From the Institute of Life Sciences, Aalborg
University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark and
¶ Agilent Technologies, Hewlett-Packardstrasse,
D-76337 Waldbronn, Germany
Received for publication, January 9, 2003, and in revised form, February 4, 2003
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ABSTRACT |
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The structure-specific recognition protein SSRP1
plays a role in transcription and replication in the chromatin context.
Mediated by its C-terminal high mobility group (HMG) box domain, SSRP1 binds DNA non-sequence specifically but recognizes certain DNA structures. Using acetic acid urea polyacrylamide gel electrophoresis and mass spectrometry, we have examined the phosphorylation of maize
SSRP1 by protein kinase CK2 In eukaryotes, the packaging of genomic DNA with histones and
other proteins into chromatin affects DNA-dependent
processes, including transcription, recombination, replication, and
repair. In many cases, chromatin represses these processes by
restricting the access of DNA binding regulatory factors to their DNA
target sites. Numerous nuclear activities have been identified that can overcome the repressive effects of chromatin. Post-translational modification of histones and ATP-dependent,
chromatin-remodeling complexes are involved in altering the chromatin
structure so that the DNA becomes more accessible to the DNA-binding
proteins required for transcription initiation (1, 2). DNA repair and
other DNA-dependent processes occurring in the chromatin
context are also coupled to the structural properties of chromatin.
DNA is frequently damaged by a variety of agents that are of endogenous
or environmental origin. For many organisms, the most important
environmental mutagen is the ultraviolet component of sunlight. The DNA
lesions induced by UV light (mainly dipyrimidine photoproducts) are
both mutagenic and cytotoxic. Therefore, constant removal and
replacement of damaged nucleotide residues by DNA repair mechanisms is
required to maintain the DNA as a carrier of stable genetic
information (3, 4). DNA damage and repair processes are
influenced to a great extent by the packaging of the DNA into
chromatin. The repair machinery is therefore dependent on the
assistance of ATP-dependent, chromatin-remodeling complexes (5-7).
In human cells, a protein complex termed FACT (facilitates
chromatin transcription) has been identified
that is a chromatin-specific elongation factor required for
transcription of chromatin templates in vitro (8). The FACT
complex interacts with the H2A/H2B dimers of the nucleosome, indicating
that it may promote transcription by nucleosome remodeling. Biochemical
analyses have revealed that the FACT of HeLa cells consists of the
general transcriptional regulator CDC68/Spt16 and the
SSRP11 protein containing an
HMG box DNA-binding domain (9). In Xenopus, a homologous
complex termed DUF (DNA unwinding
factor) has been identified that is involved in DNA
replication in cell-free extracts (10). Extensive genetic and
biochemical work in yeast has demonstrated the existence of the CP
complex consisting of CDC68 and Pob3 (which is
homologous to SSRP1) and shown that both proteins are encoded by
essential genes. The CP complex plays a role in transcription and
replication (11, 12). Yeast Pob3 lacks the HMG box DNA binding domain
of the homologous SSRP1 occurring in higher eukaryotes, and the small
HMGB-type protein NHP6 appears to provide the HMG domain function,
mediating the interaction with nucleosomes (13, 14).
In plants, SSRP1 proteins (~72 kDa) with similarity to the vertebrate
counterparts have been identified from both monocotyledonous and
dicotyledonous species (15). Mediated by its HMG box domain, maize
SSRP1 binds non-sequence specifically to linear DNA and recognizes
nucleosomes and certain DNA structures such as DNA minicircles and
supercoiled DNA. Moreover, maize SSRP1 can bend DNA and facilitate the
formation of specific nucleoprotein structures (16, 17). Here we
demonstrate that maize SSRP1 is a substrate for protein kinase CK2 and
that the phosphorylated protein (but not the non-phosphorylated
protein) recognizes UV-damaged DNA. Furthermore, we show that a yeast
nhp6a/b double mutant is sensitive toward UV
irradiation compared with the wild type strain and that the expression
of maize SSRP1 can complement the increased UV sensitivity of the yeast
Expression, Purification and Phosphorylation of SSRP1
Proteins--
The region encoding
SSRP1-(Asp455-Asp639) was amplified from the
maize SSRP1 cDNA sequence using Deep Vent DNA polymerase (New
England Biolabs) and the primers 5'-AAGGATCCGATGTTCTACGAGACACTGAT-3'
and 5'-AATTAACTGCAGCTAGTCAGACTCGTTCCCAGA-3', digested with
BamHI and PstI, and cloned into pQE9cm (providing
an N-terminal His6 tag) (18), giving
pQE9cm-SSRP1-(Asp455-Asp639). The
SSRP1-(Asp455-Asp639) protein was expressed in
Escherichia coli and purified by three-step column
chromatography (nickel nitrilotriacetic acid-agarose, S-Sepharose Fast
Flow, Resource Q) as described previously (18). Full-length SSRP1 and
the other truncated SSRP1 derivatives were expressed as
His6-tagged fusion proteins in E. coli and
purified by three-step column chromatography as described previously
(17). The recombinant SSRP1 proteins were phosphorylated by purified
recombinant maize protein kinase CK2 Mass Spectrometry of Proteins and Peptides--
Mass
spectrometric analyses of SSRP1 proteins and tryptic peptides were
performed on a Voyager-DE PRO system equipped with a nitrogen laser
(Applied Biosystems) using the linear and the reflector mode. The
system was calibrated with standards provided by the supplier. As
matrix sinapinic acid (20 mg/ml in 50% acetonitrile, 0.1%
trifluoroacetic acid) was used. Samples were mixed 1:1 with the matrix
on the target plate and air-dried.
Circular Dichroism--
CD spectra of non-phosphorylated and
CK2 Analysis of DNA Binding by Electrophoretic Mobility Shift Assays
(EMSAs)--
The purified 78-bp XbaI/KpnI
fragment of pBluescript (Stratagene) was UV irradiated with 5 or 10 J/m2 at 254 nm. The UV-induced DNA damage was analyzed by
determining the cleavability of the DNA fragment with EcoRI,
because the UV-induced dimerization of the thymine bases occurring
within the GAATTC EcoRI recognition sequence abolished the
endonuclease digestion of the fragment (21). For instance, at an UV
dose of 5 J/m2, ~50% of the fragment was cleavable with
EcoRI (data not shown). Binding reactions contained binding
buffer (10 mM Tris/HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 5%(w/v)
glycerol, 0.05% (w/v) bromphenol blue, 0.05% (w/v) xylenecyanol),
various amounts of protein, and 15 ng of the 78-bp fragment. Binding
reactions were incubated for 5 min at room temperature before loading
of the samples onto 5% polyacrylamide gels in 1× TBE buffer (90 mM Tris borate, pH 7.5, 1 mM EDTA). The
DNA was stained with SYBR Gold (Molecular Probes) and detected with the
Typhoon 8600 PhosphorImager (Amersham Biosciences). Data were analyzed
using the Image Quant software.
Analysis of the UV Sensitivity of Yeast Strains--
The coding
region for maize SSRP1 was obtained by digesting the plasmid
pQE9cm-SSRP1 (17) with PstI, creating blunt ends using the
Klenow DNA polymerase and subsequent digestion with BamHI.
The pMTL1 yeast expression vector (22) was prepared by digestion with
HindIII, creating blunt ends using the Klenow DNA polymerase
and subsequent digestion with BamHI. The SSRP1 coding region
was ligated with pMTL1, resulting in the expression plasmid pMTL1-SSRP1, which was checked by DNA sequencing. The plasmids pMTL1
and pMTL1-SSRP1 were transformed into the yeast strains Y18
(MAT Protein Kinase CK2 Phosphorylates Several Residues in
SSRP1--
Human SSRP1 can occur in an UV-induced complex with Spt16,
p53, and CK2, resulting in specific CK2-meditated Ser392
phosphorylation of p53 (26, 27). Analysis of the amino acid sequence of
maize SSRP1 using the program Phospho Base 2.0 (28) revealed that maize
SSPR1 contains a number of potential phosphorylation sites for protein
kinase CK2. Therefore, we examined whether maize SSRP1 (schematically
depicted in Fig. 1A) is also a
substrate of CK2. In contrast to most other protein kinases, CK2 can
utilize both ATP and GTP as phosphate donors (29-31). The
phosphorylation of SSRP1 by recombinant maize CK2 Phosphorylation of C-terminal Residues in SSRP1 by CK2
Because the C-terminal phosphorylation of maize chromosomal HMGB
proteins by CK2 reduces the affinity of the HMGB1 and HMGB2 proteins
for linear DNA (19), we examined whether the phosphorylation of the two
amino acid residues at the C-terminal end of SSRP1 influences the DNA
binding of SSRP1-(Lys532-Asp639). The
interaction of non-phosphorylated and CK2 SSRP1 Can Complement the UV Sensitivity of the Yeast NHP6
Mutant--
The yeast homologue of animal and plant SSRP1, termed
Pob3, lacks an HMG box DNA-binding domain. Recently, it was
demonstrated that the HMGB-type protein NHP6A/B interacts with the
CDC68/Pob3 complex (CP complex) to provide an HMG box domain so that
Pob3 and NHP6A/B function as a bipartite analogue of SSRP1 (13, 14). An
artificial SSRP1-like protein, created by fusing Pob3 and NHP6A, is
able to perform several in vivo functions (13). Yeast mutant strains lacking either NHP6A or NHP6B (88% amino acid sequence identity) grow normally, whereas the strain lacking both proteins displays, among other defects, a temperature-sensitive growth (23).
After confirming some of these published growth characteristics (data
not shown), we have monitored the sensitivity of the double mutant
strain Protein kinase CK2, typically found in a heterotetrameric
( Like the non-sequence-specific HMG domain proteins of the HMGB family,
SSRP1 appears to be involved in a variety of DNA-dependent processes such as replication and transcription (38-41). In complex with CDC68/Spt16, SSRP1 (Pob3 in yeast) can act as facilitator of
transcription and recombination in the chromatin context (9, 11).
Mediated by its HMG box domain, SSRP1 binds DNA non-sequence specifically but has the ability to recognize certain DNA structures (minicircles, cisplatin-modified DNA, four-way junctions, and supercoiled DNA) and to bend DNA (17, 42-44). As shown here, the
phosphorylation of two amino acid residues at the C-terminal end of
maize SSRP1 resulted in an increased binding to UV-damaged DNA, whereas
the interaction with untreated DNA was unchanged. SSRP1 may recognize
the bend or kink introduced by the UV-induced DNA lesion. Depending on
the type of lesion, a cyclobutane pyrimidine dimer or a pyrimidine
(4-6) pyrimidone photoproduct, bends of 7-9° or 44°,
respectively, are inserted (3, 7). In contrast to the situation with
SSRP1, HMGB proteins that are phosphorylated by CK2 C-terminal
of the HMG domain display (depending on the DNA substrate) a rather
reduced affinity for DNA (19, 45).
The yeast Pob3 protein can be considered an HMG domain-less SSRP1
protein. Pob3 interacts with the small HMGB-type protein NHP6A
(containing an HMG domain) and may form a higher eukaryote type
"bipartite SSRP1 protein" (13, 14). A yeast strain lacking both
the NHP6A and NHP6B proteins (. The kinase phosphorylated several amino
acid residues in the C-terminal part of the SSRP1 protein. Two
phosphorylation sites were mapped in the very C-terminal region next to
the HMG box domain, and about seven sites are localized within the
acidic domain. Circular dichroism showed that the phosphorylation of
the two C-terminal sites by CK2
resulted in a structural change in
the region of HMG box domain, because the negative peak of the CD
spectrum at 222 nm was decreased by ~10%. In parallel, the
phosphorylation induced the recognition of UV-damaged DNA, whereas the
non-phosphorylated protein does not discriminate between UV-damaged DNA
and control DNA. The affinity of CK2
-phosphorylated SSRP1 for
the DNA correlates with the degree of UV-induced DNA damage. Moreover,
maize SSRP1 can restore the increased UV-sensitivity of a yeast strain
lacking the NHP6A/B HMG domain proteins to levels of the control
strain. Collectively, these findings indicate a role for SSRP1 in the
UV response of eukaryotic cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
nhp6a/b strain.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
as described previously for
HMGB proteins (19). For the control experiments with non-phosphorylated
proteins, the substrate proteins were incubated in mock phosphorylation reactions lacking either ATP or the protein kinase. The phosphorylation state of the SSRP1 proteins was examined by mass spectrometry and
acetic acid urea (AU)-PAGE (20).
-phosphorylated SSRP1(K532-D639) were recorded and analyzed as
described previously (19).
ura3-52 trp1-289 his3-
1 leu2-3 112 gal2 gal10)
and Y82 (MAT
ura3-52 trp1-289 his3-
1 leu2-3 112 gal2
gal10 nhp6A-
2::URA3 nhp6B-
1::HIS3) by the lithium acetate
method. The increased generation time and the temperature sensitivity
of the
nhp6Aa/b strain (Y82) was examined and
proved to match the results published previously (23). The
UV-sensitivity of the different strains was determined by calculating
the survival rate of the cells on solid YPD medium (24, 25). Single
colonies were grown to an A600 of 1 in
selective liquid medium. The cells were harvested by centrifugation and washed with TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). After serial dilution in TE buffer, the
cells were spread onto YPD plates (strains transformed with pMTL1
plasmids on galactose-containing plates), which were irradiated using a
UV StratalinkerTM 2400 (Stratagene) with different UV
doses of UV254 nm. The plates were incubated at 30 °C
for 5 days before scoring of the surviving colonies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was analyzed for
different reaction times in the presence of [32P]GTP or
[32P]ATP (Fig. 1B). Both phosphate donors were
utilized by CK2
to phosphorylate SSRP1 with similar efficiency. To
determine which domain(s) of SSRP1 are phosphorylated, full-length
SSRP1 (residues Met1-Asp639) and the truncated
proteins SSRP1-(Asp455-Asp639), lacking the
large N-terminal domain, and
SSRP1(Lys532-Asp639), consisting essentially
of the HMG box DNA-binding domain (Fig. 1A), were tested for
phosphorylation by CK2
in the presence of [32P]ATP
(Fig. 1C). Both full-length SSRP1 and
SSRP1-(Asp455-Asp639) were readily phosphorylated by
CK2
to similar extents, suggesting that the C-terminal part of the
protein is phosphorylated. The very C-terminal end of the protein,
SSRP1-(Lys532-Asp639), is phosphorylated by
CK2
to a lesser extent (Fig. 1B), demonstrating that
SSRP1 is mainly phosphorylated within the acidic domain (residues Asp455-Gln531). To estimate the number of
amino acid residues phosphorylated in SSRP1, truncated versions of
SSRP1 were phosphorylated for different times by CK2
in the presence
of unlabeled ATP. The phosphorylated proteins were analyzed by AU-PAGE,
which can resolve proteins depending on their phosphorylation state.
Phosphorylation of the C-terminal protein constructs
SSRP1-(Lys532-Asp639) and
SSRP1-(Lys552-Asp639) resulted in the
appearance of two protein bands with reduced electrophoretic mobility
(relative to the non-phosphorylated protein) upon CK2
phosphorylation, indicating the presence of two phosphorylation sites
in these proteins (Fig. 2). The same
experiment was performed with the C-terminal portion of SSRP1 including
the acidic domain SSRP1-(Asp455-Asp639),
resulting in the appearance of several phosphorylated protein bands,
which could not be completely resolved. From the analysis of several
gels, we estimated that up to approximately nine residues are
phosphorylated by CK2
in this protein. The number of phosphorylation sites in SSRP1-(Lys552-Asp639) and
SSRP1-(Asp455-Asp639) was further examined
using MALDI/TOF mass spectrometry. Comparison of the masses determined
for non-phosphorylated and CK2
-phosphorylated SSRP1-(Lys552-Asp639) and
SSRP1-(Asp455-Asp639) revealed mass
differences of 161 and 729 Da, respectively (data not shown). This
indicates the presence of two phosphorylated residues in
SSRP1-(Lys552-Asp639) and (depending on the
number of sodium adducts, Ref. 19) up to nine phosphorylated residues
in SSRP1-(Asp455-Asp639), which is in line
with the analysis of the phosphorylated proteins by AU-PAGE. To
determine more precisely which amino acid residues of the C-terminal
part of SSRP1 are phosphorylated by CK2
, non-phosphorylated and
CK2
-phosphorylated SSRP1-(Lys552-Asp639)
were digested with trypsin, and the resulting peptides were examined by
MALDI/TOF mass spectrometry (Table I).
This analysis revealed that the very C-terminal peptide
Gly626-Asp639 (comprising the peptide
C-terminal of the HMG box domain) occurs in the double-phosphorylated
form. This peptide contains one threonine and two serine residues,
which theoretically could be phosphorylated. However, the threonine
Thr629 is unlikely to be phosphorylated, first because
serine residues are by far preferred over threonine residues as
phosphor acceptor sites of CK2 and, second, because the sequence
context of the two serine residues more closely resembles typical CK2
phosphorylation sites (29-31). Moreover, the two serine residues are
well conserved among SSRP1 proteins of different plant species, whereas
the threonine residue Thr629 occurs exclusively in maize
SSRP1 (17). Therefore, Ser634 and Ser638 are
most likely phosphorylated by CK2
. Because of the unusual acidity of
the involved peptides (which poses a problem for mass spectrometry), we
were, to date, unable to determine precisely which of the 14 possible
Ser/Thr residues (of which ~8 are situated in a CK2 consensus
sequence) in the acidic region (Asp455-Gln531)
are actually phosphorylated by CK2
.
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Fig. 1.
Protein kinase CK2
phosphorylates amino acid residues within the acidic domain and
the C-terminal region of SSRP1. A, schematic
representation of the overall structure of the maize SSRP1 protein. The
N-terminal part of SSRP1 is indicated by an open
box, while the acidic domain is indicated by a filled
box with the short basic region containing the nuclear
localization sequence (17) in dark gray and the
HMG box DNA-binding domain in light gray. The
15-amino acid residue C-terminal of the HMG domain,
Arg625-Asp639 (R625-D639), is also
indicated. The amino acid positions delineating full-length and
truncated recombinant SSRP1 proteins used in this study are depicted at
the bottom. B, autoradiography of recombinant
SSRP1 phosphorylated by CK2
in the presence of
[32P]GTP (lanes 1-5) or
[32P]ATP. SSRP1 was phosphorylated for 1 min (lanes
1 and 6), 5 min (lanes 2 and 7),
15 min (lanes 3 and 8), 30 min (lanes
4 and 9), and 60 min (lanes 5 and
10), and separated by SDS-PAGE. C,
autoradiography of full-length and truncated SSRP1 proteins (comparable
molar amounts) phosphorylated by CK2
in the presence of
[32P]ATP and separated by SDS-PAGE.
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Fig. 2.
Several amino acid residues of SSRP1 are
phosphorylated by CK2 . The C-terminal SSRP1 constructs,
SSRP1-(Lys532-Asp639),
SSRP1-(Lys552-Asp639), and
SSRP1-(Asp455-Asp639) were phosphorylated for
the indicated times by CK2
in the presence of unlabeled ATP,
separated by AU-PAGE, and stained with Coomassie Blue. In the course of
the phosphorylation reaction, the initially non-phosphorylated proteins
are increasingly phosphorylated by CK2
and display, depending on the
number of phosphorylations, a reduced electrophoretic mobility relative
to the non-phosphorylated proteins. The number of protein
phosphorylations corresponding to the differently migrating protein
bands is indicated next to the bands in the gel.
Masses of tryptic peptides derived from non-phosphorylated and
CK2-phosphorylated SSRP1-(Lys552-Asp639)
Induces
Structural Changes in the HMG Box Region and Enhances the Binding to
UV-damaged DNA--
To examine whether the phosphorylation of the two
C-terminal amino acid residues of SSRP1 results in structural changes
of the protein, we recorded CD spectra in the range of 195-260 nm. In
these wavelength scans, non-phosphorylated and CK2
-phosphorylated SSRP1-(Lys532-Asp639) were compared (Fig.
3). The decrease (~10%) of the
negative peak at 222 nm in the CD spectrum of the phosphorylated
protein (relative to the non-phosphorylated protein) indicates loss of
-helical structure. The structure of the HMG box domain is ~75%
-helical, suggesting that there may be phosphorylation-induced structural changes in the DNA-binding domain.
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Fig. 3.
Phosphorylation by CK2
induces structural changes in the HMG box region. CD spectra
of non-phosphorylated (
P) and CK2
-phosphorylated
(+PCK2)
SSRP1-(Lys532-AP639) were recorded in the
wavelength range of 195-260 nm.
-phosphorylated SSRP1-(Lys532-Asp639) with a 78-bp DNA
fragment was analyzed in electrophoretic mobility shift assays. Various
concentrations of the protein were incubated with the DNA fragment, and
the formation of protein/DNA-complexes was examined using native PAGE
(Fig. 4). The non-phosphorylated and the
CK2
-phosphorylated SSRP1-(Lys532-Asp639)
bound the DNA-fragment with similar affinity, as can be seen by the
disappearance of the unbound fragment and the appearance of a rather
diffuse complex of lower electrophoretic mobility (Fig. 4, top
panels; lanes 5 and 6). To examine whether
UV-damaged DNA is a preferred target for SSRP1, the DNA fragment was
treated with different doses of UV prior to the protein binding
studies. The UV-damage of the DNA is evident from the more diffuse and slightly reduced electrophoretic mobility of the DNA fragment (32)
relative to the untreated DNA (compare Fig. 4, lanes 1) and
from its reduced cleavability by EcoRI (data not shown). The phosphorylated protein bound the UV-treated DNA fragment at
significantly lower protein concentrations when compared with the
non-phosphorylated protein (Fig. 4), demonstrating that the
phosphorylated protein displayed a higher affinity for the UV-damaged
DNA. This effect was dependent on the UV dose used to irradiate the
DNA. When the DNA was irradiated with 5 J/m2, the
phosphorylated protein displayed a ~5-fold higher affinity for the
DNA than the non-phosphorylated protein, whereas with the DNA that was
irradiated with 10 J/m2 there was a ~10-fold difference
in affinity. Unlike the CK2-phosphorylated protein that discriminated
UV-damaged DNA and control DNA, non-phosphorylated SSRP1-(Lys532-Asp639) bound the UV-damaged DNA
and the control DNA with the same affinity.
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Fig. 4.
CK2 -mediated
phosphorylation of C-terminal amino acid residues results in the
recognition of UV-damaged DNA by SSRP1. Increasing concentrations
(0, 50, 100, 250, 500 nM, and 1 µM,
lanes 1-6, respectively) of CK2
-phosphorylated
(left panels) or non-phosphorylated
SSRP1-(Lys532-Asp639) (right
panels) were incubated with a 78-bp DNA fragment. The DNA
was either untreated (top panels) or irradiated with 5 J/m2 (middle panels) or 10 J/m2
(bottom panels). The binding reactions were
separated by native PAGE, stained with SYBR Gold, and scanned using a
PhosphorImager.
nhp6a/b (Y82) toward UV irradiation in
comparison to the control strain (Y18). The yeast strains were treated
with different doses of UV light, and the colony formation of surviving cells was scored on YPD plates. The survival rate of the
nhp6a/b strain was ~2.5-fold lower than that
of the control strain at an UV irradiation of 25 and 50 J/m2 (Fig. 5A). To
examine whether maize SSRP1 had an effect on the increased UV
sensitivity of the
nhp6a/b (Y82) strain, the
survival upon UV irradiation with 25 J/m2 of the
non-transformed Y82 strain and Y82 bearing pMTL1-SSRP1 or pMTL1 was
analyzed in comparison to the control strain Y18. Y82 bearing
pMTL1-SSRP1 displayed UV sensitivity comparable with that of the Y18
control strain (Fig. 5B), indicating that the expression of
SSRP1 could complement the lack of NHP6A/B with respect to UV
sensitivity, whereas the presence of the insert-less plasmid pMTL1 in
Y82 had no significant effect.
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Fig. 5.
The increased UV-sensitivity of the
yeast nhp6a/b strain can be complemented
by maize SSRP1. A, colonies of the
nhp6a/b strain (Y82) and the control strain (Y18) were
grown in liquid medium, spotted onto YPD plates in serial dilutions,
and irradiated with various UV doses. Following UV irradiation,
colonies were scored after 5 days of growth at 30 °C. The graphs
depict the mean values of the survival rates of at least 10 individual
clones of the two strains (relative to the non-irradiated cells). The
formation of colonies was counted from each 10 starting
colonies. The graph depicts the mean values of the survival
rates of cells of the two strains relative to the non-irradiated cells.
B, the survival rates of the
nhp6a/b strain
(Y82), the control strain (Y18), the strain Y82 bearing the insert-less
pMTL1 plasmid, and the strain Y82 bearing the pMTL-SSRP1 plasmid were
scored after UV irradiation of 25 J/m2. The mean value of
the survival rates was determined as described above, and, in the case
of Y82-pMTL1 and Y82-pMTL1-SSRP1, four clones of three independent
transformants were analyzed.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
2) complex, is a Ser/Thr kinase
conserved in a wide variety of eukaryotes, including plants, mammals,
and yeast (29-31). CK2 phosphorylates predominantly serine residues
that occur in an acidic environment within the substrate proteins and
is involved in cell growth and proliferation but also in stress
response and cell survival (33, 34). Plant CK2 from various species
occurs as heterotetrameric enzyme but also as catalytically active
monomeric CK2
(35-37). Here we have shown that CK2
phosphorylates several residues of the maize SSPR1 protein. Two
phosphorylation sites were mapped within the very C-terminal tryptic
peptide of SSRP1, which are in agreement with typical CK2
phosphorylation sites (29-31). The two serine residues are well
conserved among different plant SSRP1 proteins but not in mammalian or
Drosophila SSRP1 (17). However, the mammalian and
Drosophila SSRP1 proteins contain (in comparison to the
plant counterparts) a more extended domain C-terminal of the HMG box
domain, which contains serine residues that perfectly match CK2
consensus recognition sites. Accordingly, CK2 can phosphorylate the
C-terminal part of human SSRP1 (amino acid residues 471-709), but the
exact phosphorylation site(s) have not yet been mapped (27). Therefore,
CK2-mediated phosphorylation of residues C-terminal of the HMG box
DNA-binding domain could be a general mechanism of SSRP1 regulation.
nhp6a/b strain,
Y82) displays an increased UV-sensitivity, as evident from the reduced
survival rates of the mutant cells upon UV irradiation, relative to
control cells. Expression of maize SSRP1 in the
nhp6a/b strain restored the UV sensitivity
comparable with the levels of the control strain. An artificial
Pob3-NHP6A fusion protein proved to be functional in yeast cells (13),
and such a fusion protein has, over the entire sequence, a high degree
of amino acid sequence similarity to maize SSRP1. Moreover, the
C-terminal region of the NHP6 proteins (46) contains (similar to maize
SSRP1) a serine residue that is compatible with a CK2 phosphorylation
site. CK2-mediated phosphorylation is correlated with a UV-response in
two ways. CK2 activity can be induced by UV irradiation (47), and CK2
can associate in an UV-dependent way with SSRP1/Spt16/p53
(26, 27). The CK2-mediated phosphorylation of SSRP1 induces the
recognition of UV-damaged DNA by SSRP1 and may link SSRP1 to an UV
damage response. Therefore, in addition to its established functions in
transcription and replication (9-11, 41, 48), SSRP1 may also play a
role in DNA repair.
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ACKNOWLEDGEMENTS |
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We thank Diana J. Leeming for preparing CD samples, Hanne Krone Nielsen for excellent technical assistance and Dr. W. Hörz for the yeast strains used in this study.
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FOOTNOTES |
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* This work was supported by a grant from the Danish Research Council (to K. D. G.).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.
§ Present address: greenovation Biotech GmbH, Bötzinger Str. 29b, D-79111 Freiburg, Germany.
To whom correspondence should be addressed. Tel.:
45-9635-9126; Fax: 45-9814-1808; E-mail: kdg@bio.auc.dk.
Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M300250200
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ABBREVIATIONS |
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The abbreviations used are: SSRP1, structure-specific recognition protein 1; HMG, high mobility group; AU, acetic acid urea; CD, circular dichroism; YPD, yeast extract-peptone-dextrose; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight.
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REFERENCES |
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