The N-terminal hybrid binding domain of RNase HI from
Thermotoga maritima is important for substrate binding
and Mg
2+
-dependent activity
Nujarin Jongruja
1
, Dong-Ju You
1
, Eiko Kanaya
1
, Yuichi Koga
1
, Kazufumi Takano
1,2
and
Shigenori Kanaya
1
1 Department of Material and Life Science, Graduate School of Engineering, Osaka University, Japan
2 CRESTO, JST, Osaka, Japan
Introduction
Ribonuclease H (RNase H; EC 3.1.26.4) is an enzyme
that specifically cleaves RNA of RNA⁄ DNA hybrids
[1]. It requires divalent metal ions, such as Mg
2+
and
Mn
2+
, for activity. RNase H is widely present in bac-
teria, archaea and eukaryotes. These RNase H are
involved in DNA replication, repair and transcription

HBD. Tma-CD lost the ability to suppress the RNase H deficiency of an
E. coli rnhA mutant, indicating that the HBD is responsible for in vivo
RNase H activity. The cleavage-site specificities of Tma-RNase HI are not
significantly changed on removal of the HBD, regardless of the metal
cofactor. Binding analyses of the proteins to the substrate using surface
plasmon resonance indicate that the binding affinity of Tma-RNase HI is
greatly reduced on removal of the HBD or the mutation. These results
indicate that there is a correlation between Mg
2+
-dependent activity and
substrate binding affinity. Tma-CD was as stable as Tma-RNase HI,
indicating that the HBD is not important for stability. The HBD of
Tma-RNase HI is important not only for substrate binding, but also for
Mg
2+
-dependent activity, probably because the HBD affects the interaction
between the substrate and enzyme at the active site, such that the scissile
phosphate group of the substrate and the Mg
2+
ion are arranged ideally.
Abbreviations
Bha-RNase HI, Bacillus halodurans RNase HI; Bst-RNase HIII, Bacillus stearothermophilus RNase HIII; Bsu-RNase HII, Bacillus subtilis RNase
HII; D13-R4-D12 ⁄ D29, 29 bp DNA
13
-RNA
4
-DNA
12
⁄ DNA duplex; D15-R1-D13 ⁄ D29, 29 bp DNA
15

(type 1 and type 2 RNases H) based on the difference
in their amino acid sequences [11]. Four acidic active-
site residues are fully conserved in these RNases H,
except that from compost metagenome [12], and their
geometrical configurations are well conserved [13].
According to the crystal structures of the C-terminal
catalytic domains of Bacillus halodurans RNase HI
(Bha-RNase HI) [14] and human RNase H1 [15] in
complex with the RNA ⁄ DNA substrate, type 1 RNase
H binds to the minor groove of the substrate, such
that one depression containing the active site interacts
with the RNA backbone and the other depression con-
taining the phosphate-binding pocket interacts with
the DNA backbone. These two depressions are sepa-
rated by a ridge, which is composed of three highly
conserved Asn ⁄ Gln residues. Because two metal ions
are coordinated by the four acidic active site residues,
the scissile phosphate group of the substrate and water
molecules, a two-metal ion catalysis mechanism has
been proposed for RNase H [14,16,17]. According to
this mechanism, one metal ion is required for sub-
strate-assisted nucleophile formation and product
release, whereas the other is required to destabilize the
enzyme–substrate complex and thereby promote the
phosphoryl transfer reaction.
Thermotoga maritima is a strictly anaerobic, extre-
mely thermophilic eubacterium, isolated from various
geothermally heated locales on the sea floor, and
grows in the temperature range 55–90 °C with an opti-
mum at 80 °C [18]. Its genome sequence has been

[25,26] has also been reported for mouse and human
RNases H1, respectively.
HBD is also present in several bacterial type 1
RNases H, including Tma-RNase HI, Bha-RNase HI
and RBD-RNase HI from Shewanella sp. SIB1 [13].
However, it remains to be determined whether these
HBDs have a role similar to those of eukaryotic
RNases H1, although the isolated HBD from SIB1
RBD-RNases HI, which is renamed as SIB1 HBD-
RNase HI in the present study, has been reported to
bind to the RNA ⁄ DNA substrate [27]. Attempts to
overproduce the SIB1 HBD-RNase HI derivative lack-
ing the HBD have so far been unsuccessful, probably
as a result of the instability of the protein (T. Tadok-
oro, unpublished data). In the present study, we over-
produced, purified and biochemically characterized
Tma-RNase HI and its derivatives lacking the HBD or
RNase H domain. On the basis of the results obtained,
we discuss the role of the HBD from Tma-RNase HI.
Results
Protein preparations
The amino acid sequence of Tma-RNase HI is com-
pared with those of the representative members of type
1 RNases H, Bha-RNase HI, HBD-RNase HI from
Shewanella sp. SIB1, Saccharomyces cerevisiae RNase
H1 (Sce-RNase H1), human RNase H1 (Hsa-RNase
H1), E. coli RNase HI (Eco-RNase HI) and the
RNase H domain of HIV-1 reverse transcriptase (HIV-
1 RNase H) in Fig. 1. The HBD of Tma-RNase HI
shows relatively high amino acid sequence identities of

graphy. These values are comparable to those calculated
from the amino acid sequences (25 967 for Tma-RNase
Fig. 1. Alignment of the amino acid sequences. The amino acid sequence of Tma-RNase HI (Tma) is compared with those of Bha-RNase HI
(Bha), SIB1 HBD-RNase HI (SIB1HBD), Sce-RNase H1 (Sce), Hsa-RNase H1 (Hsa), Eco-RNase HI (Eco) and HIV-1 RNase H (HIV1). The
accession numbers are AAD36370 for Tma-RNase HI, BAF73617 for SIB1 HBD-RNase HI, DAA10134 for Sce-RNase H1, EAX01061 for Hsa-
RNase H1, P0A7Y4 for Eco-RNase HI and ABU62661 for HIV-1 RNase H. The ranges of the secondary structures of Hsa-RNase H1 are
shown above the sequence, based on the crystal structures of its HBD (Protein Data Bank code: 3BSU) and RNase H domain (Protein Data
Bank code: 2KQ9), which were independently determined in complex with the substrate. The range of HBD is also shown. The amino acid
residues, which are conserved in at least three (for HBD) or four (for RNase H domain) different proteins, are highlighted in black. The five
active-site residues are denoted by filled circles above the sequences. The amino acid residues that contact the substrate in the co-crystal
structure of the HBD of Hsa-RNase H1 with the substrate are also denoted by open circles above the sequence. The amino acid residue that
is mutated in the present study is indicated by an arrow. Gaps are denoted by dashes. The numbers represent the positions of the amino
acid residues relative to the initiator methionine for each protein.
Role of HBD from T. maritima RNase HI N. Jongruja et al.
4476 FEBS Journal 277 (2010) 4474–4489 ª 2010 The Authors Journal compilation ª 2010 FEBS
HI, 18 860 for Tma-CD and 7107 for Tma-ND), sug-
gesting that all proteins exist as a monomer in solution.
CD spectra
The far- and near-UV CD spectra of Tma-RNase HI,
Tma-CD and Tma-ND were measured at 20 °C and
pH 9.0, and comparisons are shown in Fig. 3. The far-
and near-UV CD spectra of Tma-CD are similar to
those of Tma-RNase HI, suggesting that removal
of the HBD does not significantly affect the structure
of the RNase H domain of Tma-RNase HI. The
far- and near-UV CD spectra of Tma-ND were sig-
nificantly different from those of Tma-RNase HI,
probably because the secondary structure contents and
environment of the aromatic residues are different in
these proteins. According to the crystal structures of

presence of 10 mm KCl (Fig. 4). Their enzymatic
Fig. 2. SDS ⁄ PAGE of Tma-RNase HI and its derivatives. The
purified proteins of Tma-RNase HI (lane 1), Tma-CD (lane 2) and
Tma-ND (lane 3) were subjected to electrophoresis on a 15% poly-
acrylamide gel in the presence of SDS. After electrophoresis, the
gel was stained with Coomassie Brilliant Blue. Lane M, a low-
molecular-weight marker kit (GE Healthcare, Tokyo, Japan).
Fig. 3. CD spectra of Tma-RNase HI and its derivatives. Far-UV (A)
and near-UV (B) CD spectra of Tma-RNase HI (thick solid dark line),
Tma-W22A (thin solid dark line), Tma-CD (dashed dark line) and
Tma-ND (thick solid gray line) are shown. These spectra were mea-
sured at pH 9.0 and 20 °C, as described in the Experimental proce-
dures.
N. Jongruja et al. Role of HBD from T. maritima RNase HI
FEBS Journal 277 (2010) 4474–4489 ª 2010 The Authors Journal compilation ª 2010 FEBS 4477
activities decreased to a large extent at higher
(‡ 0.2 m) salt concentrations. When the enzymatic
activity was determined in the presence of various
concentrations of MgCl
2
, MnCl
2
, NiCl
2
, ZnCl
2
,
CoCl
2
or CaCl

-dependent activity is
higher than its maximal Mn
2+
-dependent activity by
16-fold. By contrast, Tma-CD prefers Mn
2+
to Mg
2+
because its maximal Mn
2+
-dependent activity is
higher than its maximal Mg
2+
-dependent activity by
69-fold. Interestingly, the maximal Mn
2+
-dependent
activity of Tma-CD is comparable to that of Tma-
RNase HI. These results indicate that removal of the
HBD severely reduces the Mg
2+
-dependent activity of
Tma-RNase HI without significantly affecting its
Mn
2+
-dependent activity.
The kinetic parameters of Tma-CD were determined
at 30 °C in the presence of 1 mm MgCl
2
or MnCl

mined at 30 °Cin10m
M Tris ⁄ HCl (pH 9.0) containing 1 mM MgCl
2
(Tma-RNase HI) or 1 mM MnCl
2
(Tma-CD), 1 mM b-mercaptoe-
thanol, 50 lgÆmL
)1
BSA, and various concentrations of NaCl (open
circle) or KCl (closed circle), using M13 DNA ⁄ RNA hybrid as a sub-
strate. Experiments were carried out at least twice and the average
values are shown together with the errors.
Fig. 5. Metal ion dependencies of Tma-RNase HI and Tma-CD.
The enzymatic activities of Tma-RNase HI (A) and Tma-CD (B)
were determined at 30 °Cin10m
M Tris ⁄ HCl (pH 9.0) containing
50 m
M KCl (Tma-RNase HI) or 10 mM KCl (Tma-CD), 1 mM
b-mercaptoethanol, 50 lgÆmL
)1
BSA, and various concentrations of
MgCl
2
(open circle) or MnCl
2
(closed circle), using M13 DNA ⁄ RNA
hybrid as a substrate. Experiments were carried out at least twice
and the average values are shown together with the errors.
Role of HBD from T. maritima RNase HI N. Jongruja et al.
4478 FEBS Journal 277 (2010) 4474–4489 ª 2010 The Authors Journal compilation ª 2010 FEBS

DNA duplex (D13-R4-D12 ⁄ D29), 29 bp DNA
15
-
RNA
1
-DNA
13
⁄ DNA duplex (D15-R1-D13 ⁄ D29) and
18 bp RNA
9
-DNA
9
⁄ DNA duplex (R9-D9 ⁄ D18). For
comparative purposes, these substrates were cleaved
by Eco-RNase HI, Sulfolobus tokodaii RNase HI
(Sto-RNase HI) and Thermococcus kodakaraensis
RNase HII (Tk-RNase HII) as well. D13-R4-D12
and D15-R1-D13 are the chimeric oligonucleotides,
in which four and single ribonucleotides are flanked
by 12–15 bp of DNA at both sides. R9-D9 ⁄ D18 is a
Okazaki fragment-like substrate, in which the 18 base
chimeric oligonucleotide (RNA
9
-DNA
9
) is hybridized
to the 18 base complementary DNA.
Cleavage of the R12 ⁄ D12 substrate with various
RNase H enzymes is summarized in Fig. 6A,B. Tma-
RNase HI, Eco-RNase HI, Sto-RNase HI and Tk-

The cleavage sites of this substrate with Eco-RNase
HI and Tk-RNase HII are the same as those reported
previously [30]. The a16-a17 site has been reported to
be exclusively cleaved only by type 2 RNases H, except
for bacterial RNases HIII [31,32]. Therefore, Tma-
RNase HI is the first type 1 RNase H enzyme that
exclusively cleaves this substrate at this site. Tma-CD
also cleaved this substrate at a16-a17 with a similar
efficiency to that of Tma-RNase HI. However, these
enzymes cleaved this substrate only in the presence of
Mn
2+
.
Table 1. Specific activities and kinetic parameters of Tma-RNase HI and its derivatives. Hydrolysis of the M13 DNA ⁄ RNA hybrid by the
enzyme was carried out at 30 °C under the conditions described in the Experimental procedures. ND, not determined.
Protein Metal Salt
Specific activity
(UÆmg
)1
)
Relative
activity
a
(%) K
m
(lM) V
max
(UÆmg
)1
)

2
and 50 mM KCl.
N. Jongruja et al. Role of HBD from T. maritima RNase HI
FEBS Journal 277 (2010) 4474–4489 ª 2010 The Authors Journal compilation ª 2010 FEBS 4479
Role of HBD from T. maritima RNase HI N. Jongruja et al.
4480 FEBS Journal 277 (2010) 4474–4489 ª 2010 The Authors Journal compilation ª 2010 FEBS
The D15-R1-D13 ⁄ D29 substrate was used to con-
firm that Tma-RNase HI and Tma-CD do not cleave
the DNA-RNA-DNA ⁄ DNA substrate containing a
single ribonucleotide. This substrate is not cleaved by
type 1 RNases H but is cleaved by type 2 RNases H,
except for bacterial RNases HIII, at the DNA-RNA
junction [21,27,32]. As expected, this substrate was not
cleaved with Tma-RNase HI, Sto-RNase HI and Eco-
RNase HI, although it was cleaved with Tk-RNase
HII at the DNA-RNA junction (data not shown).
These results exclude the possibility that the cleavage
of the D13-R4-D12 ⁄ D29 substrate with Tma-RNase
HI at a16-a17 is caused by the contamination of a type
2 RNase H enzyme.
Cleavage of the R9-D9 ⁄ D18 substrate with various
RNase H enzymes is summarized in Fig. 6E,F. Tma-
RNase HI and Tma-CD cleaved this substrate most
preferably at g7-c8 and c8-c9, and much less preferably
at u6-g7 and c9-T10 in the presence of Mn
2+
. They
cleaved this substrate with similar site specificities in
the presence of Mg
2+

29 bp RNA ⁄ DNA hybrid (R29 ⁄ D29) were analyzed in
the absence of the metal cofactor using surface plas-
mon resonance. These proteins were injected onto the
sensor chip, on which the R29 ⁄ D29 substrate was
immobilized. The sensorgrams obtained by injecting
1 lm of these proteins are shown in Fig. 7. The disso-
ciation constants, K
D
, of the proteins for binding to
the R29 ⁄ D29 substrate, which were determined by
measuring the equilibrium-binding responses at various
concentrations of the proteins, are summarized in
Table 2. The K
D
value of Tma-ND was higher than
(although comparable to) that of Tma-RNase HI. By
Fig. 7. Binding of Tma-RNase HI and its derivatives to the sub-
strate. Sensorgrams from Biacore X showing binding of 1 l
M of
Tma-RNase HI (thick solid dark line), Tma-W22A (thin solid dark
line), Tma-CD (dashed dark line) and Tma-ND (thick solid gray line)
to the immobilized R29 ⁄ D29 substrate are shown. Injections were
performed at time zero for 60 s.
Fig. 6. Cleavage of various oligomeric substrates with various RNases H. (A, C, E) Separation of the hydrolysates by urea gel. The 5¢-end
labeled R12 ⁄ D12 (A), 5¢-end labeled D13-R4-D12 ⁄ D29 (C) and 3¢-end labeled R9-D9 ⁄ D18 (E) were hydrolyzed by the enzyme at 30 °C for
15 min and the hydrolysates were separated on a 20% polyacrylamide gel containing 7
M urea, as described in the Experimental procedures.
The concentration of the substrate was 1.0 l
M. The amount of the enzyme added to the reaction mixture (10 lL) is indicated above each
lane. The metal cofactors used to cleave these substrates with Tma-RNase HI and Tma-CD are also shown above the gel together with their

pared with one another in Fig. 8. The parameters
characterizing the thermal denaturation of these pro-
teins are summarized in Table 2. A comparison of
these parameters indicates that Tma-CD and Tma-ND
are less stable than Tma-RNase HI by 1.3 and 10.8 °C
in T
m
, respectively. These results suggest that the inter-
actions between the N- and C-terminal domains of
Tma-RNase HI do not significantly contribute to the
stabilization of its C-terminal domain but contribute
to the stabilization of its N-terminal domain. Tma-
RNase HI is thermally denatured in a single coopera-
tive fashion, probably because its N-terminal domain
is denatured immediately after its C-terminal RNase H
domain is denatured. It is noted that the DH
m
and
DS
m
values of Tma-CD are considerably higher than
those of Tma-RNase HI and Tma-ND, which are
comparable to each other. The reason why the DH
m
and DS
m
values of Tma-RNase HI increase on
removal of the N-terminal domain remains to be
clarified.
Analysis for interaction between two domains

)
Tma-RNase HI 0.16 ± 0.013 67.0 ± 0.83 – 115.9 ± 11.1 0.34 ± 0.032
Tma-W22A 3.3 ± 0.54 ND ND ND ND
Tma-CD 7.8 ± 0.47 65.7 ± 4.3 )1.3 205.7 ± 22.3 0.58 ± 0.067
Tma-ND 0.40 ± 0.083 56.2 ± 3.2 )10.8 111.7 ± 7.43 0.33 ± 0.038
a
Dissociation constant of the protein for binding to the R29 ⁄ D29 substrate was determined by measuring equilibrium-binding responses at
various concentrations of the protein using surface plasmon resonance (Biacore) as described in the Experimental procedures.
b
Parameters
characterizing thermal denaturation of the proteins were determined from the thermal denaturation curves shown in Fig. 8. The melting tem-
perature (T
m
) is temperature of the midpoint of the thermal denaturation transition. DT
m
is the difference in T
m
between the intact and trun-
cated proteins and is calculated as: T
m
(truncated) ) T
m
(intact). DH
m
and DS
m
are the enthalpy and entropy changes of unfolding at T
m
calculated by van’t Hoff analysis.
Fig. 8. Thermal denaturation curves. Thermal denaturation curves

that the HBDs of other type 1 RNases H bind to the
substrate in a manner similar to the interaction of the
HBD of Hsa-RNase H1. The mutation of Trp43 to
Ala greatly reduces both the substrate binding affinities
and enzymatic activities of Hsa-RNase H1 [22,26] and
mouse RNase H1 [24]. To examine whether the corre-
sponding tryptophan residue (Trp22) is important for
substrate binding and enzymatic activity of Tma-
RNase HI, the mutant protein, Tma-W22A, was con-
structed, overproduced and purified. The production
level and purification yield of Tma-W22A were compa-
rable to those of Tma-RNase HI. The far- and near-
UV CD spectra of Tma-W22A are similar to those of
Tma-RNase HI (Fig. 3), suggesting that the mutation
at Trp22 does not significantly affect the structure of
Tma-RNase HI.
The pH, salt and metal ion dependencies of Tma-
W22A were similar to those of Tma-RNase HI (data
not shown). Its maximal Mn
2+
-dependent activity was
also similar to that of Tma-RNase HI (Table 1). How-
ever, its maximal Mg
2+
-dependent activity was lower
than that of Tma-RNase HI by 7.5-fold (Table 1),
indicating that the mutation of Trp22 to Ala con-
siderably reduces the Mg
2+
-dependent activity of

However, on removal of the HBD, the K
m
value of
Tma-RNase HI increases by 5–7-fold, whereas its K
D
value increases by 49-fold. Because the K
m
and K
D
val-
ues are determined in the presence and absence of the
metal cofactor, these results suggest that the difference
in substrate binding affinity between Tma-RNase HI
and Tma-CD determined in the presence of the metal
cofactor is smaller than that determined in its absence.
Presumably, the HBD governs binding of Tma-RNase
HI to the substrate and its substrate binding affinity is
not significantly changed either in the presence or
absence of the metal cofactor. By contrast, the sub-
strate binding affinity of the RNase H domain proba-
bly increases in the presence of the metal cofactor
compared to that in its absence. The cleavage-site spec-
ificity of Tma-RNase HI is not significantly changed
on removal of the HBD, probably because the HBD
of Tma-RNase HI facilitates initial nonsite-specific
interactions with the substrate and promotes the site-
specific interactions between the RNase H domain of
Tma-RNase HI and substrate.
Importance of HBD for Mg
2+

strate binding, the HBD may affect the interaction
between the enzyme and substrate at the active site.
Because not only the active-site residues, but also the
substrate provide ligands for coordination of the
metal ion [14], removal of the HBD or mutation at
the HBD may alter the interaction between the sub-
strate and metal ion, such that the scissile phosphate
group of the substrate and the Mg
2+
ion are
arranged ideally. The effect of this alteration on the
coordination of the metal ion varies for Mg
2+
and
Mn
2+
, probably because Mn
2+
is a transition metal
having coordinates with a different geometry than
Mg
2+
.
Similar results have been reported for Eco-RNase
HI [34], RNase H of Moloney murine leukemia virus
reverse transcriptase (MMLV RNase H) [35] and
Bacillus stearothermophilus RNase HIII (Bst-RNase
HIII) [36]. Eco-RNase HI and Bst-RNase HIII prefer
Mg
2+

2+
. For example, Eco-RNase HI [37] and HIV-1
RNase H [38] specifically loses Mg
2+
-dependent activ-
ity by the mutation of the active-site residue (Glu48 or
Asp134 for Eco-RNase HI and Glu478 for HIV-1
RNase H). Eco-RNase HI specifically loses the Mg
2+
-
dependent activity by deletion of the last helix [39]. In
all cases, the conformation of the metal binding site is
probably slightly changed, so that it becomes unfavor-
able for binding of Mg
2+
but is kept favorable for
binding of Mn
2+
.
The finding that the D13-R4-D12 ⁄ D29 and R9-
D9 ⁄ D18 substrates cannot be effectively cleaved by
Tma-RNase HI in the presence of Mg
2+
suggests
that the RNA ⁄ DNA hybrid region in these substrates
is too short to accommodate both the HBD and the
RNase H domain. According to the crystal structure
of the catalytic domain of human RNase H1 in com-
plex with the substrate [15], the enzyme interacts with
several RNA residues preceding the scissile bond. The

greatly differ in Mg
2+
-dependent activity, the Mg
2+
-
dependent activity of Tma-RNase HI may be responsi-
ble for its RNase H activity in vivo. It has been
reported that E. coli RNase HII (Eco-RNase HII) [40]
and Bacillus subtilis RNase HII (Bsu-RNase HII) [41],
which prefer Mn
2+
to Mg
2+
for activity, complement
the temperature-sensitive growth phenotype of E. coli
MIC3001. Both proteins exhibit the highest Mn
2+
-
dependent activities ( 0.5 UÆmg
)1
) in the presence of
10 mm MnCl
2
[31], which are comparable to that of
Tma-CD (0.33 UÆmg
)1
at 1 mm MnCl
2
) (Table 1).
Eco-RNase HII and Bsu-RNase HII exhibit the high-

optimum one (Fig. 4). The surface plasmon resonance
analyses for binding to the R29 ⁄ D29 substrate indicate
that the activities of these proteins greatly decrease at
high salt concentrations because the binding affinities
of these proteins to the substrate greatly decrease.
However, the highest activities of Tma-RNase HI and
Tma-CD are observed at 50 and 10 mm KCl, respec-
tively, indicating that Tma-RNase HI requires higher
concentrations of KCl for maximal activity than does
Tma-CD. It has been reported that Hsa-RNase H1
requires a physiological salt concentration (125 mm
NaCl) for maximal activity to overcome the nonspe-
cific charge–charge interactions between HBD and
nucleic acids [22]. Because the basic residues that con-
tribute to these interactions are well conserved in the
HBD of Tma-RNase HI, Tma-RNase HI probably
requires 50 mm KCl for maximal activity to overcome
these nonspecific interactions.
Experimental procedures
Cells and plasmids
E. coli MIC3001 [F
)
, supE44, supF58, lacY1 or D(lacIZY)6,
trpR55, galK2, galT22, metB1, hsdR14(r
K
)
m
K
+
), rnhA339::

EcoRI, and ligated into the NdeI-HindIII or NdeI-EcoRI
sites of pET25b.
The pET25b derivative for overproduction of Tma-
W22A was constructed by site-directed mutagenesis using
PCR, as described previously [42]. The pET25b derivative
for overproduction of Tma-RNase HI was used as a tem-
plate. The mutagenic primers were designed such that the
codon for Trp22 (TGG) is changed to GCG for Ala. The
resultant DNA fragment was digested with NdeI and Hin-
dIII, and ligated into the NdeI-HindIII sites of pET25b.
All DNA oligomers for PCR were synthesized by Hokka-
ido System Science (Sapporo, Japan). PCR was performed
in 25 cycles using a thermal cycler (Gene Amp PCR System
2400; Applied Biosystems, Tokyo, Japan) and KOD DNA
polymerase (Toyobo Co. Ltd, Kyoto, Japan). The DNA
sequences of the genes encoding all proteins described
above were confirmed by ABI Prism 310 DNA sequencer
(Applied Biosystems).
Overproduction and purification
For overproduction of Tma-RNase HI, Tma-CD, Tma-ND
and Tma-W22A, the E. coli MIC3001(DE3) transformants
with the pET25b derivatives were grown at 30 °C. When
A
600
reached a value of approximately 0.5, 1 mm IPTG was
added to the culture medium and cultivation was continued
at 30 °C for an additional 30 min. Then, the temperature
of the growth medium was shifted to 25 °C and cultivation
was continued at 25 °C for an additional 16 h. The subse-
quent purification procedures were carried out at 4 °C.

Eco-RNase HI, 0.97 for Sto-RNase HI and 0.56 for
Tk-RNase HII. These values were calculated by using
absorption coefficients of 1576 m
)1
Æcm
)1
for Tyr and
5225 m
)1
Æcm
)1
for Trp at 280 nm [44].
Gel filtration chromatography
For estimation of the molecular masses of proteins, the
proteins were applied to a HiLoad 16 ⁄ 60 Superdex 200pg
column (GE Healthcare) equilibrated with 10 mm
Tris ⁄ HCl (pH 8.0) containing 10 mm dithiothreitol and
0.2 m NaCl. Thyroglobulin (670 kDa), bovine gamma
globulin (158 kDa), chicken ovalbumin (44 kDa), horse
myoglobin (17 kDa) and vitamin B
12
(1.35 kDa) were used
as markers.
Enzymatic activity
The RNase H activity was determined by measuring the
amount of the acid-soluble digestion product from the sub-
strate,
3
H-labeled M13 DNA ⁄ RNA hybrid, accumulated
upon incubation at 30 °C for 15 min, as described previ-

were prepared by hybridizing 1 lm of the 5¢-FAM-labeled
12 base RNA (5¢-cggagaugacgg-3¢), 29 base DNA
13
-RNA
4
-
DNA
12
(5¢-AATAGAGAAAAAGaaaaAAGATGGCAA
AG-3¢), 29 base DNA
15
-RNA
1
-DNA
13
(5¢-AATAGAGAA
AAAGAAaAAAGATGGCAAAG-3¢) and 3¢-FAM-labeled
18 base RNA
9
-DNA
9
(5¢-uugcaugccTGCAGGTCG-3¢) with
a 1.5 molar equivalent of the complementary DNA, as
described previously [41] (in these sequences, DNA and
RNA are represented by capital and lowercase letters,
respectively; FAM represents 6-carboxyfluorescein). All
oligonucleotides were synthesized by Hokkaido System
Science. Hydrolysis of the substrate at 30 °C for 15 min
and separation of the products on a 20% polyacrylamide
gel containing 7 m urea were carried out as described previ-

protein concentration was approximately 0.1 mgÆmL
)1
and
a cell with an optical path length of 2 mm was used. For
measurement of the near-UV CD spectra (250–320 mm),
the protein concentration was approximately 1.0 mgÆmL
)1
and a cell with an optical path length of 10 mm was used.
The mean residue ellipticity, h, which has the units of degÆc-
m
)2
Ædmol
)1
, was calculated by using an average amino acid
molecular weight of 110.
Binding analysis to substrate
Binding of proteins to the substrate was analyzed using
the Biacore X instrument (Biacore, Uppsala, Sweden).
R29 ⁄ D29 with the same sequence as that of D13-R4-
D12 ⁄ D29 or D15-R1-D13 ⁄ D29 was prepared so that the
RNA strand was biotinylated at the 5¢-end. The biotinylat-
ed oligonucleotide was synthesized by Hokkaido System
Science. The substrate was immobilized on the SA sensor
chip (Biacore), on which streptavidin is covalently linked,
by injecting 10 lL of NaCl ⁄ Tris buffer (10 mm Tris ⁄ HCl,
1mm EDTA, 50 mm NaCl, 1 mm b-Me, 0.005% Tween
P20, pH 9.0) containing 100 nm of biotinylated R29 ⁄ D29,
as described previously [47]. The proteins were dissolved
in NaCl ⁄ Tris buffer and injected at 25 °C at a flow rate
of 10 lLÆmin

)1
. The thermal denaturation of these proteins
was fully reversible under this condition. The temperature of
the midpoint of the transition, T
m
, was calculated from
curve fitting of the resultant CD values versus temperature
data on the basis of a least squares analysis. The enthalpy
(DH
m
) and entropy (DS
m
) changes for thermal denaturation
at T
m
were calculated by van’t Hoff analysis.
Acknowledgements
This work was supported in part by a Grant
(21380065) from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan, and by an
Industrial Technology Research Grant Program from
the New Energy and Industrial Technology Develop-
ment Organization (NEDO) of Japan.
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