allele is not specific to the synthetic lethal
phenotype because Top3 overexpression also suppressed the MMS
hypersensitivity of this allele (data not shown). The simplest
explanation for these results is that deletion of amino acids 1-158
significantly impairs the interaction between Top3 and Sgs1, and
increasing the Top3 concentration restores this interaction. Based on
this assay we conclude that the size of the interaction domain in
vivo must be larger than amino acids 1-158 and smaller than
1-322.
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Fig. 6.
Overexpression of Top3 suppresses
NH2-terminal mutations in Sgs1. Panel A,
strain NJY560 (sgs1 slx4
pJM500,
SGS1/URA3) was transformed with plasmid pNJ2566
(GAL1p-TOP3, TRP1) and the following LEU2
plasmids: pSM100 (SGS1), pJM531
(sgs1-
N50), pSM109
(sgs1-
N158), pSM110 (sgs1-
N322), pSM100-hd (sgs1-hd), and pRS415 (vector).
Transformants were streaked onto medium lacking tryptophan and leucine
but containing 5-FOA and the indicated sugar to select stains growing
in the absence of the URA3 plasmid pJM500. Panel
B, strains BSY1228 (SGS1 slx4
) and BSY1229
(sgs1-34 slx4
) were transformed with plasmid pNJ2566 and
streaked in duplicate onto selective plates lacking tryptophan but
containing either glucose (glc) or galactose
(gal) at 25 or 37 °C.
N158,
suggesting that it lacks Sgs1 NH2-terminal
function.2 As a result of
this mutation, strain BSY1229 (sgs1-34 slx4) is viable at
25 °C but not at 37 °C. We tested whether Top3 overexpression could suppress this NH2-terminal point mutation. Strain
BSY1229 was transformed with pJM2566 (GAL1p-TOP3) and the
transformants streaked on selective plates containing glucose or
galactose at 25 or 37 °C (Fig. 6B). When Top3 expression
was repressed by growth on glucose plates, the strain grew at 25 °C
but not at 37 °C. However, when Top3 was overexpressed by growth in
the presence of galactose the strain was able to grow at 37 °C (Fig.
6B). The suppression of sgs1-34 by Top3
overexpression is allele-specific, as two other SGS1
temperature-sensitive alleles whose mutations map to the DNA helicase
domain could not be suppressed (data not shown). As above, we conclude
that the sgs1-34 mutation impairs the binding of Top3 to
Sgs1 at the restrictive temperature and that increasing Top3
concentration restores the interaction.
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Fig. 7.
Sgs1 is a phosphoprotein. Extracts were
prepared from [32P]PO4-labeled yeast cells
expressing either Sgs1-HA or a 100-kDa HA-tagged control protein
(Mms4-HA). The control extract was immunoprecipitated with
anti-HA, and the Sgs1-HA extract was immunoprecipitated with anti-HA or
antiserum to RPA1 (RPA1).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
Summary of Sgs1-Top3 interactions. The
full-length Sgs1 protein and deletion derivatives are drawn
schematically as indicated on the left. The results of
coimmunoprecipitation, ELISA, and genetic suppression studies are
indicated on the right: +, Sgs1-Top3 interaction;
, no Sgs1-Top3 interaction; ND, not done. The fragment
consisting of residues 1-158 of Sgs1 (gray bars) is
consistent with the minimal Top3 binding domain based on
coimmunoprecipitation and ELISA results. The full interaction domain as
determined by genetic suppression is likely to extend somewhat further
as indicated by the black bars.
Recent evidence suggests that Top3 interacts with some, but not all,
RecQ family members. Bacterial Top3 and RecQ were shown to interact
functionally in vitro to catenate double-stranded DNA
circles (30). In S. pombe, the top3+
gene has been identified as an essential gene whose lethal phenotype is
suppressed by mutations in rqh1+, the S. pombe RecQ homolog (21). In human cells, immunolocalization studies indicate that BLM is present in promyelocytic leukemia nuclear
bodies together with Top3 (19, 50). The human RecQ5
protein was
also shown to colocalize and coimmunoprecipitate with Top3 (4). In
contrast, there is as yet no evidence that WRN interacts with Top3,
although it is associated with a large complex of replication proteins
including topoisomerase I (51).
Recently, Wu et al. (20) used a far Western blotting
technique to show that human Top3 bound BLM by direct
protein-protein interactions. These experiments demonstrated that the
NH2-terminal 212 amino acids of BLM was sufficient for
binding Top3. Our observation that Top3 binds a corresponding region of
Sgs1 (1) suggests that the NH2 termini of BLM and Sgs1
are functionally conserved. A comparison of the Sgs1 and BLM amino acid
sequences, however, reveals no obvious similarities or motifs that
might mediate the Top3 interaction. Our studies identified a single
Top3 binding domain of Sgs1, whereas human Top3 was found to bind a
second region of BLM (residues 1266-1417) (20). This difference may be
because that the far Western method included chemical cross-linking of
the two proteins, which was not used in our studies. We conclude that
this interaction is either not conserved in yeast or is not sufficiently stable to be detected by the methods used here.
While this work was in progress additional evidence of an interaction between Sgs1 and Top3 was reported. Bennett et al. showed that Sgs1 fragments bind Top3 in yeast extracts and inhibit Top3 activity in vitro (52). Maximal inhibition was obtained with a fragment of Sgs1 spanning residues 1-283. In addition, recent two-hybrid mapping studies identified a weak Top3 interaction domain between residues 1 and 116 of Sgs1 and a stronger interaction domain between residues 1 and 282 (53). Consistent with our genetic results (Fig. 6), these investigators proposed that a specific interaction between Sgs1 and Top3 is required for certain Sgs1 functions. The Top3 interacting domains identified in these studies agree closely with the Top3 binding domain identified in our experiments, as well as the NH2-terminal Top3 binding domain of BLM (20). Taken together with earlier data, it now appears that Sgs1 is more closely related to BLM than to WRN. Amino acid sequence analysis had initially shown that Sgs1 and BLM lack the exonuclease domain found in the NH2 terminus of WRN (17). Genetic complementation experiments have also shown that BLM is capable of complementing the HU hypersensitivity, top3 slow growth suppression, and premature aging phenotypes of sgs1 mutants that WRN could not (39, 54). Given that an interaction between Sgs1 and Top3 appears to be essential for Sgs1 activity, it will be of interest to determine whether the ability of human BLM to complement yeast sgs1 phenotypes depends on its ability to bind yeast Top3.
The mapping of the Top3 interaction to the region of amino acids 1-158
is significant in that small NH2-terminal truncations of
Sgs1, such as expressed by the sgs1-N158
allele, produce "hypermorphic" phenotypes that are more extreme
than the null phenotypes (16). Considering that the phenotypes of
top3 mutants are more extreme than sgs1 mutants,
one might hypothesize that the full NH2-terminal domain of
Sgs1 is required for complete Top3 activity in vivo. Although this model is consistent with the ability of overexpressed Top3 to suppress the sgs1-
N158 phenotype, it
is inconsistent with the fact that Sgs1 fragments inhibit Top3 activity
in vitro (52) and that sgs1-
N158
produces growth defects even in a top3 background (16). An
alternative model to explain these results is that a third factor
interacts with Top3 and Sgs1. Deletions of the Sgs1
NH2-terminal 158 amino acids might result in growth defects
by reducing binding to Top3 as well as this yet to be identified third factor.
The Top3·Sgs1 complex isolated from yeast is resistant to RIPA buffer, suggesting that the interaction is very stable. This affinity is retained even in a complex between Top3 and the relatively small Sgs11-158-HA fragment (Fig. 4). Given this apparent high affinity, it is surprising that multiple studies have required very sensitive methods to detect interactions between Top3 and RecQ helicases in vitro. We required a very sensitive ELISA, and it was reported that a Top3·Sgs1 complex formed in vitro was dissociated by the relatively gentle conditions of 140-250 mM NaCl (52). As mentioned above, chemical cross-linking was used to detect an interaction between Top3 and BLM (20). This suggests that the Top3·helicase complexes formed in vitro are different from those formed in vivo. This difference in affinity may simply reflect suboptimal binding conditions in vitro; higher protein concentrations and/or cotranslation might be required to form a stable complex. Alternatively, the correct interaction between Top3 and Sgs1 might depend on other cellular factors or modifications. As a test of this we found that Sgs1 is phosphorylated. The biological function of the phosphorylation is unknown, but it might regulate Sgs1 DNA helicase activity or the interaction of Sgs1 with Top3 or other proteins.
The Top3·Sgs1 complex eluted from a Superose 6 column at an approximate size of 1.3 MDa, indicating that it exists in a multimeric complex. Although we cannot rule out the possibility that a small amount of DNA mediates this complex, we feel it is unlikely for the following reasons. First, high speed extracts were used to remove bulk chromatin. Second, Top3 and Sgs1 coimmunoprecipitated despite treatment with ethidium bromide, which has been found to disrupt protein-DNA interactions. Third, we have examined the elution of double-stranded DNA by Superose 6 chromatography and found that DNA larger than 4 kilobases elutes in the void, whereas 1-kilobase DNA fragments elute at ~2 MDa. Thus, if the complex at 1.3 MDa were mediated by DNA, the fragments would have to be very small and discreet in size.
If the 1.3-MDa complex is a multimer, it might consist of a hexamer of
Top3 with a hexamer of Sgs1 which would be expected to run at that
size. Other helicases have been shown to exist as hexamers, such as
E. coli DnaB (55) and, more significantly, human BLM (56).
Alternatively, other proteins might be present in the complex as
reported recently for BLM. In addition to its presence in promyelocytic
leukemia bodies, BLM is associated with a number of human DNA repair
proteins in the BASC complex including some that are conserved in yeast
(50, 57, 58). Based on the interactions of BLM in human cells, it is
possible that Sgs1 and Top3 are associated with additional proteins
that contribute to its large native molecular weight and stable
association. Purification of the Top3·Sgs1 complex will be required
to determine which of these models is correct.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Suzanne Shanower for plasmid DNA and Hee-Sook Kim, Janet Mullen, and Marty Nemeroff for comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM55583 and AG16637.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Biology and Biochemistry, Center for Advanced Biotechnology and
Medicine, 679 Hoes Lane, Rutgers University, Piscataway, NJ 08854. Tel.: 732-235-4197; Fax: 732-235-4880; E-mail:
brill@mbcl.rutgers.edu.
Published, JBC Papers in Press, December 20, 2000, DOI 10.1074/jbc.M009719200
2 V. Kaliraman and S. J. Brill, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: Top3, DNA topoisomerase III; MMS, methyl methanesulfonate; HU, hydroxyurea; ELISA, enzyme-linked immunosorbent assay; HA, hemagglutinin; GST, glutathione S-transferase; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; IP, immunoprecipitation; RIPA, radioimmunoprecipitation assay; PBS, phosphate-buffered saline; RPA, replication protein A; FOA, fluoroorotic acid.
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