Novel Essential DNA Repair Proteins Nse1 and Nse2 Are
Subunits of the Fission Yeast Smc5-Smc6 Complex*
Received for publication, August 11, 2003, and in revised form, August 29, 2003
Published, JBC Papers in Press, September 8, 2003, DOI 10.1074/jbc.M308828200
W. Hayes McDonald‡§¶, Yelena Pavlova§?, John R. Yates, III‡**, and Michael N. Boddy?‡‡
From the Departments of ?Molecular Biology and ‡Cell Biology, The Scripps Research Institute,
La Jolla, California 92037
The structural maintenance of chromosomes (SMC)
family of proteins play essential roles in genomic stabil-
ity. SMC heterodimers are required for sister-chromatid
cohesion (Cohesin: Smc1 & Smc3), chromatin condensa-
tion (Condensin: Smc2 & Smc4), and DNA repair (Smc5
& Smc6). The SMC heterodimers do not function alone
and must associate with essential non-SMC subunits. To
gain further insight into the essential and DNA repair
roles of the Smc5–6 complex, we have purified fission
yeast Smc5 and identified by mass spectrometry the
co-precipitating proteins, Nse1 and Nse2. We show that
both Nse1 and Nse2 interact with Smc5 in vivo, as part of
the Smc5–6 complex. Nse1 and Nse2 are essential pro-
teins and conserved from yeast to man. Loss of Nse1 and
Nse2 function leads to strikingly similar terminal phe-
notypes to those observed for Smc5–6 inactivation. In
addition, cells expressing hypomorphic alleles of Nse1
and Nse2 are, like Smc5–6 mutants, hypersensitive to
DNA damage. Epistasis analysis suggests that like
Smc5–6, Nse1, and Nse2 function together with Rhp51 in
the homologous recombination repair of DNA double
strand breaks. The results of this study strongly suggest
that Nse1 and Nse2 are novel non-SMC subunits of the
fission yeast Smc5–6 DNA repair complex.
Maintenance of genomic integrity is crucial for cell viability
and suppression of cancer priming genetic alterations. The
genome cycle requires large amounts of DNA to be packaged
and structurally manipulated, during replication, mitosis and
repair. A superfamily of proteins called the structural mainte-
nance of chromosomes (SMC)1family play essential roles in all
of these processes (reviewed in Refs. 1 and 2). Eukaryotes
contain 6 SMC proteins, called SMC1–6. All SMC family pro-
teins share a similar architecture, in that they contain N- and
C-terminal Walker A and Walker B motifs, respectively, sepa-
rated by an extensive region that forms a coiled-coil structure.
These SMC proteins form specific heterodimers producing
three distinct SMC complexes involved in different aspects of
DNA metabolism. SMC1 and SMC3 form the basis of the cohe-
sin complex that tethers sister chromatids together following
passage of the replication fork. SMC2 and SMC4 are required
for chromosome condensation during mitosis, and SMC5 and
SMC6 are required for DNA repair as well as an enigmatic
essential function (reviewed in Refs. 1 and 2).
The SMC heterodimers do not function alone but require
interaction with essential proteins called non-SMC subunits.
Fission yeast cohesin (Smc1–3) requires the non-SMC sub-
units, Rad21 and psc3 (3). Rad21 is cleaved by separase (Esp1)
prior to anaphase, abolishing sister chromatid cohesion and
allowing chromosomes to segregate (3). An intriguing model
has recently been proposed in which Smc1–3 encircle sister
chromatids, with the circle held closed by interaction of Rad21
(Scc1) with the head groups of the asymmetric Smc1–3 het-
erodimer (4). Cleavage of Rad21 (Scc1) by separase (Esp1)
results in breakage of the Smc1–3 circle and loss of cohesion
Condensin requires at least three non-SMC subunits; Cnd1,
Cnd2, and Cnd3 to compact chromosomes during mitosis (5).
The non-SMC subunit requirements of the fission yeast
Smc5–6 complex are currently poorly defined. Studies in fis-
sion yeast showed that the Smc5–6 complex appears to consist
of six additional as yet unidentified non-SMC subunits (6).
Determining the function of the Smc5–6 complex would be
greatly assisted by identification and study of its non-SMC
Mutations have been isolated in SMC and non-SMC compo-
nents of cohesin and condensin that yield DNA damage-sensi-
tive phenotypes. For example, hypomorphic Rad21 mutants
display sensitivity to radiation (7). A recent study shows that
cohesin may play direct roles in DNA repair at the damage site,
rather than just maintaining chromosome cohesion (8). In
mammalian cells, cohesin is specifically recruited to the sites of
laser-induced DNA damage (8). Notably, the fission yeast con-
densin mutant cnd2-1 maintains cell viability but renders cells
sensitive to UV irradiation and replication arrest (9). How
condensin effects DNA repair is unknown.
The Smc5–6 complex is often labeled as a DNA repair com-
plex. Indeed, Smc6 (Rad18) was first identified in screens for
DNA damage-sensitive mutants (6, 10, 11). It is certainly in-
volved in DNA repair but Smc5–6 is essential even in the
absence of extrinsic DNA damage. Based on the essential na-
ture of Smc5–6 and the fact that mutants of cohesin and
condensin yield DNA damage sensitivity, it is likely that
Smc5–6 has a genome “housekeeping” role. Support for such a
role comes from the fact that in fission yeast, Smc6 (Rad18)
Recently, physical and genetic interactions were detected
between Rad60 and the Smc5–6 proteins (12, 13). Like
* This work was supported by National Institutes of Health Grant
GM068608 (to M. N. B.). The costs of publication of this article were
defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
¶ Supported by MERK-MGRI-241.
** Supported by RO1 EY1328801, MERK-MGRI-241, and CA81665
‡‡ A Research Special Fellow of the Leukemia & Lymphoma Society.
To whom correspondence should be addressed. Tel.: 858-784-7042; Fax:
858-784-2265; E-mail: email@example.com.
1The abbreviations used are: SMC, structural maintenance of chro-
mosomes; TAP, tandem affinity purification; GFP, green fluorescent
protein; MMS, methyl methanesulfonate; DAPI, 4?, 6-diamino-2-phe-
nylindole; MudPIT, multidimensional protein identification technology.
THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 278, No. 46, Issue of November 14, pp. 45460–45467, 2003
Printed in U.S.A.
This paper is available on line at http://www.jbc.org
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Smc5–6, Rad60 is essential and plays a role in DNA repair. The
similarity between the phenotypes of Smc5–6 and Rad60 mu-
tants suggests a co-dependent function of Rad60 and Smc5–6;
The identification and characterization of non-SMC subunits
of cohesin and condensin greatly facilitated functional analysis
of these complexes. Therefore, to gain insight into the func-
tion of the Smc5–6 complex and potentially Rad60, we purified
Smc5-TAP from fission yeast and applied multidimensional
protein identification technology (MudPIT) to identify co-puri-
fying proteins (14–16).
Here we report the identification of two non-SMC subunits of
the fission yeast Smc5–6 complex. Recently, the identification
of a budding yeast non-SMC component of the SMC5–6 complex
called NSE1 was described (17). We found very weak homology
between NSE1 and one of the proteins we identified. Therefore,
to maintain a unifying nomenclature we refer to this fission yeast
protein as non-SMC element Nse1 and the other as Nse2.
Gel filtration analysis shows that Nse1 and Nse2 co-migrate
with the characteristic high molecular weight Smc5–6 complex
(6). Co-immunoprecipitation studies confirmed that Nse1 and
Nse2 are indeed part of the Smc5–6 complex in vivo. Nse1 and
Nse2 are both essential genes, hypomorphic mutations of
which yield DNA damage-sensitive phenotypes. Epistasis anal-
yses suggest that, like Smc5–6, Nse1 and Nse2 function in the
homologous recombination repair of DNA double-strand breaks
with the fission yeast Rad51 homologue, Rhp51. Together,
these data strongly suggest that Nse1 and Nse2 are non-SMC
subunits of the fission yeast Smc5–6 DNA repair complex.
General Techniques—Standard fission yeast methods and media
were used in these studies (18). UV and IR sensitivity assays were
performed as described (19).
Generation of Tagged, Deleted, and Mutated Genes—Nse1 and Nse2
were deleted by replacement of the entire open reading frame (start to
stop codon) of each gene with the kanMx6 module as described in Bahler
et al. (20) producing heterozygous diploids (20). Epitope-tagged Nse1,
Nse2 and Smc5 were also generated as described in Bahler et al., 1998,
using the PCR-based method to place a Myc or TAP epitope at the C
terminus of each protein and mark the allele with the kanMx6 gene.
The tagged proteins were confirmed as fully functional.
The nse1-1 and nse2-1 alleles were generated using PCR. Genomic
DNA was isolated from yeast containing the epitope-tagged nse1-myc:
kanMx6 and nse2-myc:kanMx6 alleles. The entire genomic locus con-
taining each allele was amplified by PCR using standard conditions
(from start codon to 100-bp downstream of KanMx6). The amplified loci
were then re-amplified in 4 parallel PCR reactions. The PCR reactions
were pooled and transformed into Schizosaccharomyces pombe using
the transformation protocol described in Bahler et al. (20) and trans-
formants were selected by growth on YES media containing G418 (to
select for kanMx6) at 25 °C. Stable transformants were tested for re-
placement of the endogenous nse1 and nse2 alleles by the transformed
nse1-myc:kanMx6 and nse2-myc:kanMx6 alleles as described in Bahler
et al. (20). Stable transformants were then tested for temperature
sensitivity and drug sensitivity by plating the strains on YES media at
36 °C or on YES plates containing 5 mM hydroxyurea. Strains that
displayed temperature and/or hydroxyurea sensitivity were trans-
formed with an episomal plasmid containing the wild-type genomic
nse1 or nse2 genes to confirm that the strain defects were rescued by
and therefore, allelic to the respective genes.
Immunoblotting Gel Filtration and Microscopy Techniques—Immu-
noblotting was performed as described using extracts made from cells
lysed in a bead beater (19). Briefly, cells were lysed using in buffer A (50
mM Tris, pH 8, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 0.2% Nonidet
P-40, 5 ?g/ml each of leupeptin, pepstatin, and aprotinin, and 1 mM
phenylmethylsulfonyl fluoride) and resolved in 10% sodium dodecyl
sulfate polyacrylamide gels (SDS-PAGE). Proteins were transferred to
Immobilon membrane, blocked in 5% milk in TBS and 0.3% Tween-20,
and probed with antibodies to the epitope. Nse1-myc and Nse2-myc
were detected with anti-Myc antibody (9E10 at 1:5,000; Santa Cruz
Biotechnology). Smc5-TAP was detected with peroxidase anti-peroxi-
dase reagent (PAP at 1:2000 dilution; Sigma).
Gel filtration was performed on a Superdex 200 column calibrated
with high molecular weight markers (Amersham Biosciences). The void
volume was determined with blue dextran 2000 (Amersham Bio-
sciences). Flow rate was 0.3 ml/min and fractions were 0.6 ml. Samples
were lysed in buffer A and ?300 ?g of total protein was loaded via a
100-?l loop. For Smc5-TAP immunoprecipitation experiments, cells
were lysed in buffer A and IgG-Sepharose (Amersham Biosciences) was
added to the lysates followed by incubation at 4 °C for 1.5 h with
rotation. Complexes were collected by centrifugation and washed three
times with buffer A before resuspension in SDS-PAGE loading buffer.
Indirect immunofluorescence microscopy was performed using estab-
lished methods (21). GFP was visualized in live cells that were co-
stained with DAPI (4?,6?-diamidino-2-phenylindole) at 25 ?g/ml. Cells
were photographed with Nikon Eclipse E800 microscope equipped with
a Photometrics Quantix CCD camera.
Identification of Smc5-interacting Proteins—Proteins associating
with Smc5-TAP were identified by MudPIT using established methods
(14, 15, 22). Briefly, cells (? 50 g wet weight) expressing Smc5-TAP at
the genomic locus were frozen in liquid nitrogen and lysed using a
motorized mortar and pestle (Retsch) in buffer A (50 mM Tris, pH 8, 150
mM NaCl, 2 mM EDTA, 10% glycerol, 0.2% Nonidet P-40, 5 ?g/ml each
of leupeptin, pepstatin, and aprotinin, and 1 mM phenylmethylsulfonyl
fluoride). Smc5-TAP was purified from clarified lysate as described (23).
The final eluate was precipitated with trichloroacetic acid (25% v/v) for
1 h on ice. The precipitate was centrifuged (Eppendorf) at a relative
centrifugal force (r.c.f.) of 16. The pellet was washed twice with acetone
(?20 °C) and air-dried. The sample was reduced and alkylated using
dithothreitol and iodoacetamide and then sequentially digested with
endonuclease lyse-C (Roche Applied Science) and soluble trypsin (Roche
Applied Science) (24). The resulting peptide mixture was analyzed by
multidimensional protein identification technology (MudPIT) (15, 16)
with modifications described by McDonald and Yates (14) and MacCoss
et al. (25). Tandem mass spectra were searched against the latest
version of the pompep data base to which common contaminants such
as keratin and trypsin were added (These sequence data were produced
by the S. pombe Sequencing Group at the Sanger Centre and can
be obtained from ftp.sanger.ac.uk/pub/yeast/Pombe/Protein
Search results were filtered and grouped using the DTASelect program
and identifications confirmed through manual evaluation of spectra.
Common background proteins were also excluded by comparing the
Smc5-TAP data set to the large number of other data sets obtained by
purification of unrelated proteins in the laboratory.
Strains—All strains are ura4-D18 leu1–32 unless otherwise stated.
PR109, ura4-D18 leu1–32; NBY460, smc5:TAP:kanMx6; NBY462,
nse1::kanMx6/nse1?ade6–216/ade6–210; NBY468, nse1:myc:kanMx6;
NBY483, smc5-TAP:kanMx6 nse1:myc:kanMx6; NBY497, nse1:GFP:
kanMx6; NBY509, nse2:myc:kanMx6; NBY513, nse2::kanMx6/nse2?
ade6–216/ade6–210; NBY518, smc5:TAP:kanMx6 nse2:myc:kanMx6;
NBY526, nse1–1:myc:kanMx6; NBY527, nse2–1:myc:kanMx6; NBY530,
nse2:GFP:kanMx6; NBY536, nse2–1:myc:kanMx6 ? pE277-nse2:ura4?;
NBY537, nse2–1:myc:kanMx6 ? pE277:ura4?; NBY539, nse1–1:myc:
kanMx6 ? pE277-nse1:ura4?; NBY541, nse1–1:myc:kanMx6 ? pE277:
ura4?; NBY545, nse1–1:myc:kanMx6 rhp51::ura4?; NBY547, nse2–1:
myc:kanMx6 rhp51::ura4?; PS2345, rhp51::ura4?.
Identification of Nse1 and Nse2, Novel Smc5-associated Pro-
teins—Smc5 and Smc6 form a highly stable heterodimer that is
part of a larger complex consisting of at least six other uniden-
tified proteins (6). To gain further insight into the function of
the Smc5–6 complex of fission yeast, we purified tandem affin-
ity purification (TAP) epitope-tagged Smc5 (Smc5-TAP) and
used multidimensional protein identification technology (Mud-
PIT) to identify co-precipitating proteins (see “Experimental
Procedures” for details) (15, 23). We previously used the Mud-
PIT system to identify the Mus81-Eme1 Holliday junction re-
solving activity from fission yeast (22). In addition, we identi-
fied an interaction between the DNA repair factor Rad60 and
the Smc5-Smc6 complex (12). The major benefits of this system
are that it allows identification of proteins within a relatively
complex mixture of peptides and that it is highly sensitive.
Therefore, this system obviates the need for visualization and
excision of protein bands from gels for peptide identification.
Indeed, the MudPIT analysis of our TAP purifications typically
Smc5–6 Non-Smc Subunits
by guest on December 21, 2015
R. Yates III and Michael N. Boddy
2003, 278:45460-45467.J. Biol. Chem.
W. Hayes McDonald, Yelena Pavlova, John
and Nse2 Are Subunits of the Fission Yeast
Novel Essential DNA Repair Proteins Nse1
DNA: Replication, Repair, and
doi: 10.1074/jbc.M308828200 originally published online September 8, 2003
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