From the Biological Information Research Center,
National Institute of Advanced Industrial Science and Technology,
Higashi1-1, Tsukuba, Ibaraki 305-8566, and the ¶ National
Institute of Technology and Evaluation, Nishihara, Shibuyaku,
Tokyo 151-0066, Japan
Received for publication, July 31, 2002, and in revised form, December 6, 2002
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ABSTRACT |
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Dna2 protein plays an important role in Okazaki
fragment maturation on the lagging strand and also participates in DNA
repair in Eukarya. Herein, we report the first biochemical
characterization of a Dna2 homologue from Archaea, the hyperthermophile
Pyrococcus horikoshii (Dna2Pho). Dna2Pho has both a
RecB-like nuclease motif and seven conserved helicase motifs similar to
Dna2 from Saccharomyces cerevisiae. Dna2Pho has
single-stranded (ss) DNA-stimulated ATPase activity, DNA helicase
activity (5' to 3' direction) requiring ATP, and nuclease activity,
which prefers free 5'-ends of ssDNA as substrate. These activities
depend on MgCl2 concentrations. Dna2Pho requires a higher
concentration of MgCl2 for the nuclease than helicase
activity. Both the helicase and nuclease activities of Dna2Pho were
inhibited by substrates with RNA segments at the 5'-end of flap DNA,
whereas the nuclease activity of Dna2 from S. cerevisiae
was reported to be stimulated by RNA segments in the 5'-tail (Bae,
S.-H., and Seo, Y. S. (2000) J. Biol. Chem. 38022-38031).
DNA replication is a fundamental process that assures the
maintenance of integrity of the genome. The various steps in DNA synthesis are basically similar among all organisms in eukarya, bacteria, and archaea, although replication components differ. In
eukaryotic cells, the final processing of Okazaki fragments on the
lagging strand requires that the ribonucleotide portions as primers are
completely removed by the concerted action of three nucleases, Fen-1,
RNaseH, and Dna2. Next, the corresponding sections of the molecule are
filled by DNA polymerases. The resulting nicks on the lagging strand
are then sealed by DNA ligase (1-6). Dna2 from
Sacharomyces cerevisiae encodes a 172-kDa protein that has single-stranded (ss)1
DNA-dependent ATPase, DNA helicase, and ssDNA-specific
endonuclease activity (7, 8). The genetic and physical interactions of Dna2 with Rad27 (a yeast homologue of mammalian Fen-1) suggest that
Dna2 plays a role in Okazaki fragment metabolism (9). The endonuclease
activity associated with Dna2 preferentially cleaves ssDNA with free
ends, and the helicase activity unwinds duplex DNA in the 5' to 3'
direction. The cleavage reaction was stimulated by the presence of an
RNA segment at the 5'-end of flap DNA. The 5'-end region of the Okazaki
fragment is efficiently processed by Dna2 when it is displaced from the
template by the DNA polymerase Archaea, the third domain of life, resemble bacteria in morphology and
genomic organization (i.e. lack of a nucleus and a single
circular genome). However, archaea and eukarya likely have a common
ancestor that is separated from bacteria (12, 13). Archaeal genome
sequence analyses reveal that the cellular components for genetic
processes such as DNA replication, transcription, and translation share
many common features with eukarya, whereas those for metabolic
processes exhibit similarities to bacteria (14-18). Although the
molecular mechanism of DNA replication in archaea seems to be a
simplified version of the eukaryotic one, knowledge of archaeal DNA
replication is still rudimentary (19-21).
The gene encoding the Dna2 homologue protein (PH0109) in
Pyrococcus horikoshii (Dna2Pho) exists in an
operon containing three other ORFs that are important in DNA
replication. Although the gene encoding one ORF (PH0108) found upstream
of the Dna2Pho gene is unknown, two other downstream ORFs (PH0112 and
PH0113) are homologous to subunits of eukaryal replication factor C
that load PCNA onto the template DNA. Moreover, the Dna2Pho gene
is located only 9.5 kilobase pairs away from an operon adjacent to the
replication origin, which consists of the genes encoding the Rad51
homologue (PH0119), small and large subunits of DNA polymerase D
(PH0123 and PH0121), and origin recognition complex 1 (PH0124) (Fig. 1) (22-25). This clustering of genetically essential genes suggests that
Dna2Pho possibly plays an important role in DNA replication similar to
Dna2 in Eukarya. In the present study, we report the first biochemical
characterization of a Dna2 homologue from Archaea, the hyperthermophile
P. horikoshii (Dna2Pho).
Chemicals, Plasmids, Enzymes, and DNA
Manipulation--
Ultracompetent Escherichia coli XL2-Blue
MRF' cells and E. coli strain BL21-CodonPlus (DE3)-RIL
competent cells were obtained from Stratagene (La Jolla, CA). The
pET-21b vector was purchased from Novagen (Madison, WI). Vent DNA
polymerase and KOD polymerase were purchased from New England
Biolabs (Beverly, MA) and Toyobo (Osaka, Japan), respectively.
Restriction enzymes were bought from Takara Shuzo (Kyoto, Japan) and
Promega (Madison, WI) and used as recommended by the manufacturers. The
ligation kit was purchased from Takara Shuzo and used according to the
manufacturer's directions. T4 polynucleotide kinase was obtained from
Promega, while protease inhibitor mixture tablets (EDTA-free) were
purchased from Roche Molecular Biochemicals (Mannheim, Germany).
Chromosomal DNA of P. horikoshii OT3 was prepared from a
sarcosyl lysate of the cells as described previously (26) with slight
modification. The digestion of DNA with restriction enzymes and
analysis of DNA fragments by agarose gel electrophoresis were performed
under standard conditions (27). Transformation was carried out by the
calcium chloride procedure (27). Miniscale preparation of E. coli plasmid DNA was performed by the alkaline lysis method (27)
or with a QIAprep spin miniprep kit (Qiagen, Hilden, Germany). A
QIAquick gel extraction kit (Qiagen) was used to recover DNA fragments
from agarose gel.
Cloning of the Genes and Construction of Expression
Vectors--
The gene for Dna2Pho from P. horikoshii was
amplified by polymerase chain reaction (PCR) with the primers P1 and P2
(Table I, Fig. 1). The amplified fragment
was digested with NdeI and SalI and inserted into
an expression vector pET-21b digested with NdeI and
XhoI. The constructed plasmid was designated
pET-Dna2PhoS(21b). Since the Dna2 homologue (Dna2Pab) from
Pyrococcus abyssi was longer by 124 amino acids at the
NH2-terminal than Dna2Pho, and another initiation codon TTG
was found at the position of Dna2Pho corresponding to the initiation
codon (GTG) of Dna2Pab, the ORF coding Dna2Pho was elongated by 122 amino acids. The elongated NH2-terminal region was
amplified by PCR against the chromosomal DNA of P. horikoshii as template using the primers P3 and P4 (Table I, Fig.
1). The amplified fragment was digested with NdeI and inserted into pET-Dna2PhoS(21b) digested with NdeI and
SmaI. The newly constructed plasmid was designated
pET-Dna2PhoL(21b).
To construct the co-expression vector for PH0108 and Dna2Pho, the
PH0108 gene was amplified with primers P5 and P6 (Table I, Fig. 1). The
amplified fragment was digested with NdeI and BamHI and inserted into pET-21b digested with
NdeI and BamHI. The new plasmid was designated
pET-PH0108(21b). To add a KpnI site upstream of the ribosome
binding site (RBS) in pET-Dna2PhoL(21b), PCR was performed using
primers P4 and P7 (Table I, Fig. 1). After digestion with
KpnI, the fragment was subcloned into pUC19 digested with
KpnI and SmaI. The
PvuII-NheI fragment was inserted into
pET-Dna2PhoL(21b) digested with NdeI (The
NdeI-digested end was blunted by the Klenow fragment) and
NheI. The resulting plasmid was then digested with
KpnI and Bpu1102I and inserted into
pET-PH0108(21b) digested with KpnI and Bpu1102I.
The final co-expression vector was named pET-PH108/Dna2PhoL(21b). The
sequences of the genes were verified using an ABI PRISM kit and model
310 capillary DNA sequencer (Applied Biosystems, Foster City, CA).
Expression and Purification of Enzymes--
The newly
constructed vectors were transformed into host E. coli
BL21-CodonPlus (DE3)-RIL. The transformed cells were grown in 2× yeast
tryptone (2YT) medium (16 g of trypton, 10 g of yeast extract, and
5 g of NaCl in 1 liter of deionized water) containing ampicillin (50 µg/ml) and chloramphenicol (34 µg/ml) at 37 °C. The overnight culture was inoculated (1% inoculation) into fresh 2YT
medium containing ampicillin (100 µg/ml), and incubation was continued at 37 °C until the optical density at 660 nm reached 0.4. The inducer isopropyl- Protein Analysis--
Protein concentrations were determined by
Coomassie protein assay reagent (Pierce). The purified samples were
analyzed by 0.1% sodium dodecyl sulfate-10% polyacrylamide gel
electrophoresis (SDS-PAGE). The gel was stained by Coomassie Brilliant
Blue R-250 (Nacalai Tesque, Kyoto, Japan). NH2-terminal
peptide sequencing was examined as follows. After electrophoresis,
separated proteins were electrotransferred to a polyvinylidene
difluoride membrane (0.2 µm) (Bio-Rad) using a semidry apparatus
(Bio-Rad). The NH2-terminal amino acid sequence was
determined with an Applied Biosystems model 492 protein sequencer
(Foster City, CA).
Preparation of Radiolabeled Substrates for DNA Helicase and
Nuclease Assay--
The oligomers used in this study (Table
II) were labeled at the 5'-end with T4
polynucleotide kinase and [ ATPase Assay--
ATPase activity was measured in reaction
mixtures (20 µl) containing 50 mM HEPES (pH 7.5), 1 mM dithiothreitol, 0.01% bovine serum albumin, 0.2 nmol of
[ Helicase Assay--
The standard reaction mixture (20 µl)
contained 50 mM HEPES (pH 7.5), 1 mM
dithiothreitol, 0.01% bovine serum albumin, 2 mM ATP, 1 mM MgCl2, 32P-labeled helicase
substrate, and the purified Dna2Pho helicase. After incubation at
50 °C for 1 h, the reaction was terminated by adding 4 µl of
a solution containing 50 mM Na2EDTA, 0.5% SDS, 25% glycerol, and 0.025% bromphenol blue. The sample (10 µl) was loaded onto a 15% polyacrylamide gel in TBE buffer (89 mM
Tris borate (pH 8.2) and 2 mM EDTA) and electrophoresed at
10 mA. Gels were dried, and the helicase or nuclease products were
analyzed and quantified with the GS-525 molecular imager system. The
helicase activity was calculated with the formula X = P/(P + S), in which P is
the value of the displaced oligonucleotides, and S is the value of
nondisplaced substrates. The helicase activity was normalized with the
positive and negative controls by the following formula: DNA helicase
activity (%) = 100 × [(Xsample Nuclease Assay--
Purified Dna2Pho (130 ng) was incubated with
32P-labeled substrate at 50 °C in a 20-µl reaction
mixture the same as in the helicase reaction except that the
concentration of MgCl2 was 10 mM. For denaturing gel analysis, after mixing with 10 µl of sequencing gel
loading buffer (95% formamide, 10 mM EDTA (pH 8.0), and
0.1% (w/v) bromphenol blue), samples were boiled for 5 min and loaded onto a 15% denaturing polyacrylamide gel (7 M urea). The
gel was dried, and the radioactivity was visualized using the GS-525
molecular imager system (31).
Expression and Purification of Dna2Pho--
According to the
genomic sequence of P. horikoshii, the PH0109 gene encodes a
protein of 1188 amino acids with a predicted molecular mass of 137 kDa.
The PH0109 gene was first cloned into pET-21b to yield a construct
encoding a fusion protein tagged at the COOH terminus with VEHHHHHH
(Fig. 1). The newly constructed plasmid
was designated pET-Dna2PhoS(21b). Dna2Pho was expressed in the E. coli strain BL21-CodonPlus(DE3)-RIL. However, the majority of the
protein was in insoluble fractions. The Dna2 homologue (Dna2Pab) from
P. abyssi is longer by 124 amino acids at the
NH2-terminal region than Dna2Pho. Another initiation codon
(TTG), corresponding to the initiation codon (GTG) of Dna2Pab, is
located 366 nucleotides upstream from the initiation codon (GTG) of
Dna2Pho. Therefore, the ORF coding Dna2Pho was elongated by 122 amino
acids (Fig. 2). To obtain a soluble
recombinant protein, the elongated Dna2Pho was inserted into pET-21b to
yield a construct encoding a fusion protein tagged at the COOH terminus
with VEHHHHHH (predicted mass, 153 kDa, designated pET-Dna2PhoL(21b))
(Fig. 1). Although Dna2Pho protein was successfully expressed in the
soluble fraction using pET-Dna2PhoL(21b), only a small amount was
purified (data not shown). The gene encoding one ORF (PH0108) is found
upstream of the Dna2Pho gene. Since the subunits of DNA polymerase
(DP1Pho and DP2Pho) from P. horikoshii were successfully
overexpressed in soluble fractions using a co-expression vector as
previously described (22), to obtain large amounts of purified Dna2Pho, a co-expression vector designated pET-PH0108/Dna2Pho(21b) was constructed, although the function of PH0108 protein was unknown. In
this vector, the PH0108 and Dna2Pho genes were connected in tandem into
two ORFs. The two genes are transcribed under the control of a single
transcription promoter and terminator (T7 promoter and T7 terminator).
PH0108 and Dna2Pho are expressed as native and His-tagged forms of
proteins, respectively (Fig. 1). Both of them were successfully
expressed in soluble fractions. Dna2Pho protein was purified in larger
amounts using the co-expression system than with pET-Dna2PhoL(21b) as
described under "Experimental Procedures." Fig.
3 shows the SDS-PAGE of the purified
sample. A major protein band of His-tagged Dna2Pho around 150 kDa and a
minor protein band around 22 kDa were observed, whereas a band for
PH0108 was not detected. The amino-terminal sequence of the 22 kDa
protein was determined as MKVAKDLVVSL, which completely agreed with the
amino-terminal sequence of FKBP-type peptidyl-prolyl cis-trans-isomerase protein (FKBP-PPIase) (21 kDa) from
E. coli (32), suggesting an intimate interaction between
Dna2Pho and a chaperon-like protein, FKBP-PPIase. Since we could not
separate Dna2Pho from FKBP-PPIase with any further purification steps, this protein sample was used for the experiments in this report. We
also constructed a co-expression vector that produced PH0108 His-tagged
at the COOH terminus and the native form of Dna2Pho. SDS-PAGE analysis
showed that there was no band of Dna2Pho protein after nickel column
chromatography (data not shown). These results may indicate that
Dna2Pho does not interact with PH0108 or that the interaction between
them is weak. Therefore, pET-PH0108/Dna2Pho(21b) was used to obtain a
large amount of the purified Dna2Pho protein. It is not known what
effect addition of the His tag may have had on the activities of
Dna2Pho, although it is clear that the tagged protein has retained the
DNA helicase and the nuclease activities.
Primary Structure of Dna2Pho--
The alignment of sequences from
Dna2Pho elongated by 122 amino acids, Dna2Pab, Dna2Sce, Dna2Spo, and
Dna2Xla revealed many conserved residues over the entire length of the
protein. Helicase and RecB-like nuclease motifs are found in Dna2
homologues as shown in Fig. 2 (33, 34, 35). The Dna2 P504S protein of S. cerevisiae gives a temperature-sensitive
in vivo phenotype. This mutation affects ATPase, helicase,
and nuclease activities. The amino acid residue corresponding to
Pro504 of the Dna2Sce is not observed in Dna2Pho based on
the alignment of amino acid sequences (data not shown), whereas
Asp142, Glu155, and Tyr173 are
found in the RecB-like motif of Dna2Pho equivalent to the catalytic
residues of Dna2, Asp657, Glu675, and
Tyr693, respectively (36, 37).
The cysteine cluster conserved among eukaryotic Dna2 proteins is also
observed in archaeal Dna2 homologues. However, the function of this
cluster is still unknown (35). Dna2Pho possesses seven typical helicase
motifs (I, Ia, II, III, IV, V, and VI) (33, 34). The sequence (GTGKT)
in the walker A box, the NTP binding motif within motif I as shown in
Fig. 2, is completely conserved. Although many residues are conserved
within RecB-like nuclease and helicase motifs, no conserved residues
were identified in other regions. Dna2Pho possesses a RecB-like
nuclease motif and helicase motifs. However, the location of these
motifs in Dna2Pho is different from that in Dna2Sce (Fig. 2,
upper panel).
ATPase Activity Stimulated by a Single-stranded DNA--
Since the
inset in Fig. 4 shows that
Dna2Pho hydrolyzed ATP in a dose-dependent manner, Dna2Pho
(30 ng) was added to the reaction mixture for the ATPase assay.
Purified Dna2Pho (30 ng) was assayed for ATPase activity in the
presence of increasing concentrations of closed circular single- and
double-stranded (ds) M13 DNA. Although the optimal growth temperature
of P. horikoshii is 95 °C, ATPase assays were performed
at 50 °C because portions of double-stranded DNA are likely to
denature at high temperature. The hydrolysis of ATP by the Dna2 from
S. cerevisiae is markedly stimulated by ssDNA (8). ATPase
activity of Dna2Pho was also stimulated as the amount of ssDNA
increased. By the addition of 40 fmol of M13 ssDNA, the ATPase activity
was stimulated ~10-fold; however, ATPase activity was not stimulated
by dsDNA. These results indicate that Dna2Pho has ATPase activity
stimulated by single-stranded DNA (Fig. 4).
Nuclease and DNA Helicase Activities of Dna2Pho Need Both ATP and
MgCl2--
To confirm the existence of helicase and
nuclease activities, an M13 single-stranded DNA circle hybridized to a
30-nt oligonucleotide was used as a substrate. As shown in Fig.
5, no bands due to the displacement or
degradation of substrate were observed on varying the Mg2+
concentration in the absence of ATP. In the presence of ATP, DNA
helicase activity was detected at a lower concentration of Mg2+ (1 mM) than of ATP (2 mM).
However, bands of less than 30-mers and smear bands due to the
degradation of substrate appeared at higher Mg2+
concentrations (5 and 10 mM) than the ATP concentration (2 mM). Neither helicase nor nuclease activity was detected
when EDTA was added to the reaction mixture. These activities were not
detected when the reactions were performed with the protein sample
purified with the cell extracts of transformants harboring pET-21b
vector alone by the same purification procedure. The results indicate that Dna2Pho possesses nuclease and ATP-dependent DNA
helicase activities and Mg2+ ion is required for both.
An ATP-dependent DNA Helicase That Unwinds DNA in the
5' to 3' Direction--
Two classes of DNA helicase have been
reported. DnaB translocates in the 5' to 3' direction (32); however,
minichromosome maintenance protein translocates in the opposite
direction (from 3' to 5') (29, 38-40). In this experiment, the DNA
unwinding polarity of Dna2Pho was determined by blocking the nuclease
activity using high ratios of ATP (2 mM) to
Mg2+ (1 mM). The partial duplex DNA substrates,
consisting of linear M13 ssDNA containing either 3'-labeled or
5'-labeled 32-nucleotide (nt) fragments at both ends, were prepared by
digesting with SmaI as shown in Fig.
6 and under "Experimental
Procedures." Since the substrates comprise duplex regions at both
ends of a long linear molecule, Dna2Pho must first bind to the internal
single-stranded regions of these substrates. If the enzyme subsequently
moves from the 3' to 5' along the single-stranded DNA segment, it would displace the 5'-labeled fragment from the substrate. In contrast, the
3'-labeled fragment would be displaced, if the enzyme migrates in a 5'
to 3' direction.
As shown in Fig. 6, the 3'-labeled fragment was displaced gradually
from the substrate as the addition of Dna2Pho to the reaction mixture
increased, whereas little displaced 5'-labeled fragment was observed.
The displaced fragments were quantified and the values were normalized
as described under "Experimental Procedures." The value of DNA
helicase activity was more than 70% (closed circle) using a
3'-labeled substrate, while less than 12% (open circle) using a 5'-labeled substrate when Dna2Pho (100 ng) was added to the
reaction mixture. When a 30-mer duplex fragment, which was blunt-ended
at both ends, was assayed, the value of DNA helicase activity increased
gradually as the amount of Dna2Pho in the reaction increased. The
helicase activity was ~18% when Dna2Pho (120 ng) was added to the
reaction mixture (data not shown). These results suggest that the DNA
helicase activity using a 5'-labeled substrate was due to movement of
the enzyme in the 5' to 3' direction from the blunted end; hence,
Dna2Pho translocates in the 5' to 3' direction, similarly to the yeast
Dna2 (10).
Dna2Pho Has Nuclease Activity That Prefers the Free 5'-End to
3'-End--
To identify the nuclease activity of Dna2Pho,
single-stranded, double-stranded, 3'-overhang, and 5'-overhang DNA were
used as substrates at high ratios of Mg2+ (10 mM) to ATP (2 mM). As shown in Fig.
7A, the single-stranded DNA
substrate was rapidly degraded to oligonucleotides varying in size with
the time course of reaction, whereas the double-stranded DNA (dsDNA)
substrate was degraded slowly. The trinucleotide observed using dsDNA
substrate might be due to unwinding from the 5'-labeled end and
subsequently cleavage of the translocating side of dsDNA. These results
demonstrate that the nuclease activity of Dna2Pho prefers ssDNA. When
the 3'-overhang substrate was used for nuclease assay, the nuclease
activity was decreased, compared with the activity against the
single-stranded DNA (Fig. 7, A and B). On the
other hand, the 5'-overhang DNA substrate was more efficiently degraded
than the 3'-overhang DNA substrate, indicating that for nuclease
activity, Dna2Pho is likely to prefer ssDNA with free 5'-end as
substrate (Fig. 7, A and B).
Both Nuclease and Helicase Activities Were Inhibited by
RNA/DNA Chimeric Substrate--
The endonuclease activity
of Dna2 from S. cerevisiae is stimulated by substrate with
an RNA segment at the 5'-end, and the helicase activity enhances the
endonuclease activity. Therefore it is considered that Dna2 removes
RNA-DNA primers of Okazaki fragments with both unwinding and cleavage
activity coupled to each other during Okazaki fragment processing (10,
11, 41).
To investigate the effect of RNA/DNA chimeric substrate on the
activities, a Y-structured substrate containing a 12-nt oligonucleotide (U) segment at its 5'-end was prepared as shown in Fig.
8. In the DNA helicase assay, displaced
fragments were observed as the Dna2Pho concentration increased.
However, no displaced fragments were produced from the substrate with
an RNA segment at the 5'-end, indicating that Dna2Pho could not unwind
the substrate (Fig. 8A).
The nuclease activity was assayed on single-stranded DNA or
single-stranded RNA/DNA chimera, respectively. As shown in Fig. 8B, the single-stranded DNA was degraded into pieces varying
from 50-mer to the size of a monomer, whereas RNA-DNA oligonucleotide was not digested by the nuclease activity. The Y-structured DNA substrate was also degraded by Dna2Pho just like single-stranded DNA.
However, the Y-structured RNA-DNA chimeric substrate was not degraded
by the nuclease activity. These results demonstrate that Dna2Pho can
not displace and cleave substrates with an RNA segment at the 5'-end
and that the 5'-end moiety of ssDNA is important for the recognition or
binding of Dna2Pho to the substrate.
In this report, we have identified a number of biochemical
properties of the Dna2Pho from P. horikoshii. The gene for
Dna2Pho is encoded in an operon, in which two subunits of replication factor C are also present. Moreover, the Dna2Pho gene is located only
9.5 kilobase pairs away from an operon adjacent to the replication origin, which consists of genes encoding a Rad51 homologue, small and
large subunits of DNA polymerase D, and origin recognition complex 1 (14, 15, 23, 24). This clustering of genetically essential genes
indicates that Dna2Pho possibly plays an important role in DNA replication.
Based on the genome information of P. horikoshii (14, 15),
we cloned Dna2Pho from genomic DNA by PCR. Dna2Pho was
expressed in E. coli as a protein of 1188 amino acids, but
was unstable and difficult to purify. Therefore we expressed a protein
with an additional 122 amino acids at the NH2 terminus. The
enlarged Dna2Pho, consisting of 1310 amino acids, was successfully
produced in E. coli. Moreover, the enlarged Dna2Pho was
coexpressed with PH0108 protein, and purified in larger amounts than
Dna2Pho expressed alone in E. coli. It appears likely that
PH0108 protein participates in assisting Dna2Pho to fold into a normal
structure or stabilize the mRNA transcribed from the Dna2Pho gene.
Although further analysis of the functions of PH0108 protein is needed,
we focused on the biochemical characteristics of Dna2Pho in this report.
The Dna2Pho protein of 1310 amino acids was compared with Dna2
homologues from several species. RecB-like nuclease and helicase motifs
are found in Dna2Pho (33, 34, 35). The location of the RecB-like motif
in Dna2Pho is different from that in Dna2 from S. cerevisiae
(Dna2Sce). The NH2-terminal domain of Dna2Sce functions as
a regulator of both helicase and nuclease activities essential for the
optimal function of Dna2Sce in Okazaki fragment processing (42).
Although a sequence homologous to the NH2-terminal region
of Dna2Sce is not observed in Dna2Pho, the amino acid sequences within
RecB-like nuclease and helicase motifs are remarkably conserved among
Dna2 homologues. Dna2Pho possesses ATPase, DNA helicase, and nuclease
activities. The ATPase activity is stimulated by single-stranded DNA.
The DNA helicase activity is dependent on ATP and Mg2+ ion.
The helicase activity is detectable by blocking the nuclease activity
at a higher concentration of ATP (2 mM) than
Mg2+ (1 mM). The DNA helicase activity unwinds
DNA in the 5' to 3' direction. The nuclease prefers the free 5'-end to
the 3'-end of single-stranded DNA. These properties of Dna2Pho are
similar to those of Dna2Sce; however, a critical difference is the
specificity for substrate with an RNA segment in the 5'-tail. The
biochemical characteristics of Dna2Pho differed from those of Dna2Sce
as follows. 1) The nuclease activity of Dna2Pho could not cleave the
RNA/DNA chimeric substrate, whereas that of Dna2Sce is stimulated by
substrates with an RNA segment in the 5'-end. 2) The helicase activity
of Dna2Pho could not displace the Y-structured substrate with an RNA
segment at the 5'-end.
The eukaryotic primase is known as a DNA polymerase
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
extending the upstream primer. These
enzymatic properties of Dna2 provide a biochemical basis for a role in
Okazaki fragment maturation (10, 11).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Primers used in cloning and construction of the expression vectors
-D-thiogalactopyranoside was added (final concentration, 1 mM), and cultivation was continued
for another 4 h at 30 °C. The cells were harvested by
centrifugation (7,000 × g at 4 °C for 10 min) and
resuspended in buffer A (20 mM Tris-HCl (pH 8.0) and 20 mM NaCl) containing the protease inhibitor, DNase RQ1
(final concentration, ~1 unit/ml), and MgCl2 (final concentration, ~6 mM). The cells were disrupted by
sonication, and the resultant solution was incubated at 37 °C for 10 min. Cleared supernatants were obtained by centrifugation (24,000 × g, at 4 °C for 20 min) and then applied to the matrix
of a nickel column (Novagen) equilibrated with buffer B (20 mM Tris-HCl (pH 8.0) and 500 mM NaCl) and
washed with buffer B containing 10 mM imidazole. The
enzymes were eluted with buffer B containing 500 mM
imidazole. The eluted fraction was heated at 75 °C for 10 min and
centrifuged (24,000 × g, at 4 °C for 20 min) to
remove denatured proteins. The supernatant was supplemented with 5 M NaCl to a final concentration of 2.3 M NaCl
and applied to a HiTrap phenyl-Sepharose HP column (Amersham
Biosciences, Buckinghamshire, UK) equilibrated with buffer C (20 mM Tris-HCl (pH 8.0) and 2 M NaCl). The column was washed with the same buffer, and protein was eluted with a 2-1
M NaCl gradient. The eluted fraction was dialyzed against buffer D (20 mM Tris-HCl (pH 8.0) and 100 mM
NaCl) and stored at 4 °C.
-32P]ATP. The labeled
oligonucleotide (5 pmol) was first heated with 2 µg of M13mp18 ssDNA
or 98-mer oligonucleotide (5 pmol) in annealing buffer (20 mM Tris-HCl (pH 7.5), 10 mM MgCl2,
and 50 mM NaCl) at 100 °C for 5 min, then kept at
67 °C for 1 h, and subsequently allowed to stand at 37 °C
for 30 min (28). The annealing mixture was slowly cooled to room
temperature. For the preparation of 3'-labeled substrate, the partial
duplex substrate was prepared as described above and then labeled using
the Klenow enzyme and [
-32P]dATP.
32P-Labeled oligonucleotide substrate was purified using a
PCR purification kit (Qiagen). M13 DNA substrate was purified using a
MicroSpin S-400 column (Amersham Biosciences) and PCR purification kit
(Qiagen). To determine the direction of translocation, the 5'-labeled
or 3'-labeled substrate was digested with SmaI at 30 °C
for 6 h and purified using the PCR purification kit (Qiagen).
Oligonucleotides used in this study
-32P]ATP (15 Ci/mmol), 1 mM
MgCl2, the indicated amount of polynucleotide, and the
purified Dna2Pho helicase (30 ng). After incubation at 50 °C for 30 min, the reaction was terminated by adding 2 µl of 100 mM
Na2EDTA. An aliquot (2 µl) was spotted onto a
polyethyleneimine-cellulose thin-layer plate. The reaction products
(ATP, inorganic phosphate (Pi)) were separated by
chromatography in a 1 M formic acid, 0.5 M LiCl
solution (29). The extent of ATP hydrolysis was quantified with the
GS-525 molecular imager system (Bio-Rad).
Xn)/(Xp
Xn)], in which the Xn
value was determined by negative control assays at 50 °C without
enzyme, and Xp was obtained as a positive
control with the reaction mixture boiled for 5 min without enzyme
(30).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (38K):
[in a new window]
Fig. 1.
Schematic diagram showing the procedure for
cloning and construction of the expression vectors for Dna2Pho
from the genomic DNA of P. horikoshii. The
primers are listed in Table I.
View larger version (65K):
[in a new window]
Fig. 2.
Conserved motifs among Dna2 homologues.
The upper panel shows a schematic diagram of the Dna2
helicase from S. cerevisiae and P. horikoshii.
The area boxed by diagonal striped squares shows
the elongated amino-terminal region. The region boxed by
black and vertical striped squares shows the
RecB-like nuclease motif and helicase motifs, respectively. The
number indicates the position of the amino acid from the
amino terminus of the protein. The middle panel shows the
nucleotide and the amino acid sequences of the elongated
NH2-terminal region of Dna2 helicase from P. horikoshii. The amino acid sequence of Dna2 helicase in the data
base of P. horikoshii starts from M. The lower
panel shows the RecB-like nuclease motif and seven conserved
helicase motifs (I, Ia, II, III, IV, V, and VI). The distances from the
aligned regions to the protein termini and the distances between the
conserved blocks, where more variable regions were omitted, are
indicated. Identical amino acids are shown with a black
background. Strictly conserved residues are boxed by
black squares with asterisks. The abbreviations
used are as follows: AddABsu, ATP-dependent
nuclease subunit A from Bacillus subtilis (Swiss-Prot
accession number P23478); Ex5BEco, exodeoxyribonuclease V
chain from E. coli (Swiss-Prot accession number
P08394); Dna2Pab, Dna2 homologue from P. abyssi
(Swiss-Prot accession number B75198); Dna2Pho, Dna2
homologue from P. horikoshii (C71231); Dna2Sce,
Dna2 from S. cerevisiae (Swiss-Prot accession number
P38859); Dna2Spo, Dna2 homologue from
Schizosaccharomyces pombe (Swiss-Prot accession
number Q9URU2); Dna2Xla, Dna2 homologue from
Xenopus laevis (Swiss-Prot accession
number 2607285A).
View larger version (61K):
[in a new window]
Fig. 3.
SDS-PAGE of the purified Dna2 homologue from
P. horikoshii (Dna2Pho). M
denotes molecular mass standards (150, 100, 75, 50, 35, and 25 kDa)
purchased from Novegen. The numbers shown on the
left of the figure indicate the sizes of the markers. The
purified sample (0.5 µg of protein) was loaded onto the gel. The
arrow indicates the band of FKBP-type peptidyl-prolyl
cis-trans-isomerase co-purified with
Dna2Pho.
View larger version (38K):
[in a new window]
Fig. 4.
Dna2Pho has ATPase activity stimulated by
single-stranded DNA. The upper panel shows
DNA-stimulated ATPase activity. Dna2Pho (30 ng) was assayed for ATPase
activity in the presence of closed circular single-stranded and
double-stranded M13 DNA (5, 10, 20, 30, 40, and 50 fmol) at 50 °C
for 30 min. The lower panel indicates ATPase activity
plotted against the concentration of closed circular single-stranded
M13 (closed circle) or double-stranded M13 (open
circle). The released Pi was quantified and
calculated. The experiment was carried out three times, and the average
of the three experiments is shown with error bars as
indicated. The inset shows ATPase activity plotted against
the addition of Dna2Pho into the reaction.
View larger version (53K):
[in a new window]
Fig. 5.
Dna2Pho requires ATP for DNA helicase
activity that depends on the MgCl2 concentration.
Dna2Pho (60 ng) was assayed at 10 mM EDTA to chelate free
metal ions or at various MgCl2 concentrations (1, 5, and 10 mM) in the absence and presence of ATP (2 mM)
at 50 °C for 1 h. The substrate used in this experiment was
made by annealing oligonucleotide 2 (30-mers, Table II) to
single-stranded M13 DNA. The arrow indicates the migrated
position of the 30-mer oligonucleotide. A schematic structure of the
substrate used is shown at the left of the figure. The
asterisks indicate 32P-labeled ends.
View larger version (21K):
[in a new window]
Fig. 6.
Dna2Pho translocates along single-stranded
DNA in the 5' to 3' direction. The left
panel shows the direction of the helicase. Dna2Pho (20, 40, 60, 80, and 100 ng) was used in helicase assays. Oligonucleotide 1 (63-mers, Table II) labeled at the 5'- or 3'-end was annealed to M13
ssDNA and digested with SmaI to produce linear substrates
that were used to measure the polarity of displacement. The
arrow indicates the migration of the displaced 32-mer
oligonucleotide. The schematic structure of the substrate used is shown
at the left of the figure. The asterisks indicate
32P-labeled ends. The right panel indicates DNA
helicase activity from a single experiment of several trials using
5'-labeled (open circle) and 3'-labeled (closed
circle) oligonucleotides. Displaced 5'- and 3'-labeled
oligonucleotides were quantified with the GS-525 molecular imager
system. The values were normalized as described under "Experimental
Procedures." One-hundred percent is the value for heat-denatured
substrate.
View larger version (78K):
[in a new window]
Fig. 7.
Dna2Pho has nuclease activity and prefers the
free 5'-end of ssDNA as substrate. A, the
nuclease activity of Dna2Pho using single-stranded and double-stranded
DNA as substrate. The DNA substrates used are shown at the
top of the figures. The asterisks indicate
32P-labeled ends. The oligonucleotides used to construct
each substrate are indicated as circled numbers that are listed in
Table II. M denotes molecular size markers prepared by
labeling a synthetic mixture (2, 3, 4, 6, 40, 50, and 63-mers) and
commercial size markers ((dGATC)n, where n denotes
oligonucleotides 8-32-mers in length, Amersham Biosciences) at their
5'-ends with T4 polynucleotide kinase. The numbers shown on
the left of the figures indicate the size of the markers.
The reaction mixtures contained 50 mM HEPES (pH 7.5), 1 mM dithiothreitol, 0.01% bovine serum albumin, 2 mM ATP, 10 mM MgCl2, the
32P-labeled substrate as described under "Experimental
Procedures," and Dna2Pho (130 ng in a 20 µl-reaction mixture), at
50 °C. Samples were removed (0, 5, 10, 20, 40, 60, and 120 min),
quenched, and loaded on a 15% denaturing polyacrylamide gel (7 M urea). The gel was dried, and the radioactivity was
visualized using the GS-525 molecular imager system. B, the
nuclease activity of Dna2Pho using two partial duplex DNA (5'-overhang
and 3'-overhang) as substrate.
View larger version (44K):
[in a new window]
Fig. 8.
The helicase and nuclease activities of
Dna2Pho are inhibited by a substrate with an RNA segment in the
5'-tail. A, Dna2Pho can not unwind the Y-structured
substrate with an RNA segment in the 5'-tail. The DNA substrates used
are shown at the top of the figures. The
asterisks indicate 32P-labeled ends. The
circled numbers indicate the oligonucleotides listed in
Table II. The single-stranded 5'-tail (40 nt in length) consists of a
homopolymeric 12-nt oligonucleotide (U) segment
((U)12, bold line), and a 28-nt oligo(dT)
segment ((dT)28, thin line).
B, the left and right figures indicate
the nuclease activity of Dna2Pho using the single-stranded RNA-DNA
chimera substrate (0.2 pmol) and the Y-structured substrate with an RNA
segment in the 5'-tail, respectively. The numbers shown at
the center of the figures indicate the sizes of the markers.
These experiments were performed as described under "Experimental
Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-primase complex
composed of four subunits, p180, p70, p58, and p48. The primase
activity requires the p48 and p58 subunits, and especially, p48 is
known to be the primase catalytic subunit, which synthesizes the short
RNA segment, typically 8-12 nucleotides in length (43). The primase
complex of the hyperthermophilic archaeon, Pyrococcus furiosus, has been characterized by Liu et al. (44).
The amino acid sequences of two subunits, Pfup41 and
Pfup46, have similarity to p48 and p58 of the eukaryotic DNA
primase complex, respectively. The p41-p46 complex can synthesize both
DNA (~700 bases in length) and RNA primers (12-40 nucleotides in
length), whereas the DNA primase from yeast cannot synthesize DNA
primers. We also characterized the DNA primase from P. horikoshii (45). Surprisingly, the complex could synthesize
long DNA primers 10 times more effectively than RNA primers and also
could synthesize DNA/RNA hybrids. Liu et al. (44) speculated
that the primer synthesis was started by RNA using ATP in P. furiosus, because the p41-p46 complex can discriminate ATP from
other NTPs. We used a substrate with a poly(U)12 segment at its 5'-end in this report; therefore, further experiments to
elucidate specificity against a substrate with a poly(A) segment at the
5'-end might be necessary. Furthermore, the isolation and characterization of Okazaki fragments from Pyrococcus cells
are very interesting, since it is still unclear whether DNA primers are
synthesized in vivo or not. If the DNA primase of
Pyrococcus species could synthesize a DNA primer in
vivo, the properties of Dna2Pho might be suitable for Okazaki
fragment processing in Pyrococcus cells.
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ACKNOWLEDGEMENTS |
---|
We thank E. Yamamoto and H. Tokue for technical help during this study.
![]() |
FOOTNOTES |
---|
* 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: Inst. of Applied Biochemistry, The University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan.
To whom correspondence should be addressed. Tel.:
81-298-616142; Fax: 81-298-616151; E-mail:
ik-matsui@aist.go.jp.
Published, JBC Papers in Press, December 8, 2002, DOI 10.1074/jbc.M207748200
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ABBREVIATIONS |
---|
The abbreviations used are: ss, single-stranded; ds, double-stranded; Dna2Pho, Dna2 from P. horikoshii; ORF, open reading frame; nt, nucleotide(s); PPIase, peptidyl-prolyl cis-trans-isomerase protein.
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