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 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: malay@ccmb.res.in (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 psychrotrophic 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 helicase 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 DNAbinding activity, the mutants of motifs IV and V displayed reduced binding, 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 °C These results suggest that the requirement for RecD at lower temperatures lies in its ATP-hydrolyzing activity RecD is a 5¢ fi 3¢ helicase motor protein The primary sequence contains the characteristic seven conserved motifs (I, Ia, II, III, IV, V, and VI) of the superfamily (SF1) group of DNA helicases [1] (Fig 1) In Escherichia 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¢ fi 5¢) and (5¢ fi 3¢) polar movement, translocate the enzyme along the anti-parallel 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 intermediates [5] Interestingly, however, RecBC alone is proficient for recombination and repair of DNA, and recD-inactivated mutants of E coli 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 Abbreviations ABM, Antarctic bacterial medium; ATPc-S, adenosine 5¢-O-(thiophosphate); EMSA, electrophoretic gel mobility shift assay; SF1, superfamily FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS 1835 RecD helicase motif mutants A K Satapathy et al A B C 3′ 5′ Fig Schematic representation of P syringae RecD (A) Location of the seven conserved helicase motifs on linear RecD (shown as a horizontal 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 indicated (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 modeling 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 and of RecD as seen in the structure of the DNA-bound RecBCD complex of E coli 1836 FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS A K Satapathy et al hydrolysis-defective mutants of the helicase motif I in RecD (RecDK177Q) and RecB (RecBK29Q) of E coli, it has been concluded that there are subtle differences between the properties of RecBC, RecBCDK177Q and RecBK29QCD enzymes, and that the RecB motor is absolutely required for v recognition and RecA loading, 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 previously discovered that recD is essential for growth of the Antarctic bacterium Pseudomonas syringae Lz4W at low temperature [15] The peizophilic bacterium Photobacterium 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 recDinactivated cold-sensitive P syringae mutants accumulate DNA fragments in cells grown at °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 DNAdamaging 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 (RecDHis) and eight mutant proteins that were created by single amino acid substitutions of important residues in each of the seven conserved helicase motifs RecDHis displayed ATP-hydrolyzing as well as short DNA duplex RecD helicase motif mutants unwinding activity in vitro, but the mutations K229Q in motif I (Walker motif A), D323N and E324Q in motif 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 modest 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 temperature, 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 helicase 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 outside 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 328QLSRLTGT335 and the P syringae RecD sequence 383WLEHVSGE390 align with the helicase motif IV sequence 284QNYRSTKR291 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 415RHSRRFGEG423 in P syringae and 356QKSYRFGSD364 in E coli represent motif IV The sequences are located in a structurally 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 FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS 1837 RecD helicase motif mutants A K Satapathy et al 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 Ca 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 containing three distinct domains (domains 1–3) Domain (residues 159–417) and domain (residues 418–682) of P syringae RecD, corresponding to homologous segments (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 that constitutes 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 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 hexahistidinetagged 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 pRecDHis, a derivative of the broad host range plasmid pGL10 (see Experimental procedures) The levels of expression of the soluble form of RecD from pRecDHis 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 Ni2+-agarose by a single-step method led to the association of a few co-contaminating proteins Introduction of a heparin–Sepharose chromatographic step prior to Ni2+-agarose chromatography, as described in Experimental procedures, eliminated such contamination Typically, approximately 1–2 mg of His-tagged RecD protein were purified from 500 mL of overnight cultures of CS1(pRecDHis) by this method The finally purified protein was about 99% pure, as determined by SDS– PAGE analysis with Coomassie blue staining (supplementary 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 1838 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 RecDHis, with comparable yield and purity ATP-hydrolyzing activity of RecDHis and its mutants We first assessed the ATP-hydrolyzing activity of the recombinant wild-type RecD protein (RecDHis) of P syringae, which is an essential activity of any helicase motor The RecDHis displayed efficient ATPase activity in the presence of ssDNA Interestingly, RecD also displayed significant ATPase activity in the presence of duplex DNAs with 5¢ overhangs and forkedend 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 RecDHis, a singlestranded 40-mer oligonucleotide was used as a stimulator under identical conditions (Fig 2B) The kinetic parameters of ATPase activity, obtained from analysis of RecDHis and the various mutants, are shown in Table RecDHis hydrolyzed ATP with a maximum velocity (Vmax) of 72 lmolỈs)1 and a Km of approximately 147 lm for ATP Five of the mutant RecD proteins (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 Km values for ATP (Km(ATP)) of 217, 151 and 136 lm, respectively (Table 1) By varying the DNA concentration in the reaction, the Km(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 Vmax of ATPase activity of RecDHis in the presence of DNA oligomers of various lengths, e.g 15mer, 25-mer and 40-mer (supplementary Table S1) As expected, the Vmax of the reactions increased as the length of the oligomeric DNA chain increased (Fig 3A) However, there was a reduction in the FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS A K Satapathy et al RecD helicase motif mutants 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) ATPase (μmol·s–1) A 100 40-mer 75 ′-OV Fork-end 50 ′-OV DNA-unwinding activity of RecD and its mutants Blunt-end 25 No DNA 0 ATP conc (mM) B 100 ATPase (μmol·s–1) RecDHis T259A R419A 10 E633Q 1 ATP conc (mM) Fig ATPase activity of RecD and its mutants DNA-stimulated ATPase activity was measured spectrophotometrically by the NADH oxidation-coupled assay method (A) Activity of RecDHis protein (6.6 nM) 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) Comparison of ATPase activity between RecDHis and the three mutant RecDs (T259A, R419A, E633Q) that displayed reduced activity Assays were performed in the presence of lM 40-mer ssDNA and 6.6 nM proteins Error bars indicate the standard deviation based on a minimum of three experiments Table Kinetic parameters of ssDNA-stimulated ATPase activity of RecD The coupled NADH oxidation method with 6.4 nM protein and lM 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 Km values RecD Vmax (lmolỈs)1) Km(ATP) (lM) Km(ssDNA) (nM) Wild-type (RecDHis) T259A R419A E633Q H386D 72 52 9.5 5.6 55 147 217 151 136 133 29 30 27 29 25 ± ± ± ± ± 1.7 0.5 ± ± ± ± ± 26 19 80 65 32 ± ± ± ± ± Km(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, Four types of duplex DNA substrate (supplementary Table S1) were used in the DNA strand unwinding assay RecDHis could unwind only the 5¢ overhang substrate (25 bp duplex DNA with a 15-base 5¢ extension) and the forked-end substrate (17 bp duplex with an 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 marginally better with the forked-end substrate, characterization of helicase activity was subsequently carried out using the 5¢ overhang DNA duplex substrate The helicase activity was found to be ATP- and Mg2+-dependent, and maximum activity was observed with 2.5 mm ATP and 2.0 mm MgCl2, under our experimental conditions (data not shown) The RecD protein could catalyze the ATPase and DNA strand unwinding activities in the presence of Mg2+ or Mn2+ but not when Ca2+ or Zn2+ 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 analogue (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 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 FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS 1839 RecD helicase motif mutants A K Satapathy et al 40-mer 25-mer Fig ssDNA length-dependent ATP-hydrolyzing activity of P syringae RecD and its mutants The activity of RecDHis and three mutant proteins (T259A, R419A and E633Q) was measured spectrophotometrically using 6.6 nM protein in the presence of 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 15-mer ATPase (μmol·s–1) 100 75 50 25 A 0 5′-Overhang ATP conc (mM) RecDHis 3′ 5′ C1 C2 3′-Overhang 10 20 C1 C2 10 20 ds ATPase (μmol·s–1) 60 ss 40 3′ Blunt-end C1 C2 20 5′ 10 20 3′ Forked-end C1 C2 5′ 10 20 ds ss 0 ATP conc (mM) T259A B E 19A 33Q 60A Q N A Q 54 29 59 323 24 T E6 R4 R6 W T2 D E3 Q3 K2 C 31 31 31 23 123 31 23 10.0 ATPase (μmol·s–1) ds ss 7.5 5.0 2.5 0.0 ATP conc (mM) R419A ATPase (μmol·s–1) 7.5 5.0 2.5 0.0 E633Q 1840 Fig DNA-unwinding activity of P syringae RecD and its mutants (A) RecDHis protein (100 nM) was incubated with 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 control samples with heat-denatured ssDNA and dsDNA substrates, respectively (B) Representative phosphor image of a gel showing the DNA-unwinding activity of RecDHis (WT) and mutant RecD proteins (K229Q, T259A, D323N, E324Q, Q354E, R419A, E633Q and R660A) Assays were carried out at 25 °C using the 5¢ overhang DNA duplex substrate ATP conc (mM) (EMSA) in the presence of 32P-labeled DNA substrates (Fig 5) Wild-type RecD protein displayed stronger binding to ssDNA than to dsDNA at both assay temperatures (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 forkedend substrates were preferred (binding to approximately 80–85% of the ssDNA) compared with the FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS A K Satapathy et al RecD helicase motif mutants Table Summary of the properties of wild-type and mutant RecD proteins Biochemical activities of the wild-type protein (RecDHis) were taken as 100% for evaluation of the activities of the mutated proteins The Vmax values for ATPase activity of RecDHis at 25 and °C were 72 and 21 lmol ATPỈs)1, respectively, which were considered to be 100% for relative activity of the mutant proteins at the respective temperatures ND, not detectable under the experimental conditions Protein Complements cold-sensitivity of CS1? DNA-binding activity (%) ATPase activity at 25 °C (%) ATPase activity at °C (%) DNA unwinding at 25 °C (%) Wild-type (RecDHis) K229Q (motif I) T259A (motif Ia) D323N (motif II) E324Q (motif II) Q354E (motif III) R419A (motif IV) E633Q (motif V) R660A (motif VI) H386D + ) + ) ) ) + + ) + 100 88 84 77 135 92 52 20 83 97 100 ND 72 ND ND ND 13 ND 76 100 ND 62 ND ND ND ND 30 100 ND ND ND ND ND ND ND ND 80 A SS 5′ -OV 3′ -OV Fork Blunt C C C1 C C DNA Complex 350 300 250 200 150 100 50 T W 29 K2 b a b a b a b a b a b T2 59 A D3 23 N E3 24 Q Q3 54 R4 E 19 E6 A 33 R6 Q 60 A a b a b a b a Q DNA complex C Relative binding (%) B W T K2 29 T2 Q 59 A D3 23 E3 N 24 Q Q3 54 R4 E 19 E6 A 33 Q R6 60 A Free DNA Fig DNA-binding activity of the wild-type and mutant RecD proteins (A) Binding activity of RecDHis 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 32P-end-labeled DNA substrates (2.5 nM) alone, and lanes marked and contained labeled DNAs and RecDHis protein (250 and 500 ng, respectively) (B) Relative ssDNA binding activity of RecDHis and mutant RecD proteins Binding assays were performed with 32P-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 °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 RecDHis protein was considered as 100% for the calculations blunt-end duplex DNA (approximately 60%) Significantly, all the mutant RecD proteins retained the ability to bind the DNA substrates (Fig 5B) The efficiency of binding to DNA was, however, variable among the mutant proteins R419A and E633Q displayed 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 FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS 1841 RecD helicase motif mutants A K Satapathy et al motif II, especially E324Q, displayed a consistently higher degree of DNA-binding activity (> 2.5-fold) compared to RecDHis under identical conditions (Fig 5C) These residues in motif II are known to interact with ATP and Mg2+ for hydrolysis of the nucleotide substrate 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 proteins Genetic complementation of the cold-sensitive phenotype of CS1 by RecDHis and its mutants In vitro activities of RecD at various temperatures We reported previously that the defect in the P syringae 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 RecDHis protein to support the growth of CS1 at °C, by expressing it from the pRecDHis plasmid As expected, CS1, expressing RecDHis, grew efficiently at °C, both in ABM liquid culture and on ABM agar plates In contrast, CS1 with the empty plasmid pGL10 failed to grow in the medium at °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 complement 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 RecDHiscomplemented strain (approximately 8.5 h) It is Mutant RecD proteins (T259A, R419A and E633Q) that displayed ATP hydrolysis activity but no DNAunwinding ability in vitro were able to support growth of the cold-sensitive, recD-disrupted strain (CS1) of P syringae at °C This raises some interesting questions 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 RecDHis at various temperatures, using an enzyme-coupled NADH-oxidation assay The initial rate of ssDNA-induced ATP-hydrolyzing activity of RecDHis 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 °C by this method (data not shown) To circumvent 22 ºC ºC Empty plasmid Empty plasmid RecD HIs R660A K229Q E633Q T259A R660A RecD HIs E633Q K229Q R419A T259A R419A D323N Q354E E324Q Q354E D323N E324Q Fig Complementation of cold-sensitive growth of CS1 by wild-type and mutant RecD proteins The recD-inactivated mutant of P syringae (CS1) was transformed using empty plasmid pGL10, or pGL10-derived constructs expressing RecDHis (WT) or mutated RecD proteins (K229Q, T259A, D323N, E324Q, Q354E, R419A, E633Q and R660A) Growth of the resultant strains was determined at 22 and °C on ABM agar plates Only wild-type and mutant proteins T259A, R419A and E633Q could complement the growth defect of CS1 at °C 1842 FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS A K Satapathy et al RecD helicase motif mutants T K2 29 T2 Q 59 D3 A 23 N E3 24 Q Q3 54 E R4 19 E6 A 33 R6 Q 60 A C W W T K2 29 T2 Q 59 D3 A 23 N E3 24 Q Q3 54 E R4 19 E6 A 33 Q R6 60 A C 32Pi 32P C 100 25 °C °C 10 ATP 100 % unwound B ATPase activity (μmol·s–1) Fig 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 nM RecDHis, lM 40-mer ssDNA and 100 lM [c-32P]ATP (A) Representative phosphor images of the TLC plates from the 25 and °C assays (B) Histogram showing the relative ATPase activities of RecDHis (WT) and three mutant proteins (T259A, R419A and E633Q) at and 25 °C Error bars represent the standard deviation of the values based on three experiments (C) Relative DNAunwinding activity of RecDHis at various temperatures Reactions were carried out with 10 nM RecDHis protein on nM 5¢ overhang duplex DNA at three temperatures (37, 25 and °C) and analyzed as in Fig 4 ºC 25 ºC A 37 ºC 75 25 ºC 50 ºC 25 0 WT T259A this problem, we employed a TLC method to measure ATP hydrolysis activity using [c-32P]ATP as the substrate (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 RecDHis 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 °C, at about 25–35% of their 25 °C activity (Fig 7B) We then examined the helicase activity of P syringae RecDHis 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 °C, RecDHis (100 nm) failed to show any detectable DNA-unwinding activity When the amount of RecDHis 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 displayed approximately 30% ATPase activity at °C This suggests that the DNA strand separation assay in vitro probably underestimates RecD helicase activity at lower temperatures to a considerable extent Nonetheless, the method is robust enough to measure heli- R419A 10 20 30 Incubation time (min) E633Q 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) not affect the binding (Fig 5C) Table summarizes the key biochemical and biological activities 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¢ fi 3¢ helicase activity, like that of the mesophilic E coli RecD [2,3] However, the Vmax (72 lmolỈs)1) for the ATP-hydrolyzing activity of P syringae RecD is much higher than the reported value (5 lmolỈs)1) for mesophilic E coli RecD at 25 °C [3] The Km(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 Km(DNA) values for RecD obtained from the ATPase stimulation FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS 1843 RecD helicase motif mutants A K Satapathy et al 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 electrophoretic conditions used, and hence is likely to be less sensitive than the ATPase stimulation method for determination of the Km(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 helicase 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 functional analyses, including ATP binding and hydrolysis (motifs I and II), ssDNA binding (motifs Ia, III, IV, and V), and coupling of ATPase and helicase (DNAunwinding) 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-Mg2+ 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 1844 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 ATPassociated Mg2+ ion and active water molecule for the hydrolytic reaction, and their alteration causes reduction in the ATPase and DNA-unwinding activities [17,18] Consistent with this, the present study demonstrates that D323N and E324Q mutants of P syringae RecD 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 identical 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¢ fi 3¢ 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 (PTGKAAAR) 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 (64FTNKAAR70 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 binding 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 binding 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 °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 (corresponding to Q354 and R660 of P syringae RecD), FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS A K Satapathy et al are in contact with the c phosphate of a bound nucleotide in the crystal structure of the PcrA–DNA– adenylylimidodiphosphate ternary complex [24] Other residues in the two motifs are also involved in interaction 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 ssDNAbinding pockets for coupling ATP hydrolysis to DNAunwinding activities In the present study, we found that Q354E and R660A mutations of RecD 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 complement the cold-sensitive phenotype of the recD-inactivated CS1 strain These results are largely similar to observations with the E coli UvrD helicase, where the corresponding glutamine and arginine residue mutations (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 conserved 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 arginine residue at 284 position of motif IV in E coli UvrD helicase was changed to alanine, the mutant protein exhibited a highly increased Km 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 complement 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 Km 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 RecD helicase motif mutants 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 °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 possibly 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¢ fi 3¢) helicase How relevant are these activities for the in vivo function of RecD? It is generally 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 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 association 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 probably 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 association with the regulatory v DNA sequence It is only known that the purified E coli RecBCDK177Q mutant enzyme is less processive, and has reduced ATPase FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS 1845 RecD helicase motif mutants A K Satapathy et al activity (8–10-fold less in presence of ssDNA-binding protein) and ATP-dependent nuclease activity (4–14fold 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 suggested 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-dependent 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 RecDassociated ATPase activity required during the growth of P syringae at low temperature (4 °C) The very low (but non-zero) ATPase activity of RecD (approximately 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 temperature 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 °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 °C Experimental procedures Bacterial strains and growth conditions The Antarctic bacterium P syringae Lz4W and the coldsensitive recD mutant CS1 (recD::Tn5 tetR rifR) used in this 1846 study have been described previously [15] Depending on requirements, they were grown at 22 or °C (for high and low temperatures, respectively) in Antarctic bacterial medium (ABM) composed of gỈL)1 peptone and 2.5 gỈL)1 yeast extract, or on ABM agar (1.5%) When necessary, the ABM was supplemented with tetracycline (20 lgỈmL)1), rifampicin (100 lgỈmL)1) or kanamycin (50 lgỈmL)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 : 100, and the absorbance of the cultures at 600 nm (A600) 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 (Qiagen, Hilden, Germany) DNA sequencing reactions performed 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 syringae genomic DNA using primers PETF and RPSD (supplementary 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 confirmed 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 pRecDHis, which FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS A K Satapathy et al 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 RecDHis in CS1 was confirmed by western analysis, using anti-His-tag serum (Santa Cruz Biotechnology, 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 Ni2+nitrilotriacetic acid (Qiagen) affinity chromatography Briefly, CS1(pRecDHis) cells were grown at 22 °C in ABM culture broth Cell lysates were prepared in buffer A (20 mm Tris ⁄ Cl pH 7.5, mm dithiothreitol, 10% glycerol) by lysozyme (1 mgỈmL)1) treatment on ice, followed by gentle sonication The cell lysate was clarified by centrifugation (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 Ni2+-nitrilotriacetic acid agarose (Qiagen) column for purification 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 elution 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, mm dithiothreitol, 20% glycerol) on a PD-10 column packed with Sephadex G-25 (Amersham Biosciences) Purified RecD was stored at )70 °C, and diluted with buffer (20 mm Tris ⁄ Cl pH 7.5, 300 mm NaCl, mm dithiothreitol, 10% glycerol, 0.1 mgỈmL)1 BSA) before use Site-directed mutagenesis of helicase motifs in RecDHis Site-directed mutagenesis was carried out by overlap extension 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 Experimentally, two-step PCR reactions were carried out Briefly, in the first round, DNA encoding N- and C-terminal fragments 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 forwardstrand mutagenic primers (K229QF, T259AF, D323NF, E324QF, Q354EF, H386DF, R419AF, E633QF and R660AF) M13R and M13F are flanking primers of recD on pRecDHis, which was used as the template for amplifica- RecD helicase motif mutants tion In the second round, both N- and C-terminal fragments were purified, mixed together, and overlap PCR was performed for 10 cycles without adding any primers, followed 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 (pRecDK229Q, pRecDT259A, pRecDD323N, pRecDE324Q, pRecDQ354E, pRecDH386D, pRecDR419A, pRecDE633Q and pRecDR660A) 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 (RecDHis) 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, mm MgCl2, mm dithiothreitol, 35 mL)1 pyruvate kinase, 20 mL)1 lactate dehydrogenase, mm phosphoenol pyruvate, 0.08 mgỈmL)1 NADH, 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 (supplementary 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, Vmax (maximal rate of ATP hydrolysis), Km(ATP) (ATP concentration at the half-maximal rate of reaction) and Km(DNA) (DNA concentration at the halfmaximal rate of ATP hydrolysis) for the reactions were calculated Km(ATP) was determined at a saturating concentration (1 lm) of ssDNA, and Km(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, Darmstadt, Germany) Assays were carried out at indicated temperatures, 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, mm MgCl2, mm dithiothreitol, 0.1 mgỈmL)1 BSA, 2.5 mm ATP and the indicated concentration of ssDNA substrate (40-mer) FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS 1847 RecD helicase motif mutants A K Satapathy et al [c-32P]ATP (0.1 lCi; specific activity 3000 CiỈmmol)1) was used as a tracer in each reaction to measure the rate of ATP hydrolysis After 15 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 Pi The plates were scanned in a phosphor imager (Fuji, Tokyo, Japan), and the amounts of 32Pi and [c-32P]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-displacement method Four kinds of substrates, i.e blunt-end, 5¢ overhang, 3¢ overhang and forked-end duplex DNAs were prepared by annealing one 32P-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 performed using T4 polynucleotide kinase and [c-32P]ATP The helicase reaction mixture contained 20 mm Tris ⁄ HCl pH 7.5, 17.5 mm NaCl, mm MgCl2, mm dithiothreitol, 0.1 mgỈmL)1 BSA, 2.5 mm ATP and nm labeled DNA helicase substrate, as described previously [3] Reactions were initiated by adding RecD (100 nm, unless noted otherwise), 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 oligonucleotides ss- and dsDNA were separated by electrophoresis on a native 15% polyacrylamide gel Gels were scanned using a phosphor imager (Fuji) Band intensities corresponding to ss- and dsDNA were quantified The percentage of unwound DNA [100· ssDNA ⁄ (ssDNA + dsDNA)] was calculated, and the values were plotted using graphpad prism software To determine temperature-dependent activity, 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 doublestranded DNA (blunt-end, 5¢ overhang, 3¢ overhang and forked-end) substrates Binding assays were carried out at and 25 °C in 20 lL reaction mixtures containing 20 mm Tris ⁄ HCl pH 7.5, 17.5 mm NaCl, mm MgCl2, mm dithiothreitol, 0.1 mgỈmL)1 BSA, 2.5 nm 32P-labeled DNA substrate 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 1848 free DNA Gels were run for 60 at 10 VỈcm)1 in a cold room (4 °C) Gels were scanned and analyzed using a phosphor imager (Fuji) Genetic complementation studies For genetic complementation analysis of the cold-sensitive phenotype of the P syringae CS1 strain (recD::Tn5 tetR), His-tagged RecD-producing constructs (see above) in the broad host-range vector pGL10 (kanR) were mobilized into CS1 by conjugation Briefly, E coli S17-1 (tetS) 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 (tetR kanR) 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 °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 Biosciences) (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 FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS A K Satapathy et al RecD helicase motif mutants Acknowledgements We thank Drs Gerald Smith (Fred Hutchinson Cancer Research Center, Seattle, WA, USA), Donald Crampton (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 laboratory 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 International Senior Research Fellow in Biomedical Sciences of the Wellcome Trust, UK 11 12 13 14 15 References Gorbalenya AE & Koonin EV (1993) Helicases: amino acid sequence comparisons and structure–function relationships Curr Opin Struct Biol 3, 419–429 Chen HW, Ruan B, Yu M, Wang J & Julin DA (1997) The RecD subunit of the RecBCD enzyme from Escherichia coli is a single-stranded DNA-dependent ATPase J Biol Chem, 272, 10072–10079 Dillingham MS, Spies 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for cloning and mutagenesis of P syringae recD This material is available as part of the online article from http://www.blackwell-synergy.com RecD helicase motif mutants 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 corresponding author for the article FEBS Journal 275 (2008) 1835–1851 ª 2008 The Authors Journal compilation ª 2008 FEBS 1851 ... Nonetheless, the present study identifies the significance of the RecDassociated ATPase activity required during the growth of P syringae at low temperature (4 °C) The very low (but non-zero) ATPase. .. knowledge regarding low- temperature- adapted biology We previously discovered that recD is essential for growth of the Antarctic bacterium Pseudomonas syringae Lz4W at low temperature [15] The peizophilic... halfmaximal rate of ATP hydrolysis) for the reactions were calculated Km(ATP) was determined at a saturating concentration (1 lm) of ssDNA, and Km(DNA) was calculated at a saturating concentration of ATP