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Journal of Bacteriology, December 1998, p. 6207-6214, Vol. 180, No. 23
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Gene Cloning and Characterization of Recombinant
RNase HII from a Hyperthermophilic Archaeon
Mitsuru
Haruki,1
Keiko
Hayashi,1
Takayuki
Kochi,1
Ayumu
Muroya,1
Yuichi
Koga,1
Masaaki
Morikawa,1
Tadayuki
Imanaka,2 and
Shigenori
Kanaya1,*
Department of Material and Life Science,
Graduate School of Engineering, Osaka University, 2-1 Yamadaoka,
Suita, Osaka 565-0871,1 and
Department
of Synthetic Chemistry and Biological Chemistry, Graduate School of
Engineering, Kyoto University, Kyoto
606-8501,2 Japan
Received 21 May 1998/Accepted 22 September 1998
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ABSTRACT |
We have cloned the gene encoding RNase HII (RNase
HIIPk) from the hyperthermophilic archaeon Pyrococcus
kodakaraensis KOD1 by screening of a library for clones that
suppressed the temperature-sensitive growth phenotype of an
rnh mutant strain of Escherichia coli. This
gene was expressed in an rnh mutant strain of E. coli, the recombinant enzyme was purified, and its biochemical
properties were compared with those of E. coli
RNases HI and HII. RNase HIIPk is composed of 228 amino acid residues (molecular weight, 25,799) and acts as a monomer.
Its amino acid sequence showed little similarity to those of enzymes
that are members of the RNase HI family of proteins but showed 40, 31, and 25% identities to those of Methanococcus jannaschii, Saccharomyces cerevisiae, and
E. coli RNase HII proteins, respectively. The
enzymatic activity was determined at 30°C and pH 8.0 by use of an M13
DNA-RNA hybrid as a substrate. Under these conditions, the most
preferred metal ions were Co2+ for RNase
HIIPk, Mn2+ for E. coli
RNase HII, and Mg2+ for E. coli
RNase HI. The specific activity of RNase HIIPk
determined in the presence of the most preferred metal ion was 6.8-fold
higher than that of E. coli RNase HII and 4.5-fold
lower than that of E. coli RNase HI. Like
E. coli RNase HI, RNase HIIPk and
E. coli RNase HII cleave the RNA strand of an
RNA-DNA hybrid endonucleolytically at the P-O3' bond. In addition,
these enzymes cleave oligomeric substrates in a similar manner. These
results suggest that RNase HIIPk and E. coli RNases HI and HII are structurally and functionally related to one another.
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INTRODUCTION |
RNase H degrades only the RNA
strand of an RNA-DNA hybrid (6). The genes encoding two
types of RNase H have been cloned from various organisms (3,
7, 9, 15, 17, 18, 21, 38, 40). The rnhA gene encodes
RNase HI, and the rnhB gene encodes RNase HII.
Interestingly, some organisms contain both of these genes. However, in
contrast to the structures and functions of the RNase HI enzymes,
which have been extensively studied (5, 13, 23, 24, 32),
much less is known about those of the RNase HII enzymes. At
present, no information on the distinctive enzymatic properties, the
secondary and tertiary structures, and the types of amino acid residues
involved in catalytic function and substrate binding is available for
the RNase HII enzymes. Escherichia coli RNase HII
has been shown to specifically hydrolyze the RNA strand of an RNA-DNA
hybrid (15). The specific activity of this enzyme was
reported to be 0.4% that of E. coli RNase HI under
assay conditions that were optimal for E. coli
RNase HI (15). However, most of the enzyme accumulated
in E. coli cells in an insoluble form upon induction of
the gene (rnhB) for overexpression, probably due to its poor
solubility. This poor solubility hampers further biochemical and
enzymatic characterization of the enzyme.
Computer analyses recently showed that the genes encoding RNase HII
enzymes exist in the chromosomes of bacteria, archaea, and eucarya
(30). Because the genomes of archaea obviously contain only
genes homologous to the rnhB genes (2, 26), it
seems likely that the RNase HII enzymes are more ubiquitously
present in various organisms than are the RNase HI enzymes.
However, most of these proteins are predicted from translation of
DNA sequences. In addition to E. coli RNase
HII, only RNase HII from Streptococcus pneumoniae
has been shown to possess RNase H activity (40). Transformation with the plasmid containing the rnhB gene
encoding S. pneumoniae RNase HII complemented
the RNase H-dependent temperature-sensitive growth phenotype of an
rnhA-recC double-mutant strain of E. coli, and the enzyme expressed in E. coli showed RNase H
activity in a renaturation gel assay, suggesting that this enzyme is
functional both in vivo and in vitro. It has been suggested that
RNase HII [RNase H(35)] from Saccharomyces
cerevisiae has RNase H activity (9). However, there
is no evidence indicating that this protein has RNase H
activity. Because the enzymes produced from hyperthermophiles are
usually highly stable and because this property not only facilitates the production of recombinant enzymes in E. coli
in an amount sufficient for biochemical characterization but also
facilitates the crystallization of the enzymes for X-ray analyses, we
decided to clone the rnhB gene from the hyperthermophilic
archaeon Pyrococcus kodakaraensis KOD1.
P. kodakaraensis KOD1, which was previously designated
Pyrococcus sp. strain KOD1, was isolated from a solfatara at
a wharf on Kodakara Island, Kagoshima, Japan (33). The
growth temperature of this strain ranges from 65 to 100°C, and the
optimal temperature is 95°C. Here we report that the rnhB
gene was cloned from P. kodakaraensis KOD1 by its
ability to complement the temperature-sensitive growth phenotype of
E. coli MIC3001, with
rnh-339::cat and recB270(Ts) mutations (16). A comparison of the biochemical properties
of recombinant RNase HII from P. kodakaraensis
(RNase HIIPk) with those of E. coli
RNases HI and HII suggests that RNases HI and HII are
structurally and functionally related, despite their poor sequence similarity.
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MATERIALS AND METHODS |
Cells and plasmids.
rnhA mutant strains E. coli MIC3001 and MIC3009 (16) and plasmid pMIC2721 for
the overproduction of E. coli RNase HII
(15) were kindly donated by M. Itaya. The genotype of
E. coli MIC3009 is F
supE44 supF58
lacY1 or 
(lacIZY)6 trpR55 galK2 galT22 metB1
hsdR14(rK
mK+)
rnh-339::cat. E. coli MIC3001 has
an additional recB270(Ts) mutation. Competent cells of
E. coli HB101 [F
hsdS20(rB mB)
recA13 ara-13 proA2 lacY1 galK2 rpsL20 (Smr)
xyl-5 mtl-1 supE44 
] and plasmids pBR322
and pUC18 were from Takara Shuzo Co., Ltd. E. coli BL21
(DE3) [F ompT hsdSB(rB
mB) gal (cI857 ind1 Sam7 nin5 lacUV5-T7
gene 1) dcm(DE3)] was from Novagen. Plasmid pJLA503 was from Medac Gentechnologie. E. coli cells
were grown in Luria-Bertani medium (31) containing 100 mg of
ampicillin per liter.
Materials.
[
-32P]ATP (>5,000 Ci/mmol) and
[
-32P]ddATP (>5,000 Ci/mmol) were from Amersham.
Crotalus durissus phosphodiesterase was from Boehringer
Mannheim Biochemicals. Lysyl endopeptidase was from Wako Pure Chemical
Industries. Restriction and modifying enzymes were from either
Toyobo Co., Ltd., or Takara Shuzo Co.
General DNA manipulations.
Genomic DNA was prepared from a
Sarkosyl lysate of P. kodakaraensis KOD1 cells as
described previously (14). Digestion of DNA with restriction
enzymes, analysis of DNA fragments by agarose gel electrophoresis,
transformation by a calcium chloride procedure, and small-scale
preparation of plasmid DNA by an alkaline lysis method were performed
as described previously (36).
Construction and screening of a KOD1 DNA library.
Genomic
DNA of P. kodakaraensis KOD1 was completely digested
with BamHI or HindIII, and the resultant DNA
fragments were ligated to the alkaline phosphatase-treated
BamHI or HindIII site of pBR322. The
resultant plasmids were used to transform E. coli
MIC3001. Colonies were grown on Luria-Bertani medium plates containing 100 mg of ampicillin per liter at both 30 and 42°C. Usually, after 1 to 2 days of incubation, colonies are detected on the plate incubated
at 30°C but not on that incubated at 42°C, unless the gene
contained in the plasmid functionally complements the rnhA or recB mutation of E. coli MIC3001. Plasmid
DNAs were isolated from colonies grown at 42°C and used to transform
E. coli MIC3001 again. Cloned DNAs which repetitively
complemented the temperature-sensitive growth phenotype of
E. coli MIC3001 were selected as positive.
DNA sequence determination.
A 3-kbp BamHI
fragment cloned into pBR322 was digested with HindIII
and NruI, and the resultant 1.2-kbp
HindIII-BamHI fragment and 1-kbp
NruI-HindIII fragment were ligated to the
large BamHI-HindIII and
SmaI-HindIII fragments of pUC18,
respectively. By use of the resultant plasmids as templates, the DNA
sequences of these fragments were determined by the dideoxy chain
termination method with fluorescent dye terminators and a Prism 310 DNA
sequencer (PE Applied Biosystems).
Overproduction and purification.
The open reading frame
(ORF) for the rnhB gene (rnhBPk) was
amplified by PCR with a set of forward
(5'-TTAGGAGGTGAACATATGAAGATAGCGGGCATTGACGAGGC) and reverse
(5'-GCGCGGTCGACTCGCGGCTGCCAGTTTTGCAT) primers,
where the underlined bases show the positions of the NdeI
(for the forward primer) and SalI (for the reverse primer)
cleavage sites. PCR was performed in 25 cycles with a GeneAmp 2400 PCR
system (The Perkin-Elmer Corp.) and a GeneAmp kit (The Perkin-Elmer,
Corp.) in accordance with the procedures recommended by the supplier. All oligodeoxyribonucleotides were synthesized by Sawady Technology Co., Ltd. The resultant 700-bp NdeI-SalI fragment
was ligated into plasmid pJLA503 to construct pJAL700K. An
overproducing strain was constructed by transforming E. coli MIC3009 with pJAL700K. Overproduction was accomplished as
described previously for E. coli RNase HI
(25). Cells were then harvested by centrifugation at
6,000 × g for 10 min and subjected to purification procedures.
All purification procedures, except for heat treatment, were carried
out at 4°C. Cells were suspended in 10 mM Tris-HCl (pH 7.5)
containing 1 mM EDTA (TE buffer), disrupted by sonication with a model
450 Sonifier (Branson Ultrasonic Corp.), and centrifuged at 15,000 × g for 30 min. The supernatants were incubated at 90°C for 15 min to precipitate most of the proteins derived from host cells
and centrifuged at 30,000 × g for 30 min. The
supernatants were pooled, and the enzyme was precipitated at 70%
saturation with ammonium sulfate to remove nucleic acids. The
precipitates were collected by centrifugation at 10,000 × g for 20 min, dissolved in TE buffer, and applied to a DE-52
column equilibrated with the same buffer. The enzyme was eluted from
the column by linearly increasing the NaCl concentration from 0 to 0.5 M. Fractions containing the enzyme were combined and applied to a
HiLoad 16/60 Superdex 200 pg gel filtration column (Pharmacia) (1.6 by
60 cm) equilibrated with TE buffer containing 0.1 M NaCl. Fractions
containing the pure protein were combined and used for further
analyses. Overproduction and purification of E. coli
RNase HII were performed as described previously (15).
The purities of the enzymes were analyzed by sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (PAGE) on a 12%
polyacrylamide gel (29), followed by staining with Coomassie
brilliant blue. For estimation of the molecular weight of the enzyme by
gel filtration, bovine serum albumin, ovalbumin, chymotrypsinogen, and
RNase A were used as standard proteins.
Enzymatic activity.
The RNase H activity was determined
at 30°C for 15 min by measuring the radioactivity of the acid-soluble
digestion product from the substrate, the 3H-labeled M13
DNA-RNA hybrid, as previously described (25). The buffer was
10 mM Tris-HCl (pH 8.0) containing 50 mM NaCl, 1 mM 2-mercaptoethanol,
10 µg of bovine serum albumin per ml, and 10 mM CoCl2
(for RNase HIIPk), 10 mM MgCl2 (for
E. coli RNase HI), or 10 mM MnCl2 (for
E. coli RNase HII). One unit was defined as the
amount of enzyme producing 1 µmol of acid-soluble material/min at 30°C. The specific activity was defined as the enzymatic activity per milligram of protein.
Cleavage of oligomeric RNA-DNA and DNA-RNA-DNA-DNA
substrates.
The 29-base DNA-RNA-DNA
(5'-AATAGAGAAAAAGaaaaAAGATGGCAAAG-3'), in which DNA and RNA
are represented by uppercase and lowercase letters, respectively, and
the 29-base DNA which is complementary to this 29-base DNA-RNA-DNA were
kindly donated by ID Biomedical Corp. The 12-base RNA
(5'-cggagaugacgg-3') was chemically synthesized by Toray
Research Center Co., Ltd. The 12-base DNA which is complementary to the
12-base RNA and the 13-base DNAs with the sequences of 5'-AATAGAGAAAAAG-3' (DNA1) and
5'-AAGATGGCAAAGA-3' (DNA2), in which the
underlined sequences represent the 5'- and 3'-terminal sequences of the
29-base DNA-RNA-DNA, respectively, were synthesized by Sawady
Technology Co. Three types of hybrid duplexes, in which the 12-base RNA
was 32P labeled at the 5' end or the 29-base DNA-RNA-DNA
was 32P labeled at the 5' end or the 3' end, were
constructed. Because the 29-base DNA-RNA-DNA was 32P
labeled at the 3' end by attaching [-32P]ddATP by use
of terminal deoxynucleotidyltransferase (36), the hybrid
duplex in which the 29-base DNA-RNA-DNA was 32P labeled at
the 3' end was constructed by hybridizing the 30-base DNA-RNA-DNA to
the 29-base DNA. These hybrid duplexes (1.0 µM) were prepared by
hybridizing 32P-labeled 12-base RNA, 29-base
DNA-RNA-DNA, or 30-base DNA-RNA-DNA with 1.5 molar equivalents of
DNA oligomers.
Hydrolysis of the substrate was carried out as described for the M13
DNA-RNA hybrid. The hydrolysates were separated on a 20%
polyacrylamide gel containing 7 M urea and were analyzed with Instant
Imager (Packard). When the hybrid duplex in which RNA or DNA-RNA-DNA
was 32P labeled at the 5' end was used as a substrate, the
hydrolysates were identified by comparing their migrations on the gel
with those of oligonucleotides generated by partial digestion of
32P-labeled 12-base RNA or 29-base DNA-RNA-DNA with snake
venom phosphodiesterase (19) or that of
32P-labeled DNA1. When the hybrid duplex in which the
29-base DNA-RNA-DNA was 32P-labeled at the 3' end was used
as a substrate, the hydrolysates were identified by comparing their
migrations on the gel with that of 32P-labeled DNA2.
Protein concentration.
The protein concentration was
determined from the UV absorption, with
A2880.1% values of 0.63 for RNase
HIIPk and 0.56 for E. coli RNase HII. These values were calculated by use of an 
of 1,576 M1
cm1 for Tyr and an of 5,225 M1
cm1 for Trp at 280 nm (10).
Analyses for primary structure.
Digestion of RNase
HIIPk with lysyl endopeptidase and separation of the
peptides by reverse-phase high-performance liquid chromatography were
carried out as described previously (25). The
NH2-terminal amino acid sequence was determined with a
model 491 protein sequencer (PE Applied Biosystems). The molecular
weight of the peptide was determined with an on-line mass spectrometer (LCQ; Finnigan MAT) by electrospray ionization. Mass spectral data were
processed with Bioworks software (Finnigan MAT).
Nucleotide sequence accession number.
The nucleotide
sequence reported in this paper has been deposited in the DDBJ database
under accession no. AB012613.
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RESULTS |
Cloning of the gene encoding RNase H from P. kodakaraensis KOD1.
E. coli MIC3001 with the
rnh-339::cat and
recB270(Ts) mutations has an RNase H-dependent
temperature-sensitive growth phenotype (16). We used this
strain to isolate the DNA fragment containing the genes encoding
RNase H enzymes from P. kodakaraenis KOD1 by a
complementation assay. If KOD1 RNase H enzymes were expressed in
E. coli and exhibited RNase H activities, MIC3001
transformants with the plasmids containing the genes coding for them
could form colonies at a nonpermissive temperature, 42°C. Of 5 × 104 MIC3001 transformants with the plasmid libraries
containing either the BamHI or HindIII
fragments of the KOD1 genome, 11 transformants could grow at 42°C.
Restriction enzyme analyses indicated that all of the plasmids isolated
from these transformants were identical to one another and contained a
3-kbp BamHI fragment (data not shown).
DNA sequence analysis.
A plasmid that complements the
temperature-sensitive growth phenotype of MIC3001 does not necessarily
contain an rnh gene, because other genes may be able to
complement the rnhA or recB mutation of this
strain. Generally, a gel renaturation assay (4) has been
used to identify positive clones from those obtained by the
complementation method. However, in this experiment, we obtained only
one transformant that could grow at a nonpermissive temperature.
Therefore, we did not analyze this transformant for its RNase H
activity by a gel renaturation assay. Instead, we analyzed the DNA
sequence of the plasmid isolated from this transformant.
Because the 3-kbp
BamHI fragment of the KOD1 genome contains
an ORF encoding the amino acid sequence homologous to that of
E. coli RNase HII, we designated this ORF and the
encoded protein
rnhBPk and RNase
HII
Pk, respectively. A putative ribosome binding
site
(5'-AGGAGGTG-3'), which is complementary to the 3'-terminal
sequence of 16S rRNA from KOD1 (3'-UCCUCCAC-5') (
33), is
located
6 nucleotides upstream from the initiation codon of the ORF.
The
protein is composed of 228 amino acid residues and has a calculated
molecular weight of 25,799 and an isoelectric point of 5.43. A
HindIII site is located ~150 bp downstream from the
initiation
codon. This is the reason why we could not isolate the DNA
fragment
containing the
rnhB gene from
HindIII
libraries.
Amino acid sequence comparison.
The amino acid sequence of
RNase HIIPk was compared with those of its homologues
from archaea, eucarya, and bacteria (Fig. 1). The sequences of the RNase HII
homologues from Archaeoglobus fulgidus,
Methanobacterium thermoautotrophicum, and
Methanococcus jannaschii, to which RNase H function
has been putatively ascribed, represent those of archaeal RNase HII
enzymes. The sequences of S. cerevisiae RNase HII
and the putative RNase HII from Caenorhabditis elegans
represent those of eucaryotic RNase HII enzymes. The sequences of
E. coli RNase HII and the putative RNase HII
from Bacillus subtilis represent those of prokaryotic
RNase HII enzymes. RNase HIIPk showed
amino acid sequence identities of 42% to A. fulgidus RNase HII, 40% to M. thermoautotrophicum RNase HII
or M. jannaschii RNase HII, 31% to
S. cerevisiae RNase HII or C. elegans RNase HII, 25% to E. coli
RNase HII, and 26% to B. subtilis RNase HII. Three
motifs (motifs I to III) which are highly conserved in
various RNase HII sequences (40) were also conserved in
the RNase HIIPk sequence (Fig. 1). When the
archaeal and eucaryotic RNase HII sequences were compared, an
additional motif (motif IV) was observed (Fig. 1).
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FIG. 1.
Alignment of RNase HII sequences. The amino acid
sequences of RNase HII enzymes from bacteria, archaea, and eucarya
are compared. Gaps are denoted by dashes. Amino acid residues which are
conserved in at least three different enzymes are highlighted in black.
When two types of amino acid residues were conserved at a given
position, the most conserved one was selected; if they were conserved
equally, the residue conserved in the RNase HIIPk
sequence was selected; if they were conserved equally but either one of
them was not conserved in the RNase HIIPk sequence, one
of them was just selected randomly. Numbers represent the positions of
the amino acid residues, which start from the initiator methionine for
each enzyme. The ranges for the four sequence motifs which are well
conserved in the RNase HII sequences are shown above the sequences.
The sources of RNase HII enzymes were as follows: KOD1,
P. kodakaraensis KOD1 (DDBJ accession no. AB012613);
Afu, A. fulgidus (GenBank accession no. AE001062); Mth,
M. thermoautotrophicum (GenBank accession no. AE000875);
Mja, M. jannaschii (GenBank accession no. U67470);
Sce, S. cerevisiae (GenBank accession no. Z71348, ORF
YNL072w); Cel, C. elegans (GenBank accession no. Z66524,
product T13H5.2); Eco, E. coli (Swiss-Prot code no.
P10442); and Bsu, B. subtilis (EMBL accession no. 299112).
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Overexpression and purification of RNase
HIIPk.
Upon induction, recombinant RNase
HIIPk accumulated in cells as the most abundant protein
(Fig. 2). The level of production of the
enzyme was estimated to be about 25 mg/liter of culture. RNase
HIIPk was recovered in a soluble form after sonication
lysis and purified to homogeneity by three procedures, with a yield of
~20% (Fig. 2). Approximately 5 mg of the enzyme was purified from 1 liter of culture. The molecular weight of the enzyme was estimated to
be 28,000 from SDS-PAGE and 33,000 from gel filtration chromatography
(data not shown). These results suggest that RNase HIIPk exists in a monomeric form.
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FIG. 2.
SDS-PAGE of RNase HIIPk in each
purification step. Samples were subjected to SDS-12% PAGE and stained
with Coomassie brilliant blue. Lane 1, low-molecular-weight marker kit
(Pharmacia LKB Biotechnology) containing phosphorylase b,
albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor, and
-lactalbumin; lane 2, whole-cell lysate of E. coli
MIC3009 harboring plasmid pJAL700K; lane 3, soluble fraction extracted
from E. coli MIC3009 harboring plasmid pJAL700K; lane
4, soluble fraction obtained after heat treatment; lane 5, fractions
pooled after DE-52 column chromatography; lane 6, fractions pooled
after Superdex 200 pg column chromatography. Numbers beside the gel
represent the molecular weights of individual standard proteins (in
thousands [K]).
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Amino acid sequence analyses.
Because the molecular
weight of recombinant RNase HIIPk estimated from
SDS-PAGE and gel filtration chromatography was slightly higher than the
calculated one, we analyzed the enzyme by mass spectroscopy as well.
The molecular weight of the enzyme determined by mass spectroscopy was
25,577, which is lower than the calculated one by 222. To examine
whether a few NH2- or COOH-terminal amino acid residues are
truncated in this enzyme, the NH2- and COOH-terminal amino
acid sequences of the enzyme were determined as described in Materials
and Methods. The results showed that the COOH terminus is
Arg226 instead of Val228, indicating that
recombinant RNase HIIPk is composed of 226 amino acid residues.
Enzymatic activity of RNase HIIPk.
It has been
reported that E. coli RNase HI prefers the
Mg2+ ion for activity and exhibits 2 to 3% maximal
activity in the presence of the Mn2+ ion (1). It
has also been reported that E. coli RNase HII requires the Mg2+ ion for activity and exhibits 0.4% of
the enzymatic activity of E. coli RNase HI under
conditions which are optimal for E. coli RNase HI
(15). However, it remains to be determined whether E. coli RNase HII exhibits activity in the presence
of metal ions other than Mg2+. Therefore, we determined the
activities of these enzymes, as well as that of RNase
HIIPk, in the presence of 10 mM MgCl2,
MnCl2, BaCl2, CaCl2,
CoCl2, CuSO4, FeCl2,
FeCl3, NiCl2, SrCl2, and
ZnCl2. The results are summarized in Table
1. As expected, E. coli
RNase HI exhibited little activity in the presence of metal ions
other than Mg2+ and Mn2+. Likewise,
E. coli RNase HII exhibited activity only in the
presence of the Mg2+ or Mn2+ ion. However,
unlike E. coli RNase HI, E. coli
RNase HII preferred the Mn2+ ion for activity. In
contrast to these E. coli enzymes, RNase HIIPk showed a unique metal ion preference for activity. It
exhibited activity in the presence of the Mg2+,
Mn2+, Co2+, and Ni2+ ions but did
not exhibit activity in the presence of other metal ions. The most
preferred metal ion for RNase HIIPk was the
Co2+ ion, but the Mg2+, Mn2+, and
Ni2+ ions could be substituted for the Co2+ ion
without seriously affecting enzymatic activity. These results indicate
that the E. coli RNase HI and HII activities are
dependent on the Mg2+ and Mn2+ ions,
respectively, but that the RNase HIIPk activity is not necessarily dependent on a specific divalent cation. Instead, RNase
HIIPk exhibits rather broad metal ion specificities. Thus, at pH 8.0, the specific activity of RNase HIIPk
determined in the presence of 10 mM CoCl2 was shown to be
6.8-fold higher than that of E. coli RNase HII
determined in the presence of 10 mM MnCl2 and 4.5-fold
lower than that of E. coli RNase HI determined in
the presence of 10 mM MgCl2.
The dependence of RNase HII
Pk activity on the metal ion
concentration was analyzed for Mg
2+, Mn
2+,
Co
2+, and Ni
2+. As shown in Fig.
3, the concentrations of the metal ions
which
gave 50% maximal activity were ~5 mM for Co
2+ and
Mg
2+, ~2 mM for Ni
2+, and <1 mM for
Mn
2+. These results suggest that the Mn
2+ ion
binds most strongly to the enzyme and that the Co
2+ and
Mg
2+ ions bind most weakly to the enzyme. The dependence of
RNase
HII
Pk activity on the concentration of NaCl or
KCl was also analyzed.
The results are summarized in Table
2. These salts do not seriously
affect
enzymatic activity at concentrations lower than 100 mM
but are
inhibitory for activity at concentrations higher than
400 mM for NaCl
and 200 mM for KCl.
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FIG. 3.
Effect of metal ion concentrations on RNase
HIIPk activity. Enzymatic activity was determined with 10 mM Tris-HCl (pH 8.0) containing 50 mM NaCl, 1 mM 2-mercaptoethanol, 10 µg of bovine serum albumin per ml, and various concentrations of
MgCl2 (closed circle), MnCl2 (open square),
CoCl2 (open circle), or NiCl2 (closed
square).
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The pH dependence of RNase HII
Pk activity was not
analyzed, because the solubility of the Co
2+ ion
dramatically decreased as the pH increased beyond 9.0. However,
when
the pH dependence was analyzed in the presence of the Mg
2+
ion, the enzyme showed a pH optimum at pH 9.5 (data not shown),
indicating that RNase HII
Pk expresses activity at an
alkaline
pH, like
E. coli RNase HI. The temperature
dependence of RNase
HII
Pk activity was not analyzed
either, because the substrate
is unstable at high temperatures. We
determined the enzymatic
activity at 30°C, at which all of the
substrates used in this
experiment, including a 12-bp RNA-DNA
substrate, are
stable.
Cleavage of oligomeric substrates.
To examine
whether RNase HIIPk hydrolyzes oligomeric
substrates, we cleaved the 12-bp RNA-DNA substrate and the 29-bp
DNA-RNA-DNA-DNA substrate, both of which were 32P labeled
at the 5' ends, with the enzyme. The results are shown in Fig.
4A. The enzyme cleaved
the 12-bp RNA-DNA substrate at multiple sites, but most preferably at
a9-c10, whereas it specifically cleaved the 29-bp DNA-RNA-DNA-DNA
substrate at the phosphodiester bond between the third and fourth
adenosines (a16-a17). The specific cleavage of this DNA-RNA-DNA-DNA
substrate at a16-a17 with RNase HIIPk was further
confirmed by use of the substrate in which the 29-base DNA-RNA-DNA was
32P labeled at the 3' end (data not shown). We analyzed the
E. coli RNase HI and E. coli
RNase HII activities by using these substrates as well. The results
are summarized in Fig. 4B. E. coli RNase HI cleaved
the 12-bp RNA-DNA substrate in a manner similar to that of RNase
HIIPk. It has already been reported that E. coli RNase HI cleaves the same substrate most preferably at
a9-c10 (20). In addition, like RNase HIIPk
E. coli RNase HI cleaved the 29-bp
DNA-RNA-DNA-DNA substrate at a unique position, although it
cleaved the substrate in the middle of the tetraadenosines (a15-a16).
This result is consistent with the previous one that E. coli RNase HI specifically cleaves the 20-base DNA-RNA-DNA, which consists of tetraribonucleotides flanked on either side by 7- or 9-base DNA, only in the middle of the tetraribonucleotide in the
presence of the cDNA strand (12). E. coli
RNase HII cleaved these substrates in basically the same manner as
RNase HIIPk (data not shown). Both RNase HII
enzymes cleaved neither the 12-base RNA nor the 29-base DNA-RNA-DNA,
suggesting that these enzymes cannot cleave single-stranded RNA or DNA.
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|
FIG. 4.
Cleavage of oligomeric substrates by RNase
HIIPk. (A) Autoradiograph of cleavage reactions. Hydrolysis
of the 5'-end-labeled 12-base RNA hybridized to the 12-base DNA (a) and
the 5'-end-labeled 29-base DNA-RNA-DNA hybridized to the 29-base DNA
(b) with RNase HIIPk was carried out at 30°C for 15 min, and the hydrolysates were separated on a
20% polyacrylamide gel containing 7 M urea as described in Materials
and Methods. The concentration of the substrate was 1.0 µM. (a) Lane
1, partial digest of the 12-base RNA with snake venom
phosphodiesterase; lane 2, untreated substrate; lane 3, hydrolysate
with 0.8 ng of the enzyme; lane 4, hydrolysate with 1.6 ng of the
enzyme; lane 5, hydrolysate with 16 ng of the enzyme. (b) Lane 1, partial digest of the 29-base DNA-RNA-DNA with snake venom
phosphodiesterase; lane 2, 5'-end-labeled 13-base DNA with a sequence
which is identical to residues 1 to 13 of the 29-base DNA-RNA-DNA; lane
3, untreated substrate; lane 4, hydrolysate with 0.8 ng of the enzyme;
lane 5, hydrolysate with 1.6 ng of the enzyme; lane 6, hydrolysate with
16 ng of the enzyme. (B) Sites and extents of cleavage by RNase H. Sites of cleavage of the 12-bp RNA-DNA hybrid with RNase
HIIPk (a) and E. coli RNase HI (b) and
those of the 29-bp DNA-RNA-DNA-DNA substrate with RNase
HIIPk (c) and E. coli RNase HI (d) are
shown by arrows. Differences in the sizes of the arrows reflect the
relative cleavage intensities at the indicated positions. The sites of
cleavage of these substrates with E. coli RNase HII
were almost identical to those with RNase HIIPk. For
both panel A and panel B, deoxyribonucleotides are shown by uppercase
letters, and ribonucleotides are shown by lowercase letters.
|
|
Heat inactivation.
The stability of RNase
HIIPk against irreversible heat inactivation was analyzed
by incubating the enzyme solution (0.1 µg/ml) in 10 mM Tris-HCl (pH
7.5) containing 0.1 M NaCl, 1 mM EDTA, 10% glycerol, and 0.1 mg of
bovine serum albumin per ml at 90°C. For comparative purposes, the
stability of E. coli RNase HII against irreversible
heat inactivation was analyzed by incubating the enzyme solution (1 µg/ml) in the same buffer at 70°C. At appropriate intervals,
an aliquot of the enzyme solution was withdrawn, chilled on ice, and
analyzed for remaining activity. As shown in Fig. 5, RNase HIIPk was fairly
stable against irreversible heat inactivation at 90°C, whereas
E. coli RNase HII rapidly lost activity even at
70°C, with a half-life of 9 min.
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|
FIG. 5.
Stability against irreversible heat inactivation.
RNase HIIPk (closed circles) and E. coli RNase HII (open circles) were incubated at 90 and 70°C,
respectively, in 10 mM Tris-HCl (pH 7.5) containing 0.1 M NaCl, 1 mM
EDTA, 10% glycerol, and 0.1 mg of bovine serum albumin per ml. At
appropriate intervals, an aliquot was withdrawn and examined for
remaining activity. The concentrations of the enzymes were 0.1 µg/ml
for RNase HIIPk and 1 µg/ml for E. coli RNase HII. Enzymatic activity was determined at 30°C
for 15 min with 10 mM Tris-HCl containing 10 mM MgCl2 (for
RNase HIIPk) or MnCl2 (for E. coli RNase HII), 50 mM NaCl, 1 mM 2-mercaptoethanol, and 10 µg of bovine serum albumin per ml and with the M13 DNA-RNA hybrid as
a substrate.
|
|
| |
DISCUSSION |
Classification of RNase H families.
In this report,
we showed that RNase HIIPk, which may represent
archaeal RNase HII enzymes, has RNase H activity both in vivo and in vitro. This result supports a previous proposal that RNase H
enzymes can be classified into two families, RNase HI and
RNase HII, based on the difference in the amino acid
sequences (40). However, similarities in the
substrate cleavage patterns and the requirements for enzymatic
activity between RNase HIIPk and E. coli RNase HI suggest that RNase HI and HI enzymes are
functionally and structurally related. It has been reported that
Asp10, Glu48, Asp70, and
Asp134 form the active site of E. coli
RNase HI, to which the catalytically essential Mg2+ ion
binds (24). Three or four acidic residues may form the active site of RNase HII as well, because several acidic amino acid
residues (Asp7, Glu8, Asp40,
Asp105, Asp135, and Asp153 for
RNase HIIPk are fully conserved in RNase HII
sequences (Fig. 1). We are currently undertaking site-directed
mutagenesis and X-ray crystallographic studies to find an answer to the
question of whether RNase HI and HII enzymes form a superfamily.
Purification of recombinant RNase HIIPk.
In contrast to E. coli RNase HII, RNase
HIIPk accumulates in a soluble form in cells which
overproduce the enzyme. A comparison of the hydropathy profiles
of these enzymes suggests that E. coli RNase
HII is more hydrophobic than RNase HIIPk (data not
shown), especially at the COOH-terminal region. E. coli RNase HII has the sequence ALGTCVLILV at positions
193 to 202, whereas RNase HIIPk has the sequence
GWKTLKKIAE at the corresponding region (positions 197 to 206). Such a difference in hydrophobicity may alter the solubilities
of these enzymes.
Overproduction of RNase HII
Pk in
E. coli and purification of the recombinant enzyme yielded a
truncated enzyme in which the
COOH-terminal two residues were
eliminated. The COOH-terminal
region of RNase HII
Pk
probably assumes a disordered structure;
therefore, the peptide
bond between Arg
226 and Lys
227 is cleaved by a
protease from
E. coli, such as OmpT protease,
which is
known to specifically cleave the peptide bond between
two basic amino
acid residues (
37). Partial digestion with trypsin
or
chymotrypsin also produced a truncated protein in which the
COOH-terminal 10 to 15 residues were eliminated (
34a).
Metal ion specificity.
RNase HIIPk shows a
relatively broad metal ion specificity. In addition, other enzymes from
KOD1, such as a DNase which is associated with a RecA homologue
(35) and glycerol kinase (28), show broad metal
ion specificity. Therefore, this broad metal ion specificity may be
characteristic of the enzymes isolated from hyperthermophilic archaea.
Because hyperthermophilic archaea have been thought to retain traces of
early life forms and to produce enzymes which may represent prototypes
in the same protein family, the difference in metal ion specificity
between an archaeal enzyme and a bacterial or eucaryotic enzyme may
reflect the difference in the contents of various metal ions in the
soil or sea between the primitive earth and the modern earth.
Substrate specificity.
Good agreement in the autoradiograms of
the cleavage reactions for the 12-bp RNA-DNA substrate between
RNase HIIPk and E. coli RNase HI
strongly suggests that, like E. coli RNase HI,
RNase HIIPk cleaves the P-O3' bond of the RNA
endonucleolytically. In addition, the findings that RNase
HIIPk cleaves the 12-bp RNA-DNA substrate at multiple sites
but cannot cleave the 12-base RNA, the 29-base DNA-RNA-DNA, or the DNA
strand of the 29-bp DNA-RNA-DNA-DNA substrates clearly indicate that
the enzyme does not cleave single-stranded DNA, single-stranded RNA, or
double-stranded DNA. Because RNase HIIPk has a sequence
specificity similar to that of E. coli RNase HII
and it has been shown that E. coli RNase HII does
not cleave double-stranded RNA (15), it is highly likely
that RNase HIIPk cleaves the RNA strand only when it is
hybridized to the DNA strand.
Recently, the enzymatically active subunit of human RNase HI was
suggested to be a member of the RNase HII family of proteins
(
9). A human RNase HI from erythroleukemic cells can
cleave
DNA-RNA junctions when a DNA-RNA-DNA-DNA hybrid in which a
single
ribonucleotide is flanked on either side by DNA is used as a
substrate
(
8). This DNA-RNA junction cannot be cleaved by
E. coli RNase
HI (
12). The 29-base
DNA-RNA-DNA has two DNA-RNA junctions:
DNA-RNA and RNA-DNA junctions
from 5' to 3'. Because we used two
types of 29-bp DNA-RNA-DNA-DNA
substrates, those which were
32P labeled at the 5' end or
the 3' end, it is clear that RNase
HII
Pk cannot cleave
either the DNA-RNA junction or the RNA-DNA
junction. However, RNase
HII
Pk and
E. coli RNase HII can cleave
the 29-bp DNA-RNA-DNA-DNA substrate at the 5' end of the last
ribonucleotide at the RNA-DNA junction, whereas
E. coli
RNase
HI cannot cleave it at this site. It has been reported that
calf
thymus
E. coli RNase HI cannot cleave it at
this site. It has
been reported that calf thymus RNase HI
shows junction RNase activity,
which is responsible for cleavage at
the 5' end of the last ribonucleotide
at the RNA-DNA junction of an
Okazaki substrate regardless of
whether or not it forms an
RNA-DNA heteroduplex structure (
34).
RNase
HII
Pk and
E. coli RNase HII may have
such a junction RNase
activity and thereby facilitate the
elimination of RNA from an
Okazaki
substrate.
Stability of RNase HIIPk.
RNase
HIIPk was shown to be remarkably more stable
against irreversible heat inactivation than E. coli
RNase HII. We previously showed that E. coli
RNase HI almost fully lost activity after incubation at 70°C for
10 min (22). Therefore, there is no doubt that RNase
HIIPk is more stable than E. coli RNase
HI as well. It has been reported that an increase in the number of ion
pairs or ion-pair networks and a decrease in the volume of cavities are
responsible for the hyperstability of the enzymes from
hyperthermophilic archaea (11, 27, 39). It would be
informative to examine whether RNase HIIPk is
stabilized by similar mechanisms.
| |
ACKNOWLEDGMENTS |
We thank M. Itaya, Mitsubishi Kasei Institute of Life Sciences,
for providing E. coli MIC3001 and MIC3009 and plasmid
pMIC2721 and ID Biomedical Corp. for providing the DNA-RNA-DNA probe
for the RNase H assay. We also thank N. Ohtani and N. Hirano for
technical assistance.
This work was supported in part by a grant-in-aid for scientific
research (08455382) from the Ministry of Education, Science and Culture
of Japan.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Material and Life Science, Graduate School of Engineering, Osaka
University, 2-1, Yamadaoka, Suita, Osaka 565-0871, Japan. Phone:
81-6-879-7938. Fax: 81-6-879-7938. E-mail:
kanaya{at}chem.eng.osaka-u.ac.jp.
| |
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Abstract
Introduction
