From the Department of Biology, Faculty of Science,
Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan, the
¶ Department of Histology and Cell Biology, Nagasaki University
School of Medicine, Sakamoto, Nagasaki 852-8523, Japan, and
CREST Japan Science and Technology Corp., Kawaguchi,
332-0012, Japan
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ABSTRACT |
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We have isolated a novel nuclear protein with a
molecular mass of 49 kDa (TIP49a) from rat liver. The rat TIP49a showed
structural resemblance to several bacterial RuvBs and also displayed
Walker A and B motifs. We overproduced the recombinant TIP49a in
Escherichia coli and purified it to near homogeneity.
Biochemical investigations demonstrated that TIP49a possessed ATPase
activity that was stimulated by single-stranded DNA but neither by
double-stranded DNA nor by any forms of RNA polymers tested. Moreover,
a UV cross-linking assay indicated TIP49a specifically interacted with
ATP. Interestingly, we found that DNA duplex was unwound by the
recombinant TIP49a in the presence of ATP or dATP. Optimal
concentrations of ATP and Mg2+ for the helicase activity
were 1-2 mM and 0.25-1 mM, respectively. Displacement of the DNA strand occurred in the 3' to 5' direction with
respect to the single-stranded DNA flanking the duplex. Western blot
analysis revealed that TIP49a was abundantly expressed in testes and
moderately in spleen, thymus, and lung. In mouse seminiferous tubules,
the protein was restrictively observed in germ lineages from late
pachytene spermatocytes to round spermatids. From these observations,
we propose that TIP49a is a novel DNA helicase and may play a role in
nuclear processes such as recombination and transcription.
The unwinding of parent DNA strands is a prerequisite to basic
genetic processes including DNA replication, DNA repair, recombination, and transcription (1, 2). In each of these processes, unwinding of
duplex DNA is catalyzed by a DNA helicase, which functions to
destabilize hydrogen bonds between complementary base pairs in duplex
DNA. The energy necessary for this reaction is provided by hydrolysis
of nucleosides and deoxynucleoside 5'-triphosphates, meaning that DNA
helicase generally possesses an intrinsic ATPase activity (2).
DNA helicases are found ubiquitously from prokaryotes to eukaryotes.
Biochemical studies and computer analysis have revealed that helicases
have several characteristic motifs and that some of them are
functionally important. Two sequence signatures, so-called Walker A and
B motifs, have been identified in all helicases examined and in a wide
variety of other NTP-utilizing enzymes (3, 4). On the basis of the
primary sequence analysis of the Saccharomyces cerevisiae
genome, there are at least 41 helicase genes in the genome (5).
However, most of our biochemical knowledge about DNA helicases comes
from analyses of bacterial and phage enzymes (1, 2), because little is
known about the biochemical properties of most of the eukaryotic DNA
helicases. However, recent studies demonstrated that six established or
putative helicases are mutated in the human diseases such as xeroderma
pigmentosum, Bloom's syndrome, Cockayne's syndrome,
trichothiodystrophy, Werner's syndrome, and We originally identified a 49-kDa TATA-binding protein
(TBP)1-interacting protein
(TIP49a; termed TIP49 in previous reports) from rat liver nuclear
extracts (6, 12). TBP is one of the general transcription factors
needed for all classes of RNA polymerases (7). Although we found a
protein complex including both TIP49a and TBP in nuclear extracts (6),
it is still unclear how TIP49a is involved in transcription and why it
forms a complex with TBP. Interestingly, a computer search revealed a
high similarity between TIP49a and a prokaryotic DNA helicase, RuvB,
which is involved in branch migration of the Holliday junction (8, 9).
It is noteworthy that TIP49a contains both the Walker A and B motifs in
the RuvB homologous regions (6), suggesting that TIP49a is a putative
DNA helicase. Recently, we found highly conserved tip49a-related genes in a variety of organisms from humans
to an archaean by a data base search, implying that TIP49a plays a
fundamental and essential role throughout the archaea and eukaryotes (10). Moreover, it is generally surprising that a mammalian protein
would exhibit such extensive homology to a bacterial one. The
eukaryotic counterpart of the bacterial ruvB has not been identified so far. Demonstration and characterization of a new helicase
would be expected to help in the elucidation of eukaryotic nuclear processes.
Most recently, Qiu et al. (11) reported the identification
and characterization of a eukaryotic RuvB-like protein (RUVBL1), which
is highly homologous to bacterial RuvB. The amino acid sequence of
RUVBL1 is identical to that of human TIP49a (12) and to that of rat
TIP49a (6) with one exception; i.e. Val291 in
rat TIP49a is replaced by Ile in RUVBL1. However, they reported that
ATPase and helicase activities could not be detected in their RUVBL1
preparation. In this study, we purified the recombinant rat TIP49a
protein from Escherichia coli to near homogeneity and showed
that this protein is a DNA-stimulated ATPase and
ATP-dependent DNA helicase. We further found that TIP49a
was enriched in testes as compared with all somatic tissues examined.
Within the testes, TIP49a was localized in a subset of the germ cells
from late pachytene spermatocytes to round spermatids. Herein, the
biological significance of TIP49a will be discussed.
Plasmids for Expression of Rat TIP49a Protein--
An
NdeI site was created at translation initiation site of the
TIP49a cDNA isolated from rat liver cDNA library in Expression and Purification of Recombinant TIP49a--
The rat
TIP49a was overexpressed in E. coli BL21(DE3)pLysS strain
(13) by isopropyl-1-thio- Assay for ATP Hydrolysis--
ATPase activity was assayed by use
of activated charcoal (Sigma) as described by Armon et al.
(14). The reaction (20 µl) contained 0.3 µg of the purified TIP49a,
unless specified otherwise. The TIP49a was incubated at 37 °C for 30 min in A buffer (20 mM Tris/HCl (pH 7.5), 70 mM
KCl, 2.5 mM MgCl2, 1.5 mM
dithiothreitol, 0.1 mM ATP, and 1.25 mCi of
[ DNA Helicase Assay--
A complementary oligonucleotide
corresponding to nucleotide positions 6291-6320 in M13mp18 ssDNA was
synthesized and labeled at the 5'-end by T4 polynucleotide kinase and
[
For the DNA helicase assay, the reaction mixture (20 µl) contained 20 mM Tris/HCl (pH 7.5), 2 mM dithiothreitol, 50 mg/ml BSA, 0.5 mM MgCl2, 80 mM KCl,
1 mM ATP, and 10 ng of 32P-labeled helicase
substrate. The reactions contained 0.2 µg of the purified TIP49a
unless specified otherwise. The helicase assay was performed at
37 °C for 30 min and stopped by the addition of 5 ml of 60 mM EDTA, 0.75% SDS, and 0.1% bromphenol blue. The reaction mixture was then subjected to 10% PAGE, and the displaced oligonucleotides were visualized by autoradiography.
UV Cross-linking Assay--
UV cross-linking assays were
performed as described by Pause et al. (16). Reaction
mixture (20 µl) in a buffer (20 mM Tris/HCl (pH 7.5), 70 mM KCl, 5 mM magnesium acetate, 1.5 mM dithiothreitol, 10% glycerol, and 5 µCi of
[ Western Blotting and Immunocytochemistry--
The purified
TIP49a was used to generate polyclonal antibody in rabbits as described
by Kanemaki et al. (6). The TIP49a antibody was
affinity-purified by use of TIP49a-immobilized HiTrap NHS-activated
beads (Amersham Pharmacia Biotech). Rat tissues were homogenized in
lysis buffer (20 mM Tris/HCl (pH 7.4), 0.1% SDS, 1%
Triton X-100, 1% sodium deoxycholate, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 0.5 µg/ml
leupeptin, and 1 mM benzamidine) and then centrifuged. The
protein content of the supernatant was determined using the BCA protein
assay kit. Sample lysates (10 µg) were mixed with SDS sample buffer
and then boiled for 5 min. The protein sample was subjected to 10%
SDS-PAGE and electophoretically transferred to polyvinylidene
difluoride membranes (Millipore Corp.). The proteins were visualized by
the ECL protocol (Amersham Pharmacia Biotech).
For immunohistochemical analysis of TIP49a in mouse testes, sections
were processed as described previously (18, 19). Localization of TIP49a
was examined with anti-TIP49a antibody. Deparaffinized and rehydrated
sections were immersed in 0.3% H2O2 in
methanol and then preincubated with 0.5 mg/ml normal goat IgG in 1%
bovine serum albumin/phosphate-buffered saline for 1 h. The
sections were next reacted with rabbit anti-TIP49a antisera (1:800)
diluted with 1% bovine serum albumin in phosphate-buffered saline for
3 h. After having been washed with 0.075% Brij
35/phosphate-buffered saline, each section was incubated with
horseradish peroxidase-conjugated goat anti-rabbit IgG (1:100) for
1 h and washed with 0.075% Brij 35/phosphate-buffered saline.
Negative control sections were incubated with normal rabbit serum at a
1:800 dilution.
TIP49a Is Structurally Similar to the Bacterial RuvB--
We found
earlier that rat TIP49a showed a significant homology with RuvB, which
is a bacterial recombination factor and possesses ATPase/DNA helicase
activities (8, 9). There were mainly two conserved regions between
TIP49a and the bacterial RuvBs, and in these regions the sequence
identity and similarity were 38 and 25% and 55 and 46%, respectively
(12). The RuvB-homologous regions in the TIP49a protein described above
included Walker A and B motifs, which are responsible for ATP binding
and ATP hydrolysis, and are characteristic of RNA and DNA helicases
(Fig. 1). Therefore, we assumed that
TIP49a is a putative ATPase and also possibly a DNA or RNA helicase. A
data base search indicated the presence of TIP49a-related proteins in
various organisms such as humans and S. cerevisiae, and also
in archaea (Archaeoglobus fulgidus) (10). Amino acid
identities of the human, yeast, and archaean proteins to the rat TIP49a
protein were 99.8% (with only one amino acid substitution), 70%, and
46-48%, respectively (Fig. 1). It is remarkable that the
tip49a-related genes are so highly conserved among bacteria,
eukaryotes, and archaea, suggesting that TIP49a must play a fundamental
role in biological processes.
Purification of Recombinant TIP49a--
For biochemical
investigation of TIP49a, we overexpressed rat TIP49a protein in
E. coli. Codons for two peptide motifs, the FLAG
octapeptide, and a stretch of six histidines, were added to the 5'-end
of the TIP49a sequence to facilitate the subsequent purification of the
recombinant protein. The TIP49a carrying two tags was expressed in
E. coli (data not shown) as described under "Experimental
Procedures." The TIP49a protein was thoroughly purified by
nickel-chelate chromatography, anti-FLAG antibody-immobilized affinity
chromatography, and MonoQ column chromatography, as outlined in Fig.
2A. The final MonoQ fraction
of TIP49a protein was found to include no detectable contaminating
protein by SDS-PAGE and Coomassie Brilliant Blue staining (Fig.
2B, top).
TIP49a Is a DNA-stimulated ATPase--
The presence of the
characteristic Walker motifs in the primary TIP49a sequence predicted
that the protein would be an ATPase. Therefore, we examined whether
purified TIP49a protein had ATPase activity. The final MonoQ fractions
were incubated with [
To further confirm that the TIP49a is an ATP-utilizing enzyme, the
specific interaction between the purified TIP49a and ATP was analyzed
by a UV cross-linking assay. This reaction, in which ATP is photolyzed
by UV light in the presence of an ATP-binding protein, yields a
covalent adduct interacting with ATP and the protein. By use of
[
To examine the nucleotide specificity for the TIP49a-catalyzed
hydrolysis, we employed unlabeled nucleotides as competitors. The
addition of UTP to the reaction had a little effect on the ATPase
activity of TIP49a, whereas GTP and CTP moderately reduced the ATPase
activity (Fig. 3B). This result suggests that TIP49a is
unable to interact efficiently with nucleotides except ATP. Since it is
thought that some proteins containing the Walker motif exhibit ATPase
activity stimulated by nucleic acids, we next investigated the effects
of various nucleic acids on the TIP49a ATPase activity. Fig.
3C shows that neither homoribopolymers nor cellular RNAs affected the enzyme activity of TIP49a. The closed circular form of
plasmid DNA (pBluescript) exhibited only a weak stimulating effect,
whereas the ATPase activity of TIP49a was remarkably stimulated more
than 10-fold by the M13 ssDNA (Fig. 3C), demonstrating that TIP49a is an ssDNA-stimulated ATPase.
TIP49a Is a DNA Helicase--
Since rat TIP49a is structurally
homologous to the bacterial RuvB proteins, which possess an
ssDNA-stimulated ATPase activity and have DNA helicase activity, we
suspected that TIP49a would also be a DNA helicase. To investigate
whether the purified TIP49a protein possesses helicase activity, we
prepared a substrate for DNA helicase assay. A radiolabeled 30-mer
oligonucleotide was annealed with M13 ssDNA to construct a partial
duplex substrate. This substrate was incubated with the final MonoQ
fractions. The reaction products were separated on a polyacrylamide gel
and detected by autoradiography. In agreement with the results of the
ATPase assay (Fig. 2B), the helicase activity was also
correlated with the amount of TIP49a in each fraction (Fig.
4A), demonstrating that TIP49a
possesses a DNA helicase activity.
To determine the polarity of the DNA unwinding by TIP49a, we prepared
the following DNA substrates. For substrate preparation, a 5'- or
3'-end-labeled 54-mer oligonucleotide was annealed with M13 ssDNA. This
partial duplex DNA was digested with SmaI to yield a linear
ssDNA with either a labeled complementary 24-mer or a labeled 30-mer
bound to its ends. Following incubation of the substrates with the
TIP49a protein, no increase in unwound 24-mer oligonucleotide over
background was observed (Fig. 4B, lanes
4 and 6). The recombinant TIP49a was found to
efficiently displace the 5'-labeled 30-mer (Fig. 4B,
lane 3), showing that TIP49a has a 3' to 5'
unwinding polarity with respect to the ssDNA flanking the duplex.
Characterization of TIP49a DNA Helicase--
Next, we determined
the optimal conditions for the DNA helicase activity of TIP49a. The
optimal temperature for the helicase reaction was 37 °C, and the
optimal pH range was 7.5-8 (data not shown). For the maximal helicase
activity, TIP49a required 1-2 mM ATP and 0.25-1
mM MgCl2, and 4 mM ATP and 10 mM MgCl2 considerably inhibited the enzyme
activity (Fig. 5A). TIP49a
helicase activity was not detected in the absence of ATP or
MgCl2 (Table I),
demonstrating that TIP49a DNA helicase activity is dependent on the
presence of both Mg2+ and ATP. The helicase reaction
required the hydrolysis of a
A time course of the TIP49a-mediated strand displacement reaction under
the optimal assay conditions is shown in Fig. 5B. Percentage
of unwound fragment was determined. In the presence of 300 ng of TIP49a
protein, we found that >50% of the labeled fragments were displaced
in 5 min. The unwinding activity increased linearly up to 5 min and
reached a plateau in 20 min.
Expression of TIP49a Protein in Rat Tissues--
Finally we
examined the expression of TIP49a in various rat tissues by Western
blot analysis using affinity-purified anti-TIP49a antibody. TIP49a
protein was exclusively detected as a single band of 50 kDa (Fig.
6A). It was moderately
expressed in the thymus, spleen, and lung and abundantly in the testis.
This distribution pattern coincided well with that of Rad51, an
authentic eukaryotic recombination factor known to be involved in
mitotic and meiotic recombination (Fig. 6A) (20, 21). We
suspect that TIP49a participates in some way in recombination.
The testes contain multiple types of germ and nongerm cells. Moreover,
each type of germ cells reflects a particular stage of spermatogenesis.
We next performed immunohistochemistry to examine what kinds of
testicular cells predominantly express TIP49a proteins. Fig.
6B, a, shows that the epitope reacted with
anti-TIP49a antibody is specifically localized in a subset of the germ
cells at the stage of pachytene (stages IX-X) to round spermatids.
Immunostaining using normal serum resulted in no staining (Fig.
6B, c), demonstrating that the detected epitopes
represent TIP49a. In contrast, little signal was detected in pachytene
spermatocytes (stages IV-VI; Fig. 6B, b). These
results indicated that TIP49a is predominantly expressed in testicular
germ cells at stages from late pachytene spermatocytes to round
spermatids. No or little signal was observed in somatic Sertoli or
Leydig cells (Fig. 6B, a and b). These
observations suggest that TIP49a plays a critical role in meiosis.
TIP49a Is a Novel Eukaryotic DNA Helicase--
We originally
identified a 49-kDa TBP-interacting protein (TIP49a) from rat liver
nuclear extracts by use of an in vitro binding assay (6).
Immunofluorescence analysis using anti-TIP49a antibody revealed a
typical dot-shaped nuclear staining pattern, as previously reported
(12), indicating that TIP49a to be a nuclear protein. This finding is
consistent with a recent report that TIP49a (or NMP38) was identified
as a nuclear matrix protein (22). TIP49a contains Walker A and B
motifs, which are characteristic of DNA/RNA helicases. Thus, we
suspected it to be a putative DNA/RNA helicase. In this work, the
recombinant TIP49a was purified to near homogeneity from E. coli and was defined to be an ssDNA-stimulated ATPase and
ATP-dependent DNA helicase based on the following
biochemical evidence. (i) The ATPase activity was co-purified with the
recombinant TIP49a on the final MonoQ column (Fig. 2B), and
the same result was obtained in the case of the helicase activity (Fig.
4A). (ii) A UV cross-linking assay indicated that TIP49a
specifically interacted with ATP (Fig. 3A). Thus, it is
unlikely that a contaminating protein was involved in the
ATPase/helicase activity. (iii) The ATPase activity was remarkably
stimulated by ssDNA (Fig. 3C). (iv) The helicase activity
was dependent on the presence of ATP or dATP, but ATP
Most recently, RUVBL1, human counterpart of rat TIP49a, was isolated
from human cells. However, the purified RUVBL1 expressed in insect
cells did not possess ATPase and helicase activity, although RUVBL1 and
TIP49a are 99.8% identical (11). We also found that TIP49a
preparations purified from insect cells using the baculovirus
expression system did not hydrolyze ATP (data not shown). The failure
to detect the enzyme activity in insect cell-expressed TIP49a and
RUVBL1 might be due to the modification of the protein and/or the
presence of an inhibitory factor in insect cells.
TIP49a exhibits significant sequence similarity to bacterial RuvB,
which is a recombination factor having ATP-dependent DNA helicase activity. A functional and structural homologue of bacterial RuvB has not been found in eukaryotes yet (see below). We herein provided evidence that a mammalian RuvB-like TIP49a is an intrinsic DNA-stimulated ATPase and DNA helicase. However, we found some differences between TIP49a and RuvB in terms of biochemical properties. TIP49a required 1-2 mM ATP and 0.25-1 mM
MgCl2 for the maximal helicase activity (Fig.
5A), whereas concentrations of ATP of >0.5 mM
and MgCl2 at 10-20 mM are optimal for RuvB
helicase activity (23). The TIP49a helicase activity was considerably
inhibited at 10 mM MgCl2. Furthermore, despite
the sequence similarity between RuvB and TIP49a, TIP49a has a 3' to 5'
polarity, which is the opposite of that of RuvB. If TIP49a possesses
RuvB-like activity, this difference may be relevant to the finding that
the polarity of strand transfer promoted by Rad51 is also the opposite
of that observed in RecA (24).
What Is the in Vivo Role of TIP49a?--
Our findings that TIP49a
is a nuclear factor possessing a DNA helicase activity led us to an
assumption that TIP49a is involved in DNA-associated nuclear processes
such as recombination, transcription, and replication. Considering the
high sequence conservation in archaea and yeast as well as in higher
eukaryotes (Fig. 1), we suggest that TIP49a may play an important role
in nuclear processes. Immunological analysis indicated that Rad51,
which is responsible for an early stage in homologous recombination
(i.e. a sequential pathway up to the Holliday junction
formation), was highly expressed in the thymus, spleen, and testis, as
expected (Fig. 6A). Like Rad51, TIP49a was also
significantly enriched in tissues where recombination frequently takes
place. Moreover, it is interesting that TIP49a protein was highly
accumulated in the meiotic cells at stages from late pachytene to round
spermatids (Fig. 6B), since formation of the Holliday
junction is assumed to occur in germ cells staying at the pachytene
stage (28). Chiasmata represent the physical manifestation of crossing
over and reciprocal recombination and are cytologically detected when
homologues begin to separate at diplotene stage (29). It may be that
the TIP49a expression pattern during meiosis reflects the late stage of
recombination. Thus, we propose a hypothesis that TIP49a participates
in a recombination process like RuvB.
In bacteria, RecA generates the Holliday structure. E. coli
RuvB, which is an ATP-dependent DNA helicase, binds to the
Holliday junction, and promotes the branch migration together with RuvA during the late stage of recombination (8, 9). Rad51 in eukaryotes and
RadA in archaea were established to be RecA functional homologues (30,
31), suggesting that a recombination mechanism is conserved among the
organisms. However, in eukaryotes, RuvB-like activity has not been
identified. It was a general surprise that rat TIP49a is highly
homologous to bacterial RuvBs. Thus, with all evidence taken together,
it is conceivable that TIP49a helicase activity is involved in some
aspect of RuvB-associated recombination in eukaryotes.
Alternatively, TIP49a may function as a transcription factor, because
it was identified in a TBP-carrying complex (6). This hypothesis is
supported by a recent finding that RUVBL1, which is a human homologue
of TIP49a, is co-purified with the RNA polymerase II transcription
complex (11). We cannot exclude a possibility that TIP49a directly
participates in the transcriptional regulation. Bauer et al.
(37) reported that
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-thalassemia (5). Thus,
identification of a novel helicase is medically important.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gt11
(6), and a fragment from NdeI to BamHI (in
pBluescript vector downstream from the TIP49a cDNA) including an
entire TIP49a coding region was inserted into pET-3a vector (13).
TIP49a carrying FLAG tag and histidine tag was constructed by inserting
oligonucleotides for amino acid stretch, MDYKDDDDKHHHHHH, at the
N-terminal methionine of the rat TIP49a.
-D-galactopyranoside induction. The E. coli cells were harvested and suspended in a lysis
buffer containing 20 mM Tris/HCl (pH 7.9), 100 mM KCl, 0.1% Nonidet P-40, 1 mM
2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 10% glycerol. The cells were disrupted by sonication and centrifuged, and the supernatants were then applied onto a nickel-agarose column equilibrated with the lysis buffer. The column was washed with BC100
buffer (20 mM Tris/HCl (pH 7.9), 100 mM KCl, 1 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl
fluoride, 10% glycerol) supplemented with 20 mM
imidazole/HCl (pH 7.9). TIP49a protein was eluted with BC100 buffer
containing 300 mM imidazole/HCl (pH 7.9), and the peak
fractions were dialyzed against F buffer containing 20 mM Tris/HCl (pH 7.9), 5 mM MgCl2, 0.1% Nonidet
P-40, 500 mM KCl, and 10% glycerol. Dialyzed materials
were incubated with 5 ml of M2 anti-FLAG agarose (Eastman Kodak Co.)
for 4 h with agitation. After the column had been washed with F
buffer, the bound proteins were eluted from the M2-agarose by
incubation for 60 min with 5 ml of the FLAG peptide (Kodak) in F buffer
(0.5 mg/ml). The eluate was dialyzed against BC40 buffer. The resulting
sample was loaded onto a MonoQ (Amersham Pharmacia Biotech) column and eluted with a linear gradient of KCl from 40 to 400 mM
using BC buffer. The protein concentration was estimated by use of BCA protein assay reagent (Pierce) with bovine serum albumin as a standard.
-32P]ATP). One microgram of M13 single-stranded DNA
(ssDNA), double-stranded pBluescript DNA, RNA homopolymers (Amersham
Pharmacia Biotech), or cellular total RNA was added to as indicated.
Radioactivity was determined as Cerenkov radiation. Control reactions
without TIP49a were carried out in parallel tubes, and the control
value (radioactivity) was subtracted from each experimental one. Each assay was done in duplicate, and the results were presented as a simple
arithmetic average.
-32P]ATP. The labeled oligonucleotide was annealed
with the phage ssDNA by incubation at 95 °C for 10 min and 60 min at
37 °C. The product was purified by Suprec02 (Takara Shuzo) to remove
the unannealed oligonucleotide. In the case of the experiment to
determine the direction of DNA helicase, another substrate was
constructed as described previously (15). A complementary
oligonucleotide (54-mer) including the SmaI site,
corresponding to nucleotide positions 6226-6279 in M13mp18 ssDNA, was
synthesized and hybridized with the phage ssDNA. The oligonucleotide
was labeled with T4 DNA kinase for 5'-end labeling or with terminal
deoxynucleotidyl transferase and [
-32P]ddATP for
3'-end labeling. After SmaI digestion, this partial duplex
DNA was used as a substrate.
-32P]ATP) was irradiated by a UV cross-linker, LS1500
(Funakoshi), from a distance of 2 cm at 4 °C for 20 min. The samples
were subjected to 10% SDS-PAGE, and then the gels were stained with
Coomassie Brilliant Blue and autoradiographed.
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ABSTRACT
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RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic alignment of TIP49a homologues in
various organisms. The amino acid sequence of rat TIP49a was
aligned with those sequences of human (12), chicken (S. Takeda,
personal communication), S. cerevisiae (Y. Makino,
unpublished data), and archaeans (A. fulgidus,
GenBankTM accession number AE000977; Pyrococcus
horikoshi, DDBJ accession number AP000007) counterparts. The
percentage of identity (similarity) was calculated by the BLAST + BEAUTY program (36). Numbers indicate amino acid position.
Solid boxes and open boxes
show Walker A/B motifs and divergent regions, respectively.
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Fig. 2.
Purification of bacterially expressed
TIP49a. A, the purification scheme for rat TIP49a is
illustrated and is described under "Experimental Procedures."
B, co-elution of ATPase activity with the purified TIP49a.
The MonoQ column fractions 38-47 were separated by 10% SDS-PAGE, and
the proteins were visualized by Coomassie Brilliant Blue staining
(top). The arrow indicates the position of the
TIP49a. Corresponding fractions were assayed for ATPase activity in the
presence (0.5 µg, solid columns) and absence
(open columns) of M13 ssDNA, as described under
"Experimental Procedures" (bottom).
-32P]ATP in the presence or
absence of DNA, followed by determination of released phosphates as
described under "Experimental Procedures." Fig. 2B
(bottom) shows that the TIP49a was associated with an ATPase
activity that was strongly stimulated by the presence of ssDNA. We also
observed a weak ATPase activity in the absence of DNA (Fig.
2B). The level of ATPase activity correlated well with the
amount of TIP49a present in each fraction (Fig. 2B). A
parallel sample prepared from control E. coli had no ATPase activity (data not shown).
-32P]ATP, specific radioactive proteins were able to
be visualized by SDS-PAGE and autoradiography. As shown in Fig.
3A, illumination with UV light
resulted in the labeling of TIP49a protein (lane 2), and the labeling was strongly inhibited by cold ATP
(lane 3). Incubation of
[
-32P]ATP did not cross-link the ATP to the protein
without UV illumination (Fig. 3A, lane
1). The addition of cold UTP to the reaction had a little
effect on the UV labeling of TIP49a, whereas cold GTP and CTP
moderately reduced the labeling (Fig. 3A, lanes
4-6). These results were consistent with the effect of
nucleotides on the ATP hydrolysis by TIP49a (see below; Fig.
3B), suggesting that TIP49a does not interact
nonspecifically with radioactive ATP; i.e., the labeling is
attributed to the specific interaction between TIP49a and radioactive
ATP. From these experiments, we concluded that TIP49a possesses an
intrinsic ATPase activity.
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Fig. 3.
Characterization of TIP49a ATPase
activity. A, UV cross-linking assay for TIP49a. The
reactions containing 0.5 µg of TIP49a were subjected to UV
irradiation (+). Cold nucleoside triphosphates were added to the
mixture at a final concentration of 1 mM (lanes
3-6). The arrow indicates the position of
labeled TIP49a. Lane 1, without UV irradiation.
B, nucleoside triphosphate specificity for TIP49a ATPase
activity. The ATPase activity was assayed in the presence of M13 ssDNA.
Cold nucleoside triphosphates were added to the mixture at a final
concentration of 3 mM. Relative ATPase activities are
shown. C, effect of nucleic acids on TIP49a ATPase activity.
The ATPase activity was assayed in the presence or absence ( ) of
nucleic acids as indicated. One microgram of M13 ssDNA, pBluescript as
a double-stranded DNA, cellular total RNA prepared from rat liver (17),
or various RNA homopolymers (Amersham Pharmacia Biotech) was added to
the reaction mixture.
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Fig. 4.
TIP49a possesses DNA helicase activity.
A, co-elution of DNA helicase activity with TIP49a protein.
The MonoQ column fractions 38-47 shown in Fig. 2B
(top) were examined for DNA helicase activity, as described
under "Experimental Procedures." Corresponding fractions were
incubated with a radiolabeled 30-mer oligonucleotide annealed to M13
ssDNA used as a substrate. The reaction products were analyzed by 10%
polyacrylamide gel. For comparison, the substrate that was heated or
incubated with buffer alone is also indicated. The arrow
indicates the position of the displaced oligonucleotides. B,
direction of unwinding by TIP49a. The bottom shows the
structure of the linear substrate used. The asterisks denote
the 32P-labeled end. As a control, the substrate was
incubated with buffer alone (lanes 1 and
4) or heated at 95 °C for 3 min (lanes
2 and 5). The arrow indicates the
position of the displaced oligonucleotides.
-
bond of ATP, since the poorly
hydrolyzable analog ATP
S, ADP, and AMP did not exhibit the cofactor
function (Table I). Compared with ATP, other nucleoside
5'-triphosphates were inert as a co-factor, with the exception of dATP,
which supported more than 90% of the activity of ATP (Table I). We
next examined the requirement of various divalent cations for TIP49a
helicase activity. Although the presence of Mn2+ at a 1 mM concentration resulted in 75% of the activity of
Mg2+, all other tested cations such as Ca2+,
Cu2+, and Zn2+ could not replace
Mg2+ for the enzyme activity (Table I). MgCl2
was able to be replaced by either MgSO4 or
Mg(CH3COO)2.
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Fig. 5.
Characterization of TIP49a DNA helicase.
A, effect of ATP and MgCl2 concentration on the
DNA helicase activity. For determination of the optimal ATP
concentration (left), the reactions contained 0.5 mM MgCl2. Right, the reactions
contained 1 mM ATP. The arrow indicates the
position of the displaced oligonucleotides. B, time course
of strand displacement reaction mediated by TIP49a. The time of
reaction is indicated at the top of each lane.
The arrow indicates the position of the displaced
oligonucleotides.
Reaction requirements of TIP49a
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Fig. 6.
Expression of TIP49a protein in tissues.
A, tissue-specific distribution of TIP49a in various rat
tissues. Ten micrograms of total protein of lysates was used for
Western blot analysis using anti-TIP49a antibody (top) or
anti-Rad51 antibody (bottom). B,
immunohistochemical staining of mouse testis sections. Sections were
reacted with anti-TIP49a antibody (a and b) or
normal rabbit serum (c). Stages of seminiferous tubule
sections in a and b are IX-X and IV-VI,
respectively. P, pachytene spermatocyte; RS,
round spermatid; Sr, Sertoli cell; Ly, Leydig
cell.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S could not
substitute for ATP (Table I).
-catenin and TBP were binding partners of
the human TIP49a (Pontin52). As another possibility, TIP49a may
involved in a transcription-recombination coupling reaction. Indeed,
several subunits of the TFIIH complex, which is a general transcription
factor, are involved in nucleotide excision repair (32). Rad51 and
BRCA1 are found to be components of the RNA polymerase II-holoenzyme
complex (33, 34), and BRCA1 has a potential to enhance transcription
(35). These facts suggest that some DNA recombination-repair proteins
may be associated with the transcriptional apparatus. It is possible
that recombination-repair may be linked to transcription. If this is
the case, TIP49a may play a pleiotropic role in DNA metabolism. Further
investigation of TIP49a should provide new insight into the nuclear
events that transpire in eukaryotes.
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ACKNOWLEDGEMENTS |
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We thank Dr. T. Ohta for the generous gift of anti-Rad51 antibody. We also thank Drs. T. Ogawa and H. Shinagawa for valuable discussions.
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FOOTNOTES |
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* This work was supported by grants-in-aid for scientific research on priority areas from the Japanese Ministry of Education, Science, Sports, and Culture.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.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AB002406.
§ A research fellow of the Japan Society for the Promotion of Science.
** To whom correspondence should be addressed. Tel.: 81-43-290-2823; Fax: 81-43-290-2824; E-mail: btamura{at}nature.s.chiba-u.ac.jp.
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ABBREVIATIONS |
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The abbreviations used are:
TBP, TATA-binding
protein;
TIP, TBP-interacting protein;
RUVBL1, RuvB-like protein 1;
PAGE, polyacrylamide gel electrophoresis;
ssDNA, single-stranded DNA;
ATPS, adenosine 5'-O-(thiotriphosphate).
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REFERENCES |
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