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Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 335-341 |
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doi:10.1111/j.1745-7270.2006.00165.x |
Characterization of ATPase
Activity of Recombinant Human Pif1
Yu HUANG, Deng-Hong ZHANG, and
Jin-Qiu ZHOU*
Max-Planck
Junior Research Group at the State Key Laboratory of Molecular Biology,
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, Shanghai 200031, China
Received: December
2, 2005�������
Accepted: January
16, 2006
This work was
supported by a grant from the National Natural Science Foundation of China (No.
30125010)
*Corresponding
author: Tel, 86-21-54921078; Fax, 86-21-54921076; Email,
[email protected]
Abstract������� Saccharomyces cerevisiae Pif1p helicase is the
founding member of the Pif1 subfamily that is conserved from yeast to human.
The potential human homolog of the yeast PIF1 gene has been cloned from
the cDNA library of the Hek293 cell line. Here, we described a purification
procedure of glutathione S-transferase (GST)-fused N terminal truncated human
Pif1 protein (hPif1DN) from yeast and
characterized the enzymatic kinetics of its ATP hydrolysis activity. The ATPase
activity of human Pif1 is dependent on divalent cation, such as Mg2+, Ca2+ and
single-stranded DNA. Km for ATP for the ATPase activity is approximately
200 mM. As the ATPase activity is
essential for hPif1's helicase activity, these results will facilitate the
further investigation on hPif1.
Key words������� Pif1; helicase; telomere; telomerase; ATPase
Telomeres, the natural ends of
eukaryotic chromosomes, are the DNA protein-structures that are essential for
the protection of chromosomes from end-to-end fusion, recombination and
shortening [1-5]. Telomerase is a specific
reverse transcriptase (RNA-dependent DNA polymerase) that catalyses the
synthesis and extension of telomeric DNA by adding telomeric sequence repeats
onto the chromosome ends. In human, telomerase reverse transcriptase associates
with telomerase RNA to generate a functional core complex [6]. The RNA
component of telomerase provides the sequence template for telomeric repeat
synthesis [7]. Most human somatic cells have little or no telomerase activity
[8,9], and their telomeres are subjected to gradual shortening following each
cell division due to the "end replication problem" [10,11]. The
regulation of telomerase activity has been widely studied and, so far, many
proteins have been found involving in the regulation of telomerase activity.
The PIF1 gene was first found in Saccharomyces cerevisiae, involved in the repair and recombination of mitochondrial DNA [12,14]. Database analysis shows that Pif1 is conserved among many species [15], and its homologs have been found in Caenorhabditis elegans, Drosophila malanogaster, and Homo sapiens [16], constituting a big Pif1 subfamily of DNA helicases. Yeast S. cerevisiae Pif1 protein yPif1p is a 5'�3' DNA helicase [17,18] that has been identified as a negative regulator of telomerase [16]. Overexpression of enzymatically active yPif1p causes telomere shortening. yPif1p is associated with telomeric DNA in vivo, and its effect on telomeres depends on its helicase activity [16], yPif1p inhibits telomere elongation by removing the telomerase from telomere ends [19]. Another member of the Pif1 DNA helicase subfamily in S. cerevisiae, Rrm3p, also shows helicase activity [20]. Both Rrm3p and yPif1p are rDNA-associated, but they have opposite effects on replication fork progression in ribosomal DNA [21]. Pfh1 helicase is the Pif1 homolog in Saccharomyces pombe, which is related to the maintenance of telomeric DNA [22].
In human, many helicases have
been studied, of which the most thoroughly investigated is the helicase
subfamily RecQ. Some helicases of the RecQ subfamily are known to cause
diseases upon mutation. Werner's syndrome, Bloom's syndrome and Rothmund-Tomson
syndrome are caused by or related to deletion or mutation of helicases in the
RecQ subfamily [18,23,24].
The human homolog of the PIF1 gene (hPIF1) encodes a potential nuclear protein which contains the ATPase and helicase domains I, Ia, II, III, IV, V and VI, and has an identity greater than 30% to the S. cerevisiae homolog in these regions [16]. We have cloned the hPif1 cDNA, and the helicase activity appeared to be important for the function of hPif1, whereas the ATPase activity is essential for its helicase activity. In this paper, we describe a procedure of purification of GST-fused and N-terminal truncated hPif1 (hPif1DN) and characterization of its ATPase activity. The biochemical studies of hPif1 protein will provide insight to the functional study of hPif1 on telomere length regulation.
Material and Methods
Yeast strain
Yeast strain BCY123 (MATa, Can1, ade2, trp1, Ura3-52, his3, leu2-3, 112, pep4::his+, prb1::leu2+, bar1::HisG+, lys2::pGAL1/10-GAL4+) was used as the host strain to overexpress GST-hPif1DN and GST-hPif1DN(K234A) [25].
Cloning of hPIF1 gene
The full-length human Pif1
coding sequence was amplified by polymerase chain reaction (PCR) from the cDNA
library of the HEK293 cell line and introduced into the EcoRI site of
pUC19. hPIF1DN (498-1926 bp), which encoded a polypeptide
of 476 amino acids, was also cloned into the expression vectors. An invariant
lysine in motif I, the ATP-binding domain, was changed to alanine (K234A) using
site-directed mutagenesis (Clontech, San Jose, USA).
Expression and purification of
recombinant GST-hPif1DN protein
The hPif1DN protein was expressed in bacteria, a
baculovirus system and yeast. To express hPif1DN
in Escherichia coli, hPIF1DN was
cloned in to the EcoRI and SmaI sites of pGEX-4T-1 (Pharmacia,
Uppsala, Sweden) and transformed into BL21. Expression was induced by 300 mM
isopropyl-D-thiogalactoside and purified with glutathione Sepharose 4B
(Amersham Pharmacia Biotech, Uppsala, Sweden). The glutathione Sepharose 4B
column purified protein was further purified by loading it onto Q-Sepharose
(Amersham Pharmacia Biotech) equilibrated in Tris buffer, pH 7.8, and eluted
with a linear gradient (total 200 ml) of 50-600
mM NaCl [in 10 mM Tris (pH 7.8), 5 mM dithiothreitol (DTT), 4 mM
phenylmethylsulfonyl fluoride]. To express hPif1DN
in a baculovirus system, the Bac-to-Bac Baculovirus Expression System (Gibco,
Carlsbad, USA) was applied. His(6)-tagged protein was purified by
phosphocellulose (Amersham Pharmacia Biotech) and Ni-NTA (Gibco) columns. To
express hPif1DN and hPif1DN(K234A) in yeast, hPIF1DN and hPif1DN(K234A)
were subcloned into the BamHI and HindIII sites of pEG(KT) [26]
to generate pEG(KT)-hPif1DN and pEG(KT)-hPif1DN(K234A), and transformed into BCY123, a
protease-deficient S. cerevisiae strain [25]. In pEG(KT), proteins are
expressed as carboxy terminal fusions to GST and are expressed under the
control of the galactose-inducible GAL1 promoter. Expression was carried
out using the methods described by Bennett et al. [25], with minor
modifications. Cells were harvested, washed, and resuspended in seven volumes
of ice-cold lysis buffer (50 mM Tris, pH 7.8; 150 mM NaCl, 2 mM MgCl2, 0.01%
Triton X-100, 0.004% octanol, 5 mM DTT) and a mixture of protease inhibitors.
The resuspended cells were disrupted by passing them twice through a cell
disruptor (EmulsiFlex-C5; Avestin, Ottawa, Canada). After it
was centrifuged
at 19,000 rpm for 30 min, the supernatant was brought to 50% saturation with
ammonium sulfate and left on ice for 30 min. The precipitate was collected by
centrifugation at 20,000 rpm for 30 min, and suspended in 15 ml of 1�phosphate-buffered saline
(PBS) (3% glycerol, 0.1% Triton X-100, 10 mM DTT, 0.2 mM phenylmethylsulfonyl
fluoride). The soluble fraction was recovered by centrifugation, and was loaded
onto a glutathione Sepharose 4B column equilibrated with PBS. The column was
washed serially, twice with 1�PBS (1% Triton X-100, 3% glycerol), once
with 1�PBS
(1 M NaCl, 1% Triton X-100, 3% glycerol), then once 1�PBS (1% Triton X-100, 3% glycerol).
Protein was eluted with 10 ml elution buffer (10 mM Tris, pH 8.8; 1 mM EDTA, 5%
glycerol, 0.01% Triton X-100, 50 mM NaCl, 0.0005% octanol, 5 mM DTT, 20 mM
glutathione). The eluate was dialyzed with 1�PBS (1% Triton X-100, 30%
glycerol).
Production of anti-hPif1-C
antibody
3'-terminal fragment of
the hPIF1 gene (encoding amino acid 439-550)
was inserted into pGEX-4T-1 for expression of the GST-fused protein in E.
coli. Recombinant hPif1 fragments were purified with glutathione Sepharose
4B. Two rabbits were injected for immunization then, 1 month later, they were
injected four times at 1-week time intervals. For the first injection, ~200 mg of the fusion protein was emulsified
with 1 ml of Freund's complete adjuvant (1:1). For subsequent injections, ~200 mg
of fusion protein was mixed with an equal volume of incomplete adjuvant. The
anti-hPif1 antibodies were purified with antigen affinity column as reported
previously [27].
Characterization of hPif1DN DNA-dependent ATPase activity
ATPase reactions were carried
out in 20 ml of ATPase buffer [25 mM
HEPES, pH 7.6; 5 mM MgCl2, 2 mM ATP, 1 mM DTT, 100 mg/ml bovine serum albumin (BSA), 200 mg/ml salmon sperm DNA, 100 ng of
recombinant hPif1DN protein or 100 ng of yPif1p]
at 37 �C for 30 min. Each reaction mixture contained 0.5 mCi of [g-32P]ATP.
Reaction was stopped by the addition of 1 ml
of 0.5 M EDTA, and 0.5 ml of each reaction was spotted
on a polyethylimine cellulose plate. The plate was developed in 0.8 M LiCl and
dried in hot air. The ATP hydrolysis was visualized on a Molecular Dynamics
PhosphorImager (Amersham Pharmacia Biotech).
Results
Overexpression of hPif1DN and hPif1DN(K234A)
In order to characterize hPif1
activity in vitro, we tried to overexpress and purify the full-length
recombinant hPif1 protein. However, the full-length hPif1 was difficult to
overexpress or purify even in heterologous expression systems, including those
utilizing bacteria, yeast and insect cells. According to the sequence alignment
of Pif1 subfamily, the N-terminals of these proteins are not conserved,
suggesting that they may not be essential for the conserved enzymatic activity.
Therefore, we cloned a truncated version, which encoded a polypeptide of 476
amino acids including all the seven helicase motifs, and was thus named hPif1DN. The point-mutated form hPif1DN(K234A) (in the first helicase motif)
expected to have no ATPase activity of the enzyme, was also cloned. We
overexpressed the recombinant hPif1DN in E.
coli, insect cells and yeast, but failed to purify it in either E. coli
or insect cells. Therefore, we put our effort on the yeast expression system.
In yeast, the overexpression of hPif1DN or
hPif1DN(K234A) was under the control
of a galactose inducible promoter. The hPif1DN
or hPif1DN(K234A) was not detected when
the cells were cultured in glucose medium (Fig. 1, uninduced). The hPif1DN or hPif1DN(K234A)
with an appropriate molecular weight was overexpressed with galactose induction
[Fig. 1(A,C), induced total], and cross-reacted with anti-hPif1
antibodies [Fig. 1(B,D), induced total].
Purification of hPif1DN and hPif1DN(K234A)
To purify GST-hPif1DN for subsequent study, typically 4
liters of cells were used. The soluble cell lysate was precipitated with
ammonium sulfate to 50% saturation [Fig. 1(A,B), 50% (NH4)2SO4]. The
precipitate was resuspended and subjected to glutathione sepharose 4B affinity chromatography.
The recombinant GST-hPif1DN protein was eluted and
dialyzed (see "Materials and Methods"). The purified recombinant
GST-hPif1DN protein was examined with
Coomassie blue-stained SDS-PAGE and western
blot with antibodies against hPif1 [Fig. 1(A,B), GST column]. The
recombinant GST-hPif1DN (K234A) was also purified in
parallel [Fig. 1(C,D), GST column]. The GST-hPif1DN and GST-hPif1DN
(K234A) proteins were purified at about 90% and 85% purity respectively, and
quantified to have concentration of 600 ng/ml
and 160 ng/ml, respectively.
hPif1DN
possesses a DNA-dependent ATPase activity
DNA helicases, with associated
DNA-dependent ATPase activity, are presumed to use the hydrolysis of ATP to
translocate on ssDNA and to subsequently break the hydrogen bonds of duplex
DNA. To characterize its ATPase activity, the traditional ATPase activity was
performed. The purified hPif1DN [Fig. 2(A), hPiflDN], but not hPif1DN(K234A)
[Fig. 2(A), hPif1DN(K234A)], had ATPase
activity. This result indicated, as expected, that the ATPase activity of hPif1
depends on motif I, usually referred to as the Walker A
motif, an ATP binding motif. Furthermore, it suggested that the N-terminal of
the protein is not essential for ATPase activity.
ATPase activity of hPif1 requires Mg2+ and ssDNA
like other ATPase, the ATPase
activity of hPif1DN was dependent on both Mg2+ and
ssDNA, because in the absence of either Mg2+ or ssDNA, the hydrolysis of
ATP was not efficient [Fig. 2(B), ssDNA, Mg2+]. To further analyze the
effect of single-stranded DNA on its ATPase activity, we measured the APTase activity
with different concentrations of ssDNA [Fig. 3(A,B)]. When the ssDNA
concentration was below 80 ng/ml, the ATPase activity showed
a proportional enhancement with the increase of the ssDNA, whereas
concentrations of ssDNA higher than 80 ng/ml
did not inhibit ATPase activity, but brought it to saturation. The stimulation
effects of ssDNAs with different lengths and various double-stranded DNAs
(dsDNAs) were tested [Fig. 3(C,D)]. Compared with dsDNAs, all the ssDNAs
tested showed much higher stimulation effects on ATPase activity. Of these, the
25-mer oligonucleotide showed the highest stimulation effect.
effect of
divalent cation on the ATPase activity of hPif1
ATPase activity always
strictly requires divalent cation. To test whether hPif1 prefer Mg2+ to
other divalent cations, we performed the ATPase activity in the presence of Mg2+, Ca2+, Mn2+ and Co2+. both Mg2+ and Ca2+ could
support efficient hydrolysis of ATP, whereas Mn2+ and Co2+ were
much less effective (Fig. 4). Under the optimal conditions, the Km(ATP)
for ATPase activity was determined to be approximately 200 mM (Fig. 5).
Discussion
Sequence analysis data have
revealed that the primary structure of hPif1 is related to that of several
helicases and that it possesses seven consensus sequence motifs of a large
family of DNA helicases [15]. The sequence similarity brought about the
speculation that human Pif1 protein might share the same enzymatic property
with the so far studied Pif1 subfamily members yPif1 [16,18], Rrm3 [20] in S.
cerevisiae and Pfh1 in S. pombe [22]. The C terminal 476
amino acid of hPif1 (hPif1DN) contained all these seven
helicase motifs. In order to assay hPif1 for its ATPase and helicase activity,
it was first necessary to purify the enzyme. However, the concentration of the
native hPif1 protein is so low in cells that it has been difficult to detect
the protein by western blot
analysis (data not shown). Therefore, we overexpressed and purified the
GST-fused N-terminal truncated hPif1 (GST-hPif1DN)
(Fig. 1).
ATPase assay was performed to
view the ATP hydrolysis activity. hPif1DN
is able to hydrolyze ATP (Fig. 2). The stimulation of this activity
appeared to be most efficient in the presence of ssDNA [Fig. 3(C,D)] and
the stimulation effect is proportional to the concentration of ssDNA before
saturation [Fig. 3(A,B)]. dsDNA is also able to stimulate ATPase
activity [Fig. 3(C,D)], suggesting that the binding affinity of hPif1DN for duplex DNA is much lower than that
for ssDNA. Thus, like yeast Pif1p, hPif1 protein possesses ssDNA-dependent
ATPase activity. Like most of the ATPases, its activity is stimulated by
divalent cation, among which Mg2+ has the highest stimulation effect (Fig.
5).
Helicases play key roles in essentially every function that involves DNA and RNA, including DNA replication, repair and recombination, and RNA transcription, processing and translation. Many helicases appear to be multifunctional, for example, yPif1p is involved in telomere length regulation, rDNA replication and mitochondrial DNA recombination [16,21,28,29]. The members of Pif1 helicase subfamily studied so far all appear involved in telomere replication.� In S. pombe, mutation of the Pfh1p results in telomere shortening [30]. In S. cerevisiae, the Rrm3 helicase promotes the passage of the replication fork through telomere and sub-telomere regions [20]. Pif1p inhibits telomere elongation by reducing the processivity of the telomerase and by removing the telomerase from telomere ends [31]. In all cases, the effects of these proteins on telomeres require ATPase activity as a point mutation, which abolishes Pif1's ATPase activity will eliminate its physiological function. It will be interesting to further investigate whether hPif1 plays any role in telomere replication.
Acknowledgement
We thank Lu-Xia Xu for constructing pEG(KT)-hPif1DN (K234A).
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