ATPase activity of RecD is essential for growth of the
Antarctic Pseudomonas syringae Lz4W at low temperature
Ajit K. Satapathy, Theetha L. Pavankumar, Sumana Bhattacharjya, Rajan Sankaranarayanan and
Malay K. Ray
Centre for Cellular and Molecular Biology, Hyderabad, India
RecD is a 5¢fi3¢ helicase motor protein. The primary
sequence contains the characteristic seven conserved
motifs (I, Ia, II, III, IV, V, and VI) of the superfam-
ily 1 (SF1) group of DNA helicases [1] (Fig. 1). In Esc-
herichia coli, RecD displays ssDNA-dependent ATPase
and helicase activity in vitro [2,3]. In vivo, it functions
as a component of the RecBCD complex (also known
as exonuclease V) that is involved in DNA repair and
recombination in many bacteria [4]. RecBCD is a
highly processive helicase⁄ nuclease enzyme with dual
motor activity, in which RecB and RecD subunits,
with their respective (3¢fi5¢) and (5¢fi3¢) polar
movement, translocate the enzyme along the anti-par-
allel strands of dsDNA. DNA unwinding by helicase
activity is accompanied by degradation of the strands
until the enzyme encounters the recombination hotspot
v (chi) sequence (5¢-GCTGGTGG-3¢). This changes
the nuclease property of the enzyme, leading to the
generation of 3¢-extended ssDNA and loading of RecA
onto the DNA for homologous pairing and DNA
strand exchange, producing recombination inter-
mediates [5]. Interestingly, however, RecBC alone is
proficient for recombination and repair of DNA, and
recD-inactivated mutants of E. coli do not show any
growth defects [6,7]. Thus, the contribution of the
RecD subunit is thought to be of less significance

in vivo. Remarkably, RecD inactivation leads to the
loss of exonuclease V activity in cells, despite the
fact that the only nuclease catalytic center of
RecBCD complex lies in the RecB subunit [8]. Hence,
a role for RecD in regulating the nuclease activity of
RecBCD has been advocated. Recently, using ATP
Keywords
cold adaptation; Pseudomonas syringae;
RecBCD enzyme; RecD ATPase; RecD
helicase
Correspondence
M. K. Ray, Centre for Cellular and Molecular
Biology, Uppal Road, Hyderabad 500007,
India
Fax: +91 40 2716 0591
Tel: +91 40 2719 2512
E-mail:
(Received 10 October 2007, revised 12
February 2008, accepted 18 February 2008)
doi:10.1111/j.1742-4658.2008.06342.x
RecD is essential for growth at low temperature in the Antarctic psychro-
trophic bacterium Pseudomonas syringae Lz4W. To examine the essential
nature of its activity, we analyzed wild-type and mutant RecD proteins
with substitutions of important residues in each of the seven conserved
helicase motifs. The wild-type RecD displayed DNA-dependent ATPase
and helicase activity in vitro, with the ability to unwind short DNA
duplexes containing only 5¢ overhangs or forked ends. Five of the mutant
proteins, K229Q (in motif I), D323N and E324Q (in motif II), Q354E (in
motif III) and R660A (in motif VI) completely lost both ATPase and heli-
case activities. Three other mutants, T259A in motif Ia, R419A in motif IV

and E633Q in motif V exhibited various degrees of reduction in ATPase
activity, but had no helicase activity. While all RecD proteins had DNA-
binding activity, the mutants of motifs IV and V displayed reduced bind-
ing, and the motif II mutant showed a higher degree of binding to ssDNA.
Significantly, only RecD variants with in vitro ATPase activity could
complement the cold-sensitive growth of a recD-inactivated strain of
P. syringae at 4 °C. These results suggest that the requirement for RecD
at lower temperatures lies in its ATP-hydrolyzing activity.
Abbreviations
ABM, Antarctic bacterial medium; ATPc-S, adenosine 5¢-O-(thiophosphate); EMSA, electrophoretic gel mobility shift assay;
SF1, superfamily 1.
FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS 1835
A
B
C
3′
5′
Fig. 1. Schematic representation of P. syringae RecD. (A) Location of the seven conserved helicase motifs on linear RecD (shown as a hori-
zontal bar) are indicated by shaded boxes, except motifs I and II, known as Walker motifs A and B, which are shown in black. The amino
acid substitutions (in single-letter code) that were introduced into the helicase motifs are shown above the bar, with the position number of
the residues between the wild-type and mutated amino acids. The location of the H386D mutation between motifs III and IV is also indi-
cated. (B) Alignment of the amino acids of the seven helicase motifs of RecD from P. syringae (Ps) and E. coli (Ec) and other well-studied
members of DNA helicases belonging to SF1 (Rep of E. coli, PcrA of Bacillus stearothermophilus (Bs) and UvrD of E. coli), indicating the
conserved nature of the residues. The mutated residues of P. syringae RecD are underlined. Asterisks indicate amino acids that are identical
to the residue in P. syringae RecD. (C) Ribbon diagram of the structural model of P. syringae RecD. The model was built by homology mod-
eling using the coordinates of the E. coli RecD (D-chain of the RecBCD complex, Protein Data Bank code 1W36). The three domains of
RecD, and the residues that were mutated in the seven conserved motifs, in addition to residue H386, are indicated. The arrowheads mark
the positions of the putative insertion sequences in P. syringae RecD, which are absent from E. coli RecD. The 5¢-end of the DNA substrate
has also been shown schematically to indicate the relative positions of domains 2 and 3 of RecD as seen in the structure of the DNA-bound
RecBCD complex of E. coli.

RecD helicase motif mutants A. K. Satapathy et al.
1836 FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS
hydrolysis-defective mutants of the helicase motif I in
RecD (RecD
K177Q
) and RecB (RecB
K29Q
)ofE. coli,it
has been concluded that there are subtle differences
between the properties of RecBC, RecBCD
K177Q
and
RecB
K29Q
CD enzymes, and that the RecB motor is
absolutely required for v recognition and RecA load-
ing, while the RecD subunit is dispensable for motor
activity of the complex [9].
Psychrophilic and psychrotrophic bacteria from
Antarctica have evolved various novel adaptive features
that allow them to survive and grow at a very low
temperature [10–14]. A molecular understanding of
these features would be important to our knowledge
regarding low-temperature-adapted biology. We previ-
ously discovered that recD is essential for growth of the
Antarctic bacterium Pseudomonas syringae Lz4W at
low temperature [15]. The peizophilic bacterium Photo-
bacterium profundum also required RecD function
during growth under high pressure [16]. These two
studies suggested that the RecD protein might be

required for growth of bacteria under stress conditions,
as E. coli does not show any growth defect due to recD
inactivation. In addition, we observed that the recD-
inactivated cold-sensitive P. syringae mutants accumu-
late DNA fragments in cells grown at 4 °C but not at
22 °C [15]. Concurrently, the recD mutants were also
sensitive to DNA-damaging agents, such as UV and
mitomycin C, unlike in the case of mesophilic E. coli
[6,7]. This led us to believe that the Antarctic bacteria
are probably subjected to greater DNA damage at low
temperature, and RecD might play a direct role in
the RecBCD-dependent repair of such damage. As
P. syringae possesses genes for the RecB (recB) and
RecC (recC) subunits, we have initiated studies to
examine their role in cold adaptation. A recent genetic
study (T. L. Pavankumar and M. K. Ray, unpublished
results) indicated that the recB and recC mutants of
P. syringae also are cold-sensitive like the recD
mutants, suggesting that function of the entire RecBCD
machinery is important for growth. In the case of
E. coli, mutations in the recB or recC genes impair
homologous recombination, and the mutant cells have
reduced cell viability and reduced resistance to DNA-
damaging agents [4,6].
As a first step towards gaining an insight into why
RecD is essential in P. syringae, we have analyzed the
in vitro biochemical activities of this protein. We report
here the comparative activities of the C-terminally
hexahistidine-tagged form of wild-type RecD (RecD
His

)
and eight mutant proteins that were created by single
amino acid substitutions of important residues in each
of the seven conserved helicase motifs. RecD
His
dis-
played ATP-hydrolyzing as well as short DNA duplex
unwinding activity in vitro, but the mutations K229Q in
motif I (Walker motif A), D323N and E324Q in moti-
f II (Walker motif B), Q354E in motif III and R660A
in motif VI caused complete loss of these activities in
RecD. However, the three mutant proteins of motifs Ia
(T259A), IV (R419A) and V (E633Q) retained reduced
ATPase activity to varying degrees, but showed no
DNA-unwinding activity. In the biological activity
assay, only the wild-type and the three mutant proteins
retaining ATPase activity were able to complement
the growth defect of a recD-disrupted strain (CS1) of
P. syringae. These results suggest that RecD with mod-
est ATP-hydrolyzing activity, which does not support
DNA unwinding in vitro, is sufficient for growth of the
Antarctic P. syringae at low temperature.
Results
Selection of amino acid residues for mutational
analysis of P. syringae RecD
To dissect the biochemical activities of RecD with
regard to its requirement during growth at low temper-
ature, we used a mutational approach, assessing the
roles of conserved amino acids in various helicase
motifs of the RecD motor protein (Fig. 1). Eight of

the conserved residues (K229, T259, D323, E324,
Q354, R419A, E633Q and R660) chosen in this study
for mutational analysis are located on the seven heli-
case motifs (I, Ia, II, III, IV, V, and VI) whose roles
have been assessed in other helicases [17,18]. One other
residue, H386, which was mutated to D, is located out-
side the conserved motifs of RecD (Fig. 1), although
it was putatively identified to be on motif IV in a
previous study [19]. The incorrect identification was
primarily due to blast and clustal w alignments of
amino acid sequences of RecD proteins showing that
the E. coli RecD sequence 328QLS
RLTGT335 and
the P. syringae RecD sequence 383WLE
HVSGE390
align with the helicase motif IV sequence
284QNY
RSTKR291 of PcrA and 281QNYRSTSN288
of UvrD, respectively [19–21].Using the recent crystal
structure of RecD in the RecBCD complex obtained
from E. coli [22], we built a structural model of the
P. syringae RecD protein (Fig. 1C), which establishes
that the RecD sequences 415RHSR
RFGEG423 in
P. syringae and 356QKSYRFGSD364 in E. coli repre-
sent motif IV. The sequences are located in a structur-
ally similar region of Rep and PcrA helicases [23,24].
The H386D mutation located outside the conserved
helicase motifs nevertheless gave us an opportunity to
compare its biochemical and biological activities with

those of the wild-type and other mutated proteins.
A. K. Satapathy et al. RecD helicase motif mutants
FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS 1837
The structural model of the P. syringae RecD
(Fig. 1C) was built by homology modeling, using
E. coli RecD [22] as the template. The two proteins are
highly homologous, given that 519 C
a
atoms across the
length of the proteins could be superimposed with an
rmsd value of 1.21 A
˚
(supplementary Fig. S1). They are
also similar in their domain architectures, each contain-
ing three distinct domains (domains 1–3). Domain 2
(residues 159–417) and domain 3 (residues 418–682) of
P. syringae RecD, corresponding to homologous seg-
ments (residues 110–358 and 359–593) of E. coli RecD,
represent the motor domains 1A and 2A of other SF1
helicases [23,24]. The N-terminal domain 1 that consti-
tutes the main interface between RecD and RecC in the
RecBCD complex is a little longer in P. syringae (1–159
residues) compared to E. coli RecD (1–110 residues).
Two more extra segments of amino acids within
domain 3 of P. syringae RecD are also present (marked
by arrowheads in Fig. 1C).
Expression and purification of RecD in soluble
form
To assess its biochemical activity, P. syringae RecD
was initially expressed as a C-terminally hexahistidine-

tagged protein from the high-level expression vector
pET21D-His in E. coli, in which it formed inclusion
bodies. Therefore, the protein was subsequently
expressed in soluble form in Antarctic P. syringae
(Fig. S2) using the plasmid pRecD
His
, a derivative of
the broad host range plasmid pGL10 (see Experimen-
tal procedures). The levels of expression of the soluble
form of RecD from pRecD
His
in the recD-null mutant
of P. syringae (CS1), although much lower than the
amount expressed from pET21D-His in E. coli, were
satisfactory for purification under native conditions.
Hence, the recombinant P. syringae RecD was mainly
purified from the CS1 strain. However, purification of
His-tagged RecD on Ni
2+
-agarose by a single-step
method led to the association of a few co-contaminat-
ing proteins. Introduction of a heparin–Sepharose
chromatographic step prior to Ni
2+
-agarose chroma-
tography, as described in Experimental procedures,
eliminated such contamination. Typically, approxi-
mately 1–2 mg of His-tagged RecD protein were
purified from 500 mL of overnight cultures of
CS1(pRecD

His
) by this method. The finally purified
protein was about 99% pure, as determined by SDS–
PAGE analysis with Coomassie blue staining (supple-
mentary Fig. S1). Gel-filtration chromatography on a
Superose HR 10 ⁄ 30 column demonstrated that the
protein elutes as a discrete peak at about 76 kDa,
corresponding to the monomer form of the protein.
All eight helicase motif mutants (K229Q, T259A,
D323N, E324Q, Q354E, R419A, E633Q and R660A)
and one (H386D) outside the conserved motifs of the
P. syringae RecD protein (Fig. 1) reported in this
study were also expressed in CS1 cells. Expression
of the proteins was confirmed by western analyses
using anti-RecD and anti-His serum (supplementary
Fig. S1). Mutant proteins were purified by an identical
method to that followed for RecD
His
, with comparable
yield and purity.
ATP-hydrolyzing activity of RecD
His
and its
mutants
We first assessed the ATP-hydrolyzing activity of the
recombinant wild-type RecD protein (RecD
His
)of
P. syringae, which is an essential activity of any heli-
case motor. The RecD

His
displayed efficient ATPase
activity in the presence of ssDNA. Interestingly, RecD
also displayed significant ATPase activity in the pres-
ence of duplex DNAs with 5¢ overhangs and forked-
end substrates, but much reduced ATPase activity with
3¢ overhangs and blunt-ended DNA (Fig. 2A). No
detectable intrinsic ATPase activity was associated
with the protein.
To compare the ATP-hydrolyzing activities of the
mutant RecD proteins with those of RecD
His
, a single-
stranded 40-mer oligonucleotide was used as a stimula-
tor under identical conditions (Fig. 2B). The kinetic
parameters of ATPase activity, obtained from analysis
of RecD
His
and the various mutants, are shown in
Table 1. RecD
His
hydrolyzed ATP with a maximum
velocity (V
max
)of72lmolÆs
)1
and a K
m
of approxi-
mately 147 lm for ATP. Five of the mutant RecD pro-

teins (K229Q, D323N, E324Q, Q354E, and R660A)
had barely detectable ATPase activity. However, the
mutant proteins T259A, R419A, and E633Q exhibited
reduced ATPase activity, about 72, 13 and 7% of the
wild-type value, with K
m
values for ATP (K
m(ATP)
)
of 217, 151 and 136 lm, respectively (Table 1). By
varying the DNA concentration in the reaction, the
K
m(DNA)
values for ATPase stimulation were also
determined, and were roughly similar (27–30 nm)to
each other.
It is generally believed that the ssDNA-dependent
ATP hydrolysis activity of helicases is related to its
translocation along the DNA strand. Therefore, we
tested the V
max
of ATPase activity of RecD
His
in the
presence of DNA oligomers of various lengths, e.g. 15-
mer, 25-mer and 40-mer (supplementary Table S1). As
expected, the V
max
of the reactions increased as the
length of the oligomeric DNA chain increased

(Fig. 3A). However, there was a reduction in the
RecD helicase motif mutants A. K. Satapathy et al.
1838 FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS
K
m(DNA)
values with the increase in DNA chain length,
which might be related to the increased residence time
of the proteins on longer DNA substrates. Importantly,
the three mutated RecD proteins (T259A, R419A and
E633Q) that had reduced ssDNA-dependent ATPase
activities also showed a DNA chain-length-dependent
increase in ATP hydrolysis (Fig. 3B–D).
DNA-unwinding activity of RecD and its mutants
Four types of duplex DNA substrate (supplementary
Table S1) were used in the DNA strand unwinding
assay. RecD
His
could unwind only the 5¢ overhang
substrate (25 bp duplex DNA with a 15-base 5¢ exten-
sion) and the forked-end substrate (17 bp duplex with
an 8 bp unpaired extension) (Fig. 4A). Unwinding
activity was barely detectable in assays with a 25 bp
blunt-end DNA duplex or with the 25 bp duplex
containing a 3¢ ssDNA tail (3¢ overhang substrate).
Although the activity of P. syringae RecD was mar-
ginally better with the forked-end substrate, charac-
terization of helicase activity was subsequently carried
out using the 5¢ overhang DNA duplex substrate.
The helicase activity was found to be ATP- and
Mg

2+
-dependent, and maximum activity was
observed with 2.5 mm ATP and 2.0 mm MgCl
2
, under
our experimental conditions (data not shown). The
RecD protein could catalyze the ATPase and DNA
strand unwinding activities in the presence of Mg
2+
or Mn
2+
but not when Ca
2+
or Zn
2+
were used in
the assays. Addition of EDTA or removal of ATP
from the reaction mixture abolished the DNA strand
separation activity. The non-hydrolysable ATP ana-
logue (ATPc-S) also did not support the activity
(data not shown).
To assess the importance of conserved residues in
the helicase motifs on DNA-unwinding activity,
mutant RecD proteins were tested for their ability to
unwind the 5¢ overhang duplex DNA substrate at
25 °C. None of the eight mutants of RecD helicase
motifs (K229Q, T259A, D323N, E324Q, Q354E,
R419A, E633Q and R660A) showed any measurable
helicase activity (Fig. 4B). However, the mutant
(H386D) that had an alteration outside the conserved

helicase motifs showed approximately 76% of the
wild-type ATPase activity and approximately 80% of
the helicase activity in vitro, under identical conditions
(Tables 1 and 2).
DNA-binding activity of the mutant RecD
proteins
To further examine whether the loss or reduction in
activities of the mutant RecD proteins are due to their
inability to bind DNA, the binding activity was
assessed by an electrophoretic gel mobility shift assay
Table 1. Kinetic parameters of ssDNA-stimulated ATPase activity
of RecD. The coupled NADH oxidation method with 6.4 n
M protein
and 1 l
M 40-mer ssDNA was used to determine ATPase activity at
25 °C. The activities of the RecD mutant proteins K229Q, D323N,
E324Q and Q354E, which were very low (0.41, 1.0, 0.43, and
0.86 lmol ATP hydrolysed per lmol RecD per second, respectively)
are not listed, and were not used for calculation of the K
m
values.
RecD
V
max
(lmolÆs
)1
)
K
m(ATP)
(lM)

K
m(ssDNA)
(nM)
Wild-type (RecD
His
) 72 ± 7 147 ± 26 29 ± 2
T259A 52 ± 5 217 ± 19 30 ± 1
R419A 9.5 ± 1.7 151 ± 80 27 ± 4
E633Q 5.6 ± 0.5 136 ± 65 29 ± 3
H386D 55 ± 6 133 ± 32 25 ± 7
0 1 2 3 4 5
0
25
50
75
100
40-mer
5 ′-OV
Fork-end
3 ′-OV
Blunt-end
No DNA
ATP conc (mM)
ATPase (
μ
mol·s
–1
)
0 1 2 3 4 5
1

10
100
RecD
His
T259A
R419A
E633Q
ATP conc (mM)
ATPase (
μ
mol·s
–1
)
A

B
Fig. 2. ATPase activity of RecD and its mutants. DNA-stimulated
ATPase activity was measured spectrophotometrically by the
NADH oxidation-coupled assay method. (A) Activity of RecD
His
pro-
tein (6.6 n
M) in the presence of ssDNA (40-mer) and dsDNA with
various end structures (5¢ overhang, 3¢ overhang, blunt-end and
forked-end, as indicated in Supplementary Table S2). (B) Compari-
son of ATPase activity between RecD
His
and the three mutant
RecDs (T259A, R419A, E633Q) that displayed reduced activity.
Assays were performed in the presence of 1 l

M 40-mer ssDNA
and 6.6 n
M proteins. Error bars indicate the standard deviation
based on a minimum of three experiments.
A. K. Satapathy et al. RecD helicase motif mutants
FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS 1839
(EMSA) in the presence of
32
P-labeled DNA substrates
(Fig. 5). Wild-type RecD protein displayed stronger
binding to ssDNA than to dsDNA at both assay tem-
peratures (4 and 25 °C). From quantification of the
band intensities in EMSA (Fig. 5A), it appears that
the DNA duplexes with a 5¢ or 3¢ overhang or forked-
end substrates were preferred (binding to approxi-
mately 80–85% of the ssDNA) compared with the
Fig. 3. ssDNA length-dependent ATP-hydrolyzing activity of P. sy-
ringae RecD and its mutants. The activity of RecD
His
and three
mutant proteins (T259A, R419A and E633Q) was measured spec-
trophotometrically using 6.6 n
M protein in the presence of 1 lM
ssDNA of various lengths (15-, 25- and 40-mer). No activity was
observed in the absence of ssDNA (not shown). The curves were
obtained by nonlinear fitting of data using
GRAPHPAD PRISM software.
The data are the mean of three independent experiments.
0 1 2 3 4 5
0

25
50
75
100
40-mer
25-mer 15-mer
RecD
His
ATP conc (mM)
ATPase (
μ
mol·s
–1
)ATPase (
μ
mol·s
–1
)ATPase (
μ
mol·s
–1
)ATPase (
μ
mol·s
–1
)
0 1 2 3 4 5
0
20
40

60
T259A
ATP conc (mM)
0 1 2 3 4 5
0.0
2.5
5.0
7.5
10.0
R419A
ATP conc (mM)
0 1 2 3 4 5 6
0.0
2.5
5.0
7.5
E633
Q

ATP conc (mM)
C 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
WT
K229Q
T259A
D323N
E324Q
Q354E
R419A
E633Q
R660A

ds
ss
5 ′- Overhang
5′
3′- Overhang
3′
C1 C2 2 5 10 20 min
ds
ss
C1 C2 2 5 10 20
Blunt - end
5′
3′ 3 ′
Forked - end
5′
C1 C2 2 5 10 20
min
ds
ss
C1 C2 2 5 10 20
A
B
Fig. 4. DNA-unwinding activity of P. syringae RecD and its
mutants. (A) RecD
His
protein (100 nM) was incubated with 1 nM
32
P-labeled duplex DNA of various types (5¢ overhang, 3¢ overhang,
blunt-end and forked-end, as shown in supplementary Table S2).
Reactions were carried out at 25 °C and analyzed by EMSA on

native 15% polyacrylamide gel. Shown here are the phosphor
images of the gels. The lanes marked as C1 and C2 contained con-
trol samples with heat-denatured ssDNA and dsDNA substrates,
respectively. (B) Representative phosphor image of a gel showing
the DNA-unwinding activity of RecD
His
(WT) and mutant RecD pro-
teins (K229Q, T259A, D323N, E324Q, Q354E, R419A, E633Q and
R660A). Assays were carried out at 25 °C using the 5¢ overhang
DNA duplex substrate.
RecD helicase motif mutants A. K. Satapathy et al.
1840 FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS
blunt-end duplex DNA (approximately 60%). Signifi-
cantly, all the mutant RecD proteins retained the abil-
ity to bind the DNA substrates (Fig. 5B). The
efficiency of binding to DNA was, however, variable
among the mutant proteins. R419A and E633Q dis-
played weaker ssDNA binding activity (2- and 5-fold
less, respectively) compared to the wild-type RecD
protein (Fig. 5C). As these two mutant proteins also
showed reduced ATPase activity, the reduction might
be related to the weaker DNA binding. On the other
hand, the lack of or defective ATPase activity in the
remaining mutants, such as K229Q, T259A, D323N,
E324Q, Q354E and R660A, could not be related to
any DNA-binding defect. Surprisingly, two mutants of
Table 2. Summary of the properties of wild-type and mutant RecD proteins. Biochemical activities of the wild-type protein (RecD
His
) were
taken as 100% for evaluation of the activities of the mutated proteins. The V

max
values for ATPase activity of RecD
His
at 25 and 4 °C were
72 and 21 lmol ATPÆs
)1,
respectively, which were considered to be 100% for relative activity of the mutant proteins at the respective tem-
peratures. ND, not detectable under the experimental conditions.
Protein
Complements
cold-sensitivity of CS1?
DNA-binding
activity (%)
ATPase
activity at
25 °C (%)
ATPase
activity
at 4 °C (%)
DNA unwinding
at 25 °C (%)
Wild-type (RecD
His
) + 100 100 100 100
K229Q (motif I) ) 88 ND ND ND
T259A (motif Ia) + 84 72 62 ND
D323N (motif II) ) 77 ND ND ND
E324Q (motif II) ) 135 ND ND ND
Q354E (motif III) ) 92 ND ND ND
R419A (motif IV) + 52 13 7 ND

E633Q (motif V) + 20 8 6 ND
R660A (motif VI) ) 83 ND ND ND
H386D + 97 76 30 80
C 1 2 C 1 2 C 1 2 C 1 2 C 1 2
SS 5′-OV 3′- OV Fork Blunt
DNA
Complex
Free DNA
0
50
100
150
200
250
300
350
a b a b a b a b a b a b a b a b a b
Relative binding (%)
WT
K229Q
T259A
E324Q
D323N
Q354E
R419A
E633Q
R660A
WT
K229Q
T259A

DNA
complex
E324Q
D323N
Q354E
R419A
E633Q
R660A
A
B C
Fig. 5. DNA-binding activity of the wild-type and mutant RecD proteins. (A) Binding activity of RecD
His
. Single-stranded and double-stranded
oligonucleotides with various end structures [5¢ overhang (5¢-OV), 3¢ overhang (3¢-OV), blunt-end and forked-end] were analyzed by EMSA on
8% polyacrylamide gel. Lanes marked ‘C’ contained
32
P-end-labeled DNA substrates (2.5 nM) alone, and lanes marked 1 and 2 contained
labeled DNAs and RecD
His
protein (250 and 500 ng, respectively). (B) Relative ssDNA binding activity of RecD
His
and mutant RecD proteins.
Binding assays were performed with
32
P-labeled 25-mer single-stranded oligonucleotides (2.5 nM) and 500 ng of RecD proteins, and analyzed
by EMSA as in (A). (C) Histogram showing the relative binding activity of various RecD proteins to ssDNA at 4 °C (bar ‘a’) and 25 °C (bar ‘b’).
Binding values were obtained by quantifying the band intensities on gel phosphor images. Error bars represent the standard deviation of the
values obtained from three independent experiments. The ssDNA binding activity of 500 ng RecD
His
protein was considered as 100% for the

calculations.
A. K. Satapathy et al. RecD helicase motif mutants
FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS 1841
motif II, especially E324Q, displayed a consistently
higher degree of DNA-binding activity (> 2.5-fold)
compared to RecD
His
under identical conditions
(Fig. 5C). These residues in motif II are known to
interact with ATP and Mg
2+
for hydrolysis of the
nucleotide substrate.
Genetic complementation of the cold-sensitive
phenotype of CS1 by RecD
His
and its mutants
We reported previously that the defect in the P. syrin-
gae recD mutant CS1, which does not grow at low
temperature (4 °C), is complemented by the wild-type
recD gene in trans [15]. We have tested the ability of
the recombinant RecD
His
protein to support the
growth of CS1 at 4 °C, by expressing it from the
pRecD
His
plasmid. As expected, CS1, expressing
RecD
His

, grew efficiently at 4 °C, both in ABM liquid
culture and on ABM agar plates. In contrast, CS1 with
the empty plasmid pGL10 failed to grow in the med-
ium at 4 °C. When the eight mutant RecD proteins
were tested in the trans complementation assay, five
(K229Q, D323N, E324Q, Q354E and R660A) failed to
support growth of CS1 at low temperature. Only the
three mutant proteins (T259A, R419A and E633Q)
that displayed ATPase activity in vitro could comple-
ment the low-temperature-sensitive growth of the CS1
strain (Fig. 6). The generation times (9–11 h) of the
complemented strains expressing the three mutant
proteins were roughly similar to that of the RecD
His
-
complemented strain (approximately 8.5 h). It is
important to note that the levels of expression of all
RecD proteins in CS1 were observed to be similar by
western analysis (supplementary Fig. S2). This rules
out quantitative differences as an explanation for the
observed difference in biological activities of the pro-
teins.
In vitro activities of RecD at various temperatures
Mutant RecD proteins (T259A, R419A and E633Q)
that displayed ATP hydrolysis activity but no DNA-
unwinding ability in vitro were able to support growth
of the cold-sensitive, recD-disrupted strain (CS1) of
P. syringae at 4 °C. This raises some interesting ques-
tions about the contribution of these two enzymatic
activities to RecD function during growth at low

temperature. While it is impossible to directly assess
these enzymatic activities of RecD in vivo, the relative
in vitro activities of the two enzyme reactions at low
temperature (4 °C) could be compared between the
proteins to detect any correlation and ⁄ or their relative
importance during growth. Towards this goal, we
measured the ssDNA-dependent ATPase activity of
wild-type RecD
His
at various temperatures, using an
enzyme-coupled NADH-oxidation assay. The initial
rate of ssDNA-induced ATP-hydrolyzing activity of
RecD
His
was highest at 37 °C, as seen for many other
enzymes from the bacterium [10,25,26]. However,
ATPase activity dropped sharply to about 2% of the
activity at 25 °C, and could not be measured below
7 °C by this method (data not shown). To circumvent
Empty
plasmid
Empty
plasmid
RecD
HIs
RecD
HIs
K229Q
T259A
D323N

D323N
E324Q E324Q
Q354E
Q354E
R419A
R419A
E633Q
E633Q
R660A
R660A
22 ºC
4 ºC
Empty
plasmid
Empty
plasmid
RecD
HIs
RecD
HIs
K229Q
K229Q
T259A
T259A
D323N
D323N
E324Q E324Q
Q354E
Q354E
R419A

R419A
E633Q
E633Q
R660A
R660A
Fig. 6. Complementation of cold-sensitive growth of CS1 by wild-type and mutant RecD proteins. The recD-inactivated mutant of P. syrin-
gae (CS1) was transformed using empty plasmid pGL10, or pGL10-derived constructs expressing RecD
His
(WT) or mutated RecD proteins
(K229Q, T259A, D323N, E324Q, Q354E, R419A, E633Q and R660A). Growth of the resultant strains was determined at 22 and 4 °Con
ABM agar plates. Only wild-type and mutant proteins T259A, R419A and E633Q could complement the growth defect of CS1 at 4 °C.
RecD helicase motif mutants A. K. Satapathy et al.
1842 FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS
this problem, we employed a TLC method to measure
ATP hydrolysis activity using [c-
32
P]ATP as the sub-
strate (Fig. 7A). This method was also suitable for
measuring ssDNA-dependent ATPase activity under
identical buffer and salt conditions to those employed
in the DNA-unwinding assays in vitro. Results from
TLC assays also demonstrated that wild-type RecD
His
has the highest activity at 37 °C (data not shown), but,
more importantly, that RecD displayed about 30% of
the 25 °C activity even at a lower temperature (4 °C)
in vitro. Like the wild-type, the mutant RecD proteins
(T259A, R419A and E633Q) also hydrolyzed ATP
efficiently at 4 °C, at about 25–35% of their 25 °C
activity (Fig. 7B).

We then examined the helicase activity of P. syrin-
gae RecD
His
in vitro at various temperatures, using
identical buffer conditions to the TLC-based ATPase
assay method. Again, maximum DNA unwinding was
observed at 37 °C, and was about 10-fold higher than
that at 25 °C (Fig. 7C). However, at 4 °C, RecD
His
(100 nm) failed to show any detectable DNA-unwind-
ing activity. When the amount of RecD
His
was
increased up to 800 nm and the reaction time to
30 min, the protein could unwind DNA duplex at only
0.8–1% of the 25 °C activity (data not shown). This is
surprising, considering the fact that the protein dis-
played approximately 30% ATPase activity at 4 °C.
This suggests that the DNA strand separation assay
in vitro probably underestimates RecD helicase activity
at lower temperatures to a considerable extent. None-
theless, the method is robust enough to measure heli-
case activity at higher temperatures (25–37 °C), and is
sufficient to establish that the T259A, R419A and
E633Q mutants lack helicase activity, at least under
these in vitro conditions.
With regard to the DNA-binding activity of the
RecD proteins at various temperatures, it appears that,
by and large, the in vitro assay temperatures (4 and
25 °C) do not affect the binding (Fig. 5C). Table 2

summarizes the key biochemical and biological activi-
ties of the wild-type and mutated RecD proteins
obtained in this study.
Discussion
Our results establish that recombinantly produced
P. syringae RecD has ssDNA-dependent ATPase and
5¢fi3¢ helicase activity, like that of the mesophilic
E. coli RecD [2,3]. However, the V
max
(72 lmolÆs
)1
)
for the ATP-hydrolyzing activity of P. syringae RecD
is much higher than the reported value (5 lmolÆs
)1
) for
mesophilic E. coli RecD at 25 °C [3]. The K
m(DNA)
(29 nm) for the P. syringae RecD towards ss-DNA, for
stimulation of ATPase activity, is lower than the
reported value (9 lm) for E. coli RecD. The higher
activity of RecD from P. syringae could be due either
to its inherent efficient activity or due to its isolation
in native soluble form, unlike the insoluble form of
E. coli RecD that required unfolding and refolding in
order to recover the active protein [3]. It is perhaps
important to point out here that the K
m(DNA)
values
for RecD obtained from the ATPase stimulation

0 10 20 30
0
25
50
75
100
37 ºC
25 ºC
4 ºC
Incubation time (min)
% unwound
C
32
Pi
32
P ATP
25 ºC
4 ºC
WT
K229Q
T259A
E324Q
D323N
Q354E
R419A
E633Q
R660A
C
WT
K229Q

T259A
E324Q
D323N
Q354E
R419A
E633Q
R660A
WT T259A R419A E633Q
1
10
100
25 °C
4 °C
ATPase activity (μmol·s
–1
)
A
BC
Fig. 7. Activity of P. syringae RecD protein
at various temperatures. ATPase assays
were performed by a TLC method at various
temperatures in the presence of 16.6 n
M
RecD
His
,1lM 40-mer ssDNA and 100 lM
[c-
32
P]ATP. (A) Representative phosphor
images of the TLC plates from the 25 and

4 °C assays. (B) Histogram showing the rel-
ative ATPase activities of RecD
His
(WT) and
three mutant proteins (T259A, R419A and
E633Q) at 4 and 25 °C. Error bars represent
the standard deviation of the values based
on three experiments. (C) Relative DNA-
unwinding activity of RecD
His
at various
temperatures. Reactions were carried out
with 10 n
M RecD
His
protein on 1 nM 5¢ over-
hang duplex DNA at three temperatures (37,
25 and 4 °C) and analyzed as in Fig. 4.
A. K. Satapathy et al. RecD helicase motif mutants
FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS 1843
experiments are much lower than the apparent values
calculated from the EMSA data, for which 2.5 nm
DNA was used to bind to 66.6–333 nm of RecD. We
believe that the EMSA method depends largely on
stability of the DNA–protein complex under the elec-
trophoretic conditions used, and hence is likely to be
less sensitive than the ATPase stimulation method for
determination of the K
m(DNA)
value.

Our study also shows that wild-type RecD of
P. syringae is very active in unwinding 5¢ overhangs
and forked-end short DNA duplexes (15–25 bp)
in vitro. However, RecD fails to unwind duplexes of
> 100 bp (A. K. Satapathy & M. K. Ray, unpublished
results), suggesting that RecD, on its own, is not a
‘strong’ helicase in vitro. The helicase activities of RecD
from P. syringae and E. coli could not be compared
due to the different DNA substrates used in the studies
with E. coli [3]. Additionally, detailed analysis on the
helicase activity of E. coli RecD protein alone has not
been reported. However, the ability of P. syringae
RecD to unwind both 5¢ overhangs and forked-end
duplex DNAs is similar to the behavior of RecD
protein from the radio-resistant bacterium Deinococcus
radiodurans [27]. However, the RecD of this bacterium
belongs to RecD2 subgroup, which is present in
bacteria lacking RecBC protein homologues [28].
Mutational effects of conserved residues in the
helicase motifs of RecD
One conserved residue from each of the seven heli-
case motifs (except motif II in which two residues
were changed) has been altered in the present study
to dissect the biochemical activities of RecD. The
roles of these conserved residues have been assessed
previously in other helicases by structural and func-
tional analyses, including ATP binding and hydrolysis
(motifs I and II), ssDNA binding (motifs Ia, III, IV,
and V), and coupling of ATPase and helicase (DNA-
unwinding) activities to translocation on ssDNA

(motifs III, IV, V, and VI) [17,18]. In the context of
RecD, only the role of the conserved lysine residue
in motif I (Walker motif A) has been investigated
previously in E. coli [2,3,9]. The lysine residue in
other helicases, including PcrA and UvrD, makes
contact with the b-phosphate of ATP-Mg
2+
and
thereby plays a role in the catalytic reaction [17,18].
Consistent with these results, our data show that the
K229 residue is essential for ATP hydrolysis and
DNA-unwinding activities, and, as expected, the
K229Q mutant protein is biologically inactive with
respect to support of the growth of P. syringae at
low temperature.
Similar to K229, the residues (D323 and E324) in
motif II (Walker motif B) are also conserved in RecD.
In other helicases, these residues co-ordinate the ATP-
associated Mg
2+
ion and active water molecule for the
hydrolytic reaction, and their alteration causes reduc-
tion in the ATPase and DNA-unwinding activities
[17,18]. Consistent with this, the present study demon-
strates that D323N and E324Q mutants of P. syringae
RecD do not display ATPase and helicase activities
in vitro. However, a surprising finding here is that the
D323N and E324Q mutant proteins bind ssDNA
2.5–3.0-fold more than the wild-type RecD under iden-
tical conditions (Fig. 5). The implication is that these

residues might normally interfere with binding of DNA.
In the crystal structure of the DNA-bound RecBCD
complex of E. coli [22], the 5¢-end of the bound DNA
molecule was not in the vicinity of the DNA-binding
pocket of RecD. Therefore, it is not clear how the
invariant D and E residues of the ATP-binding pocket
would affect nucleic acid binding. Interestingly, Walker
motif A and B mutants of RuvB, a 5¢fi3¢ hexameric
helicase, were also reported to be defective in DNA
binding in addition to the ATP-binding defect [29,30].
The T259A mutation was created in RecD based on
an analysis showing that the RecD sequence
(P
TGKAAAR) from both P. syringae and E. coli is
found in similar locations in PcrA and Rep helicase,
and they include a conserved threonine residue in their
Ia motifs (64F
TNKAAR70 and 55FTNKAAR61,
respectively). The conserved threonine in PcrA and
Rep proteins was shown to interact with the phosphate
backbone of ssDNA [23,24]. Although the DNA bind-
ing role of residues in motif Ia has been corroborated
by mutational analysis of the UL9 protein (SF2 group
of helicases) of HSV-1 virus [31], our study shows that
the RecD T259A mutant protein retains ssDNA bind-
ing. However, the protein displays reduced ATPase
activity (72%) and lacks DNA-unwinding activity
in vitro. This might result from the uncoupling of ATP
hydrolysis and DNA unwinding, which has been
shown previously in the case of the RecBCD enzyme

when inter-strand cross-linked DNA duplexes or
DNA:RNA hybrids were used as substrates [32,33].
Importantly, however, T259A is active in supporting
growth of CS1 at 4 °C. Retention of the biological
activity suggests that the uncoupled ATPase activity in
the mutant protein might have other significance, as
discussed below. Only a limited number of mutational
studies have been carried out on the residues in motif
Ia from various helicases [34,35].
Two highly conserved residues, a glutamine in motif
III and an arginine in motif VI of PcrA helicase (cor-
responding to Q354 and R660 of P. syringae RecD),
RecD helicase motif mutants A. K. Satapathy et al.
1844 FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS
are in contact with the c phosphate of a bound nucleo-
tide in the crystal structure of the PcrA–DNA–
adenylylimidodiphosphate ternary complex [24]. Other
residues in the two motifs are also involved in inter-
action between the two motifs, and with the residues
of motif II [17]. In fact, motif III is thought to act as
an interface between the ATP-binding and ssDNA-
binding pockets for coupling ATP hydrolysis to DNA-
unwinding activities. In the present study, we found
that Q354E and R660A mutations of RecD do not
affect DNA-binding activity, but lead to a complete
loss of both ssDNA-dependent ATPase and helicase
activities. The mutant proteins also failed to comple-
ment the cold-sensitive phenotype of the recD-inacti-
vated CS1 strain. These results are largely similar to
observations with the E. coli UvrD helicase, where the

corresponding glutamine and arginine residue muta-
tions (Q251E and R605A) were efficient in binding to
ssDNA, but not in the ssDNA-dependent stimulation
of ATPase activity, and the mutant proteins failed
to complement the DNA repair function of the
uvrD-inactivated strain [36,37].
In contrast, mutational analysis of two other con-
served residues, an arginine (R419) and a glutamic acid
(E633) of P. syringae RecD (corresponding to the
conserved R and E residues in helicase motifs IV and
V, respectively) led to an interesting insight into the
properties of RecD protein. Structurally, residues in
the motif IV, as shown with PcrA helicase, form a
bridge connecting the two large domains of the protein
at the bottom of the nucleotide-binding pocket [24].
Corroborating this finding, when the conserved argi-
nine residue at 284 position of motif IV in E. coli
UvrD helicase was changed to alanine, the mutant
protein exhibited a highly increased K
m
value for ATP
but normal DNA binding. The defect in ATP binding
also resulted in a complete loss of ATPase as well as
helicase activity, and the mutant protein failed to com-
plement the uvrD function in vivo [20]. Therefore, it is
significant that mutant R419A, with mutation of the
equivalent arginine residue to alanine in P. syringae
RecD, could complement the cold-sensitive growth
of CS1 strain. The mutated RecD R419A exhibited
reduced ATPase activity (only approximately 13% of

the wild-type activity) in vitro, but without any effect
on the K
m
for ATP. The R419A protein retained
almost 50% of the DNA-binding activity, but was
devoid of any DNA helicase activity in vitro. Similarly,
the RecD E633Q mutant protein displayed lower
DNA-binding activity (nearly 3-fold less), reduced
ssDNA-dependent ATPase activity (approximately 8%
of the wild-type) and a total lack of DNA helicase
activity. This is consistent with the known role of the
residues in this motif in other helicases, including PcrA
and Rep proteins. Therefore, it is remarkable that this
mutant RecD with lower ATPase activity was able to
complement the cold-sensitive phenotype of CS1 at
4 °C. While the in vivo role of the residual ATPase
activity in R419A and E633Q proteins is not known,
we observed that this activity is enhanced with the
increased length of the oligonucleotides in vitro
(Fig. 3), suggesting that the mutant protein has
retained the translocation activity along ssDNA. This
property might be critical for its in vivo role, most pos-
sibly in RecBCD-dependent DNA repair, during
growth at low temperature.
Relevance of the in vitro activities of RecD for
in vivo function
The present study provides a detailed analysis of the
effects of point mutations in all seven helicase motifs
on the biochemical activities of isolated RecD protein,
which is a (5¢fi3¢) helicase. How relevant are these

activities for the in vivo function of RecD? It is gener-
ally believed that RecD never functions alone, which is
at least true for those homologues that belong to the
RecD1 group where the protein works as a subunit of
the RecBCD complex, as opposed to proteins of the
RecD2 group, found in bacteria that do not contain
homologues of RecB and RecC [28]. The Pseudomonas
RecD belongs to the RecD1 group of proteins, and
therefore it could be argued that the in vitro activities
of the isolated protein studied here might have limited
physiological significance. However, this argument
excludes the possibility of identifying any subtle but
crucial function of the protein during adaptation of
bacteria to specific environment. Hence, dissection of
the various biochemical activities of RecD might be
relevant for its in vivo function in the protein complex.
The RecBCD complex in vivo initiates the repair of
double-strand DNA breaks (DSBs) by its coordinated
ATP-dependent helicase and nuclease activities in asso-
ciation with the regulatory v sequence and RecA
recombinase in the cell [4,38,39]. Although all the
subunits of RecBCD contribute towards the activities
of the complex, the RecD subunit in E. coli is required
for the exonuclease activity of the RecBCD enzyme,
both in vitro and in vivo [3,5]. The nuclease domain,
which resides only within the RecB subunit, is proba-
bly regulated by RecD in the complex [8]. However, it
is far from clear how RecD regulates the nuclease
domain of RecB either in the complex or during asso-
ciation with the regulatory v DNA sequence. It is only

known that the purified E. coli RecBCD
K177Q
mutant
enzyme is less processive, and has reduced ATPase
A. K. Satapathy et al. RecD helicase motif mutants
FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS 1845
activity (8–10-fold less in presence of ssDNA-binding
protein) and ATP-dependent nuclease activity (4–14-
fold less) compared to the wild-type enzyme [40,41]
but remains capable of loading RecA protein onto
v-specific ssDNA [9]. An in vivo study previously sug-
gested that the exonuclease activity imparted by RecD
is important in cellular physiology, as recJ-encoded
exonuclease could functionally complement the recD
mutants of E. coli and Salmonella sp. for survival in
the hosts [42,43]. In this context, our observations that
the recD-inactivated strains of P. syringae accumulate
DNA fragments inside the cells [15] and that recBC
null mutants are also cold-sensitive (T. L. Pavankumar
& M. K. Ray, unpublished results) are significant,
indicating a defect of the DNA degradation ⁄ repair
process in mutants. How or whether the RecD-depen-
dent helicase ⁄ nuclease activity of RecBCD complex is
related to the ATPase activity of the altered RecD
subunits (T259A, R419A or E633Q) is an important
question that needs to be addressed. Nonetheless, the
present study identifies the significance of the RecD-
associated ATPase activity required during the growth
of P. syringae at low temperature (4 °C). The very low
(but non-zero) ATPase activity of RecD (approxi-

mately 8–10% of the wild-type) is able to perform the
RecBCD-dependent function at low temperature.
Probably, in the RecBCD protein, which has dual
motors, when the RecB motor activity is intact and
sufficient for DNA strand unwinding [9], the simple
translocation of mutant RecD along ssDNA using
reduced ATPase activity is sufficient for the overall
processivity of the RecBCD enzyme, even at low tem-
perature. Alternatively, the ATP-hydrolyzing activity
of RecD, in addition to its role in providing energy for
DNA unwinding, might participate in a crucial step of
DNA degradation ⁄ processing (e.g. by regulating the
nuclease activity of RecBCD complex) during growth
at 4 °C, which perhaps occurs in the cells with the
T259A, R419A and E633Q mutant proteins.
To conclude, this study, for the first time, reports on
genetic and biochemical analyses of conserved residues
in all seven helicase motifs of RecD, and suggests that
RecD with a reduced amount of ssDNA-dependent
ATPase activity uncoupled from DNA unwinding
in vitro is sufficient for its in vivo function, allowing
growth of the Antarctic P. syringae at 4 °C.
Experimental procedures
Bacterial strains and growth conditions
The Antarctic bacterium P. syringae Lz4W and the cold-
sensitive recD mutant CS1 (recD::Tn5 tet
R
rif
R
) used in this

study have been described previously [15]. Depending on
requirements, they were grown at 22 or 4 °C (for high and
low temperatures, respectively) in Antarctic bacterial med-
ium (ABM) composed of 5 gÆL
)1
peptone and 2.5 gÆL
)1
yeast extract, or on ABM agar (1.5%). When necessary,
the ABM was supplemented with tetracycline (20 lgÆmL
)1
),
rifampicin (100 lgÆmL
)1
) or kanamycin (50 lgÆmL
)1
).
E. coli cells were grown in Luria–ertani medium [44]. For
growth analysis, bacterial cells from overnight cultures were
inoculated into fresh medium at a dilution of 1 : 100, and
the absorbance of the cultures at 600 nm (A
600
) was
measured at various time intervals.
Enzymes and reagents
Chemicals were of analytical reagent grade. Pyruvate kinase,
lactate dehydrogenase, phosphoenol pyruvate, NADH, ATP
and the ATP analogue ATP-cS were purchased from Roche
Diagnostics (Mannheim, Germany). Restriction enzymes, T4
polynucleotide kinase and other DNA-modifying enzymes
were purchased either from New England Biolabs (Ipswich,

MA, USA) or from Promega (Madison, WI, USA), unless
otherwise noted. Oligonucleotides were synthesized at the
in-house facility, or purchased from a commercial source
(Bioserve Biotechnology, Hyderabad, India).
General recombinant DNA methods
General molecular biology techniques, including isolation
of genomic DNA, cloning, Southern hybridization, PCR
etc. were performed as described by Sambrook et al. [44].
Plasmids were isolated using a plasmid isolation kit (Qia-
gen, Hilden, Germany). DNA sequencing reactions per-
formed using double-stranded plasmid DNA as templates
and the ABI PRISM dye terminator cycle sequencing
method (Perkin-Elmer, Boston, MA, USA), and analyzed
on an automated DNA sequencer (ABI model 377).
Cloning, expression and purification of
P. syringae RecD
The full-length recD gene was PCR-amplified from P. syrin-
gae genomic DNA using primers PETF and RPSD (supple-
mentary Table S2), and cloned initially into the NdeI and
HindIII sites of pET21a (Novagen, San Diego, CA, USA) to
generate pT21D-His. This construct produced RecD protein
with a C-terminal 6x His tag. The reading frame was con-
firmed by DNA sequencing, and by N-terminal amino acid
sequencing of the expressed protein. However, expression in
E. coli BL21(DE3) produced RecD in inclusion bodies, as
experienced by others for E. coli RecD [2,3]. To obtain the
native soluble form, we cloned the 6x His-RecD reading
frame from pT21D-His into the plasmid vector pGL10 [16],
which has a broad host range, to generate pRecD
His

, which
RecD helicase motif mutants A. K. Satapathy et al.
1846 FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS
was used to express the protein in a recD-null strain (CS1) of
P. syringae. The reading frame was cloned between the
XbaI ⁄ SmaI sites of pGL10. This strategy fulfilled at least
two purposes: first, RecD was purified in native soluble form,
and second, the same plasmid construct was used for genetic
complementation analysis of P. syringae recD mutants. The
expression of RecD
His
in CS1 was confirmed by western
analysis, using anti-His-tag serum (Santa Cruz Biotechnol-
ogy, Santa Cruz, CA, USA) and anti-RecD serum raised
against P. syringae RecD.
RecD purification was carried out in two steps, including
heparin–Sepharose chromatography, followed by Ni
2+
-
nitrilotriacetic acid (Qiagen) affinity chromatography.
Briefly, CS1(pRecD
His
) cells were grown at 22 °C in ABM
culture broth. Cell lysates were prepared in buffer A
(20 mm Tris ⁄ Cl pH 7.5, 2 mm dithiothreitol, 10% glycerol)
by lysozyme (1 mgÆmL
)1
) treatment on ice, followed by
gentle sonication. The cell lysate was clarified by centrifuga-
tion (10 000 g for 30 min) and then loaded onto a heparin–

Sepharose column (Amersham Biosciences, Uppsala,
Sweden). The column was thoroughly washed with buffer B
(buffer A containing 50 mm NaCl), and the matrix-bound
proteins were eluted with buffer C (buffer A containing
500 mm NaCl). These were then directly loaded onto an
Ni
2+
-nitrilotriacetic acid agarose (Qiagen) column for puri-
fication of the recombinant RecD under native conditions,
using the Qiagen protein purification protocol with a few
minor modifications. This involved washing of column with
buffer D (buffer C containing 50 mm imidazole) and elu-
tion of bound protein with buffer E (buffer C containing
300 mm imidazole). Finally, protein solution was exchanged
with storage buffer (20 mm Tris ⁄ Cl pH 7.5, 300 mm NaCl,
2mm dithiothreitol, 20% glycerol) on a PD-10 column
packed with Sephadex G-25 (Amersham Biosciences). Puri-
fied RecD was stored at )70 °C, and diluted with buffer
(20 mm Tris ⁄ Cl pH 7.5, 300 mm NaCl, 5 mm dithiothreitol,
10% glycerol, 0.1 mgÆmL
)1
BSA) before use.
Site-directed mutagenesis of helicase motifs
in RecD
His
Site-directed mutagenesis was carried out by overlap exten-
sion PCR [45] using the mutagenic oligonucleotide primers
listed in supplementary Table S2. The positions of the
mutated residues in RecD are shown in Fig. 1A. Experi-
mentally, two-step PCR reactions were carried out. Briefly,

in the first round, DNA encoding N- and C-terminal frag-
ments was amplified separately for each protein using
M13R and reverse-strand mutagenic primers (K229QR,
T259AR, D323NR, E324QR, Q354ER, H386DR,
R419AR, E633QR and R660AR) and M13F and forward-
strand mutagenic primers (K229QF, T259AF, D323NF,
E324QF, Q354EF, H386DF, R419AF, E633QF and
R660AF). M13R and M13F are flanking primers of recD
on pRecD
His
, which was used as the template for amplifica-
tion. In the second round, both N- and C-terminal frag-
ments were purified, mixed together, and overlap PCR was
performed for 10 cycles without adding any primers, fol-
lowed by 25 cycles of reactions with flanking M13R and
M13F primers to amplify the full-length genes containing
targeted changes. Amplified DNA was then digested using
PstI and EcoRI and cloned in corresponding sites of
pGL10. Mutated constructs were confirmed by DNA
sequence analysis of the inserts. All eight mutated RecD
proteins (K229Q, T259A, D323N, E324Q, Q354E, H386D,
R419A, E633Q and R660A) were expressed in the recD-null
strain (CS1) from the respective plasmids (pRecD
K229Q
,
pRecD
T259A
, pRecD
D323N
, pRecD

E324Q
, pRecD
Q354E
,
pRecD
H386D
, pRecD
R419A
, pRecD
E633Q
and pRecD
R660A
)
such that all of them contained a C-terminal 6x His tag.
Mutant proteins were purified from CS1 as described above
for the His-tagged wild-type protein (RecD
His
).
ATPase activity assay
ATPase activity of RecD was assessed by two methods.
The spectrophotometric assay was based on coupling of
the ATP hydrolysis to NADH oxidation, in a coupled
enzymatic reaction system that measures the decrease in
absorbance at 340 nm per minute [3]. Assays were carried
out at 25 °C, or other indicated temperatures, using
6.6 nm RecD proteins (increased to 100 nm for mutant
proteins) in a 100 lL reaction mixture containing 20 mm
Tris ⁄ HCl pH 7.5, 50 mm NaCl, 3 mm MgCl
2
,4mm di-

thiothreitol, 35 UÆmL
)1
pyruvate kinase, 20 UÆmL
)1
lactate
dehydrogenase, 2 mm phosphoenol pyruvate, 0.08 mgÆmL
)1
NADH, 2 mm ATP (or at the indicated concentration) in
the presence of single-stranded (15-, 18-, 20-, 25- and
40-mer) or double-stranded 25 bp DNA substrates (supple-
mentary Table S1). Activity was expressed as lmol of
ATP hydrolyzed per lmol RecD per second. Data were
fitted to the Michaelis–Menten equation using the program
graphpad prism 4.0 (Graphpad Software, San Diego, CA,
USA). In each case, V
max
(maximal rate of ATP hydroly-
sis), K
m(ATP)
(ATP concentration at the half-maximal rate
of reaction) and K
m(DNA)
(DNA concentration at the half-
maximal rate of ATP hydrolysis) for the reactions were
calculated. K
m(ATP)
was determined at a saturating concen-
tration (1 lm) of ssDNA, and K
m(DNA)
was calculated at a

saturating concentration of ATP (2 mm). The values
obtained with 40-mer ssDNA were used for comparison of
activity between the proteins.
In the second method, ATPase activity was assayed by
TLC on poly(ethyleneimine)–cellulose plates (Merck, Darm-
stadt, Germany). Assays were carried out at indicated tem-
peratures, with 25 ng of RecD protein (16.6 nm final
concentration) in a 20 lL reaction mixture containing
20 mm Tris⁄ HCl pH 7.5, 17.5 mm NaCl, 2 mm MgCl
2
,
4mm dithiothreitol, 0.1 mgÆmL
)1
BSA, 2.5 mm ATP and
the indicated concentration of ssDNA substrate (40-mer).
A. K. Satapathy et al. RecD helicase motif mutants
FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS 1847
[c-
32
P]ATP (0.1 lCi; specific activity 3000 CiÆmmol
)1
) was
used as a tracer in each reaction to measure the rate of
ATP hydrolysis. After 15 min of reaction, 0.5 lL aliquots
of the samples were spotted on TLC plates, which were
developed in a solvent containing 0.5 m formic acid and
0.5 m lithium chloride to resolve ATP and hydrolyzed P
i
.
The plates were scanned in a phosphor imager (Fuji,

Tokyo, Japan), and the amounts of
32
P
i
and [c -
32
P]ATP
were quantified using imagegauge software (Fuji). Data
were analyzed and plotted using graphpad prism 4.0
software. The assay buffer for determination of ATPase
activity by the TLC method was identical to that used for
helicase and DNA-binding assays (see below).
DNA helicase assay
DNA helicase assays were carried out by a strand-displace-
ment method. Four kinds of substrates, i.e. blunt-end,
5¢ overhang, 3¢ overhang and forked-end duplex DNAs
were prepared by annealing one
32
P-labeled oligonucleotide
with its complementary ‘cold’ oligonucleotide partner of
different length, as listed in supplementary Table S1.
32
P-labeling of the 5¢ ends of oligonucleotides was per-
formed using T4 polynucleotide kinase and [c-
32
P]ATP.
The helicase reaction mixture contained 20 mm Tris ⁄ HCl
pH 7.5, 17.5 mm NaCl, 2 mm MgCl
2
,4mm dithiothreitol,

0.1 mgÆmL
)1
BSA, 2.5 mm ATP and 1 nm labeled DNA
helicase substrate, as described previously [3]. Reactions
were initiated by adding RecD (100 nm, unless noted other-
wise), and terminated using 0.4% w ⁄ v SDS, 40 mm EDTA,
8% v ⁄ v glycerol, 0.1% w ⁄ v bromophenol blue and
50 nm‘cold’ oligonucleotides (same strand as the labeled
one) to prevent re-annealing of unwound labeled oligonu-
cleotides. ss- and dsDNA were separated by electrophoresis
on a native 15% polyacrylamide gel. Gels were scanned
using a phosphor imager (Fuji). Band intensities corre-
sponding to ss- and dsDNA were quantified. The percent-
age of unwound DNA [100· ssDNA ⁄ (ssDNA + dsDNA)]
was calculated, and the values were plotted using graphpad
prism software. To determine temperature-dependent activ-
ity, assays were performed using 5¢ overhang duplex DNA
substrates only.
DNA-binding assay
The DNA-binding activity of RecD proteins was measured
by EMSA, using single-stranded (25-mer) and double-
stranded DNA (blunt-end, 5¢ overhang, 3¢ overhang and
forked-end) substrates. Binding assays were carried out at 4
and 25 °Cin20lL reaction mixtures containing 20 mm
Tris ⁄ HCl pH 7.5, 17.5 mm NaCl, 2 mm MgCl
2
,4mm dith-
iothreitol, 0.1 mgÆmL
)1
BSA, 2.5 nm

32
P-labeled DNA sub-
strate and the indicated concentration of RecD protein.
Samples were analyzed on 6–8% native polyacrylamide gels
for separation of the DNA–protein complex from unbound
free DNA. Gels were run for 60 min at 10 VÆcm
)1
in a cold
room (4 °C). Gels were scanned and analyzed using a phos-
phor imager (Fuji).
Genetic complementation studies
For genetic complementation analysis of the cold-sensitive
phenotype of the P. syringae CS1 strain (recD::Tn5 tet
R
),
His-tagged RecD-producing constructs (see above) in the
broad host-range vector pGL10 (kan
R
) were mobilized
into CS1 by conjugation. Briefly, E. coli S17-1 (tet
S
) was
first transformed with the plasmid constructs, and the
transformants were then conjugated with CS1 by
bi-parental mating [46]. Following 48 h of incubation at
22 °C, the transconjugants (tet
R
kan
R
) were selected on

ABM agar plates containing appropriate antibiotics.
Positive clones were further confirmed by colony PCR,
and western analysis using anti-His-tag and anti-RecD
serum. The ability of the selected strains to grow at
22 °C and 4 °C was monitored, both in ABM broth and
on ABM agar plates. Generation times were calculated
from the growth curves of the complemented strains in
ABM broth.
Other biochemical methods
Proteins were quantified by the dye binding method of
Bradford [47], using BSA as the standard. SDS–PAGE and
transfer of proteins onto poly(vinylidene) difluoride (for
sequencing) and Hybond C membrane (Amersham Bio-
sciences) (for western analyses) were performed as described
previously [14]. The protein blots were probed with either
P. syringae RecD-specific polyclonal antibodies, which were
raised in rabbit against purified recombinant RecD protein
in the laboratory, or with commercially available polyclonal
anti-His-Tag serum (Santa Cruz Biotechnology). Alkaline
phosphatase-conjugated secondary antibodies were used
for chromogenic detection of the immuno-cross-reactive
proteins.
Molecular modeling of P. syringae RecD
The 3D-PSSSM protein fold recognition server [48]
predicted the structure of amino acid residues 30–694 of
P. syringae RecD to be similar to the RecD structural fold
in the E. coli RecBCD complex (Protein Data Bank code
1W36). The 3D-JIGSAW protein comparative modeling
server [49] was then used to obtain the final model of
P. syringae RecD. The model was viewed and checked

using the program O [50], and was compared with that of
the template and related structures. The program O was
also used for manual superimposition of RecD structure on
that of PcrA helicase (Protein Data Bank code 2PJR) for
comparison.
RecD helicase motif mutants A. K. Satapathy et al.
1848 FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS
Acknowledgements
We thank Drs Gerald Smith (Fred Hutchinson Cancer
Research Center, Seattle, WA, USA), Donald Cramp-
ton (Harvard Medical School, Boston, MA, USA) and
D. P. Kasbekar (Centre for Cellular and Molecular
Biology, Hyderabad, India) for reading the manuscript
and helpful suggestions. Research in M. K. R.’s labo-
ratory is supported by the Council of Scientific and
Industrial Research (CSIR), Government of India.
A. K. S. and T. L. P. gratefully acknowledge the CSIR
and the ICMR (Indian Council of Medical Research),
respectively, for research fellowships. S. B. participated
in the research as a summer trainee. R. S. is an Inter-
national Senior Research Fellow in Biomedical
Sciences of the Wellcome Trust, UK.
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Supplementary material
The following supplementary material is available for
this article online:
Fig. S1. Structural homology between P. syringae and
E. coli RecD proteins.
Fig. S2. Expression and purification of hexa-histidine-
tagged RecD and its mutant variants from P. syringae.
RecD helicase motif mutants A. K. Satapathy et al.
1850 FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS
Table S1. Oligonucleotides used in this study for stim-
ulation of ATPase and preparation of various helicase
substrates for RecD.
Table S2. Primers used in this study for cloning and
mutagenesis of P. syringae recD.
This material is available as part of the online article
from .
Please note: Blackwell publishing are not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
A. K. Satapathy et al. RecD helicase motif mutants
FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS 1851