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Originally published In Press as doi:10.1074/jbc.M408645200 on October 4, 2004

J. Biol. Chem., Vol. 279, Issue 50, 52024-52032, December 10, 2004
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DNA Helicase Activity of the RecD Protein from Deinococcus radiodurans*

Jianlei Wang and Douglas A. Julin{ddagger}

From the Department of Chemistry & Biochemistry, University of Maryland, College Park, Maryland 20742

Received for publication, July 29, 2004 , and in revised form, October 4, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The bacterium Deinococcus radiodurans is extremely resistant to high levels of DNA-damaging agents, including gamma rays and ultraviolet light that can lead to double-stranded DNA breaks. Surprisingly, the organism does not appear to have a RecBCD enzyme, an enzyme that is critical for double-strand break repair in many other bacteria. The D. radiodurans genome does encode a protein whose closest characterized homologues are RecD subunits of RecBCD enzymes in other bacteria. We have purified this novel D. radiodurans RecD protein and characterized its biochemical activities. The D. radiodurans RecD protein is a DNA helicase that unwinds short (20 base pairs) DNA duplexes with either a 5'-single-stranded tail or a forked end, but not blunt-ended or 3'-tailed duplexes. Duplexes with 10–12 nucleotide (nt) 5'-tails are good unwinding substrates and are bound tightly, while DNA with shorter tails (4–8 nt) are poor unwinding substrates and are bound much less tightly. The RecD protein is much less efficient at unwinding slightly longer substrates (52 or 76 base pairs, with 12 nt 5'-tails). Unwinding of the longer substrates is stimulated somewhat (4–5-fold) by the single-stranded DNA-binding protein from D. radiodurans. These results show that the D. radiodurans RecD protein is a DNA helicase with 5'-3' polarity and low processivity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Double-strand DNA (dsDNA)1 breaks in bacterial cells are generally thought to be repaired by homologous recombination, in which the broken chromosome is reattached to a homologous chromosome, or to a partially replicated chromosome. This process, understood in greatest detail from studies in Escherichia coli, depends critically on the RecA and RecBCD enzymes (reviewed in Refs. 1 and 2). The RecBCD helicase/nuclease binds to a double-stranded DNA end and initially degrades the DNA using its exonuclease activity. The nuclease activity is modified when the enzyme encounters a Chi sequence (5'-GCTGGTGG in E. coli) so that it produces a 3'-terminated single-stranded DNA end. RecBCD then loads RecA protein onto this 3'-terminated strand, and RecA can carry out a strand invasion of the homologous duplex to initiate the recombination reaction.

The results from numerous genome sequencing projects as well as from biochemical studies show that many bacteria have an enzyme homologous to the RecBCD enzyme of E. coli. These enzymes consist of three protein subunits encoded by recB, recC, and recD genes. Some bacteria have a two-subunit helicase/nuclease enzyme (called e.g. AddAB in Bacillus subtilis) that appears to function in a largely similar reaction path, using the cognate RecA protein and organism-specific Chi-sequence (3). One subunit (AddA) is similar to RecB of E. coli and other bacteria, while the other subunit (AddB) is not similar to either RecC or RecD. A surprising exception is the bacterium Deinococcus radiodurans. This organism is remarkably resistant to ionizing radiation and other DNA-damaging agents. Its DNA can suffer many double-strand breaks upon irradiation with x-rays or {gamma}-rays, and the organism has the remarkable ability to reassemble its genome and survive in conditions where other bacteria (e.g. E. coli) are killed very quickly (4–6). The enzymatic basis for the radiation resistance is not well understood. The complete D. radiodurans genome has been sequenced, and orthologues of many familiar DNA repair proteins are readily identifiable (7). For example, a D. radiodurans recA gene is found, and the RecA protein has been characterized biochemically (8, 9). However, the D. radiodurans genome apparently has neither recB nor recC genes, nor are there addAB-like genes (7). Thus it is not clear how the organism is able to withstand such a high level of double-strand DNA breaks, without a RecBCD-like enzyme.

The D. radiodurans genome does have a predicted open reading frame that would encode a protein whose closest characterized homologues are other bacterial RecD proteins (7). The gene was thus annotated as the D. radiodurans recD gene. Based on amino acid sequence analysis, as reported in the Cluster of Orthologous Groups (COG) data base (Refs. 10 and 11; see www.ncbi.nlm.nih.gov/COG/new), this putative D. radiodurans RecD protein was included in a RecD protein family (COG0507). Many other members of this family are clearly RecD subunits of a RecBCD enzyme. However, the predicted D. radiodurans RecD protein is most similar to a subgroup of this family, which includes predicted proteins in genomes of several other Gram-positive bacteria, including B. subtilis, Lactococcus lactis, Streptococcus pyogenes, and several Chlamydia species (Fig. 1). Interestingly, B. subtilis, L. lactis, and several other bacteria in this subgroup have the two-subunit AddAB-like enzyme (which lacks a RecD-like subunit), while the Chlamydia genomes have two so-called recD genes (12–14): one encodes a likely subunit of the Chlamydial RecBCD enzyme whereas the second Chlamydial RecD-like protein groups with the D. radiodurans RecD protein. Because of these considerations, the two subgroups are listed as separate families in the TIGR Protein Families data base (Ref. 15; see www.tigr.org/TIGRFAMs/). The RecD subunits are grouped in the TIGR01447 family, while the RecD-like proteins from organisms that do not have RecB and RecC, including D. radiodurans, are grouped in the TIGR01448 family.



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  		FIG. 1.
Schematic structure of RecD proteins. Rectangles represent the RecD proteins from D. radiodurans (GenBankTM accession number: NP_295625 [GenBank] ), close relatives of D. radiodurans RecD from Bacillus subtilis (YrrC, NP_390625 [GenBank] ), Lactococcus lactis (NP_267896 [GenBank] ), Streptococcus pyogenes (NP_269843 [GenBank] ), and C. muridarum (RecD (1), NP_296681 [GenBank] ), and the RecD subunit of the E. coli RecBCD enzyme (NP_417296 [GenBank] ). N terminus is on the left. Vertical bars represent the seven amino acid motifs that are conserved among Superfamily I helicases.

 
The function of the putative D. radiodurans RecD protein and its close relatives is unknown, because the only known function of RecD in other bacteria is as a subunit of a RecBCD enzyme. The greatest amino acid sequence similarity between D. radiodurans RecD and the other RecD family members is in regions that are similar to the Superfamily I helicases, of which RecD and RecB are considered members (16, 17). Indeed, the RecD protein from E. coli is both a DNA-dependent ATPase (18) and a DNA helicase that moves with 5'-3' polarity on single-stranded DNA (19). The helicase superfamily is defined by seven conserved helicase motifs, and these are the sequences that align most strongly among the RecD family members (Fig. 1). The D. radiodurans-type RecD proteins are larger than the RecD subunit proteins (715–865 and 493–721 amino acids residues, respectively). The additional amino acids are found in the long N-terminal region (~360–380 residues in the D. radiodurans-like RecDs) that precedes the first helicase motif (the "Walker A" ATP-binding motif, Ref. 20). These N-terminal regions are less conserved among the RecD family members and do not contain sequences found in other helicases.

The RecD-like proteins in D. radiodurans and other bacteria may serve a novel function in these organisms. We have cloned, expressed, and purified the novel RecD protein from D. radiodurans as a first step toward learning about its biological function and whether it plays a role in the remarkable resistance to DNA damage of this organism. The purified protein is a DNA helicase that readily unwinds short (20 bp) double-stranded DNA substrates having either a 5'-single-stranded terminal extension or a fork of single-stranded DNA at the end. The protein is much less efficient at unwinding slightly longer duplexes (52 or 76 bp), suggesting that it has low processivity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Synthetic DNA oligonucleotides were purchased from Invitrogen and were purified from denaturing polyacrylamide gels using a MERmaid kit (BIO101 Corp.). Purified D. radiodurans SSB (DrSSB) protein (22) was a generous gift from Professor Mike Cox, University of Wisconsin. E. coli single-stranded DNA-binding protein was purchased from US Biochemicals Corp. or was purified as in Ref. 21.

D. radiodurans recD Gene Cloning—D. radiodurans strain R1 was obtained from the American Type Culture Collection, Manassas, VA. D. radiodurans genomic DNA was isolated as described (23). A 2192-bp fragment that includes the recD coding region (locus DR1902, GenBankTM accession number AE002029 [GenBank] .1) was amplified from the genomic DNA by PCR using as primers: (upstream) 5'-CGC GGA ATT CAT ATG TCT GCT GCC CTG CC and (downstream) 5'-AGT GGG ATC CCT GAC AGA ACT CTT AAG GCG TCT TAA TG. The amplified DNA product was digested with EcoRI and BamHI and ligated to the plasmid pTZ19R (24) cut with the same enzymes, to produce pDr-recD.ptz. The complete sequence of the D. radiodurans DNA insert, determined at the DNA sequencing facility in the Center for Biosystems Research, University of Maryland, was identical to that found in GenBankTM.

D. radiodurans RecD Protein Expression—We first transferred the cloned recD gene to the plasmid pET-15b (Novagen Corp.) for protein expression with an N-terminal His tag. A soluble protein of about the expected size (78.6 kDa) was seen in cell extracts analyzed by SDS-PAGE when expression was induced by adding isopropyl-1-thio-{beta}-D-galactopyranoside (data not shown). However, the N-terminal His-tagged RecD protein had low affinity for a nickel-containing resin and could not be purified under native conditions by this method.

We then transferred the recD gene to pET-21a (Novagen Corp.) for expression with a C-terminal His tag. Two primers were designed to amplify a ~1500-base pair fragment from the 3'-end of the recD gene in pDr-recD.ptz. The upstream primer (5'-GAC AAG CTC TGG CAG GCC CGC GG) included the SacII site within the recD gene and the downstream primer (5'-CTT GGA TCC TCG AGA TGC GCC CTG ATT CTT TCC) introduced new XhoI and BamHI sites after the last codon of the RecD coding region, and replaced the natural stop codon. The PCR product was digested with SacII and BamHI and the 1535 base pair fragment was ligated to pDr-recD.ptz cut with the same enzymes, to replace the native SacII-BamHI fragment. The modified recD gene was excised from this new plasmid by cleavage with NdeI and XhoI, and ligated to pET-21a cut with the same enzymes, to make pDr-recD-21. This plasmid encodes the D. radiodurans RecD protein with the peptide Leu-Glu-His-His-His-His-His-His appended to its C terminus. The complete sequence of the recD gene insert in pDr-recD-21 was verified.

D. radiodurans RecD Protein Purification—The C-terminal His-tagged RecD protein was expressed in E. coli strain BL21(DE3) transformed with pDr-recD-21. Cells were grown in 2 liters of LB medium containing 50 µg/ml ampicillin at 37 °C with vigorous shaking, until the OD600 = 0.5. Isopropyl-1-thio-{beta}-D-galactopyranoside was then added to 0.5 mM final concentration, and the cells were incubated at 30 °C for 3 h and then harvested by centrifugation. The cell pellets (4 g) were stored at –80 °C overnight and then thawed on ice and suspended in 50 ml of native binding buffer (20 mM sodium phosphate, pH 7.8, 500 mM NaCl). Phenylmethylsulfonyl fluoride (600 µl of 1 M stock) and 300 µl of a protease inhibitor mixture for polyhistidine-tagged proteins (Sigma, Inc.) were added to the cell suspension. The cells were lysed by sonication, and the cell debris was removed by centrifugation at 16,000 x g for 30 min at 4 °C.

The crude cell extract was applied to a 10-ml Ni2+-NTA column (ProBond resin, Invitrogen Corp.) in native binding buffer. The column was washed with 30 ml of native binding buffer, 100 ml of native wash buffer (20 mM sodium phosphate, pH 6.0, 500 mM NaCl) containing 60 mM imidazole, and the RecD protein was eluted in a 140-ml gradient of 60–400 mM imidazole in native wash buffer, followed by 10 ml of native wash buffer with 500 mM imidazole.

The fractions containing RecD protein, based on SDS-PAGE, were collected and dialyzed against Buffer A (20 mM potassium phosphate, pH 7.5, 1 mM EDTA, 1 mM DTT, 10% (v/v) glycerol). The dialyzed pool was applied to a 5-ml ssDNA cellulose column (Sigma) in Buffer A. The column was washed with ten volumes of Buffer A containing 50 mM NaCl, followed by 30 ml each of Buffer A with 0.6 M NaCl, 1 M NaCl, and 1.5 M NaCl. The bulk of the RecD eluted in the 1.0 M and 1.5 M NaCl washes. The fractions containing RecD were pooled, dialyzed against Buffer A containing 10% (v/v) glycerol, concentrated by ultrafiltration (Amicon), and then dialyzed against Buffer A containing 50% glycerol. The resulting protein solution (> 90% pure) was aliquoted and stored at –80 °C. The purified protein retained its activity for at least 2 years under this storage condition. The RecD concentration was determined from the absorbance at 280 nm, using {epsilon}280 = 52,060 M–1 cm–1, calculated for His-tagged RecD using the program ProtParam (ca.expasy.org/tools/protparam.html). The concentrated RecD stock was diluted in 25 mM potassium phosphate, pH 7.5, 0.1 mM EDTA, 0.1 mM DTT, 0.14 mg/ml BSA, and 10% glycerol and kept on ice, for all enzymatic assays described below.

ATP Hydrolysis Assays—ATP hydrolysis was measured by thin layer chromatography on polyethyleneimine (PEI)-cellulose plates (J. T. Baker) as described (18). The reaction mixtures (20 µl of total volume) contained 50 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 10 µM (nt residues) DNA or no DNA, 1 mM DTT, 5% glycerol, 0.1 mg/ml BSA, 10 µM [{gamma}-32P]ATP (30 Ci/mmol), and 0.2 nM RecD enzyme. The reactions were incubated at 37 °C and 0.5-µl aliquots were spotted on the TLC plates. The plates were developed and analyzed using a Storm PhosphorImager and ImageQuant software (Amersham Biosciences) as described (18).

DNA Unwinding Assays—The oligonucleotides used as helicase substrates are listed in Table I. These oligonucleotides can be annealed in pairs to form DNA duplexes with various end structures (Table I). One oligonucleotide of each double-stranded molecule was labeled at its 5'-end with [32P] as described (25). The labeled oligomers were purified using a QIAquick Nucleotide Removal Kit (Qiagen Corp.), and their concentrations were determined from scintillation counting of samples removed before and after the purification procedure. The two oligomers (20 µM each) were mixed in 50 mM NaCl, 20 mM Tris acetate, pH 7.5, 1 mM magnesium acetate, heated to 95 °C, and then cooled slowly to room temperature to allow them to anneal.


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  		TABLE I
Oligodeoxyribonucleotides

 
Most unwinding reaction mixtures contained 25 mM Tris acetate, pH 7.5, 10 mM magnesium acetate, 1 mM ATP, 1 mM DTT, 0.1 mg/ml BSA, and 1 nM duplex substrate. Reactions at pH other than 7.5 were done as described in the figure legends. The reaction mixtures were incubated at 30 °C for 5 min, a 5-µl aliquot was taken (0 time point), and the reactions were started by adding RecD enzyme. Aliquots (5 µl) were quenched with 1.5 µl of 4x quenching buffer (40% glycerol, 2.4% (w/v) SDS, 100 mM EDTA, 0.12% (w/v) bromphenol blue, and 15 nM unlabeled oligonucleotide corresponding to the labeled strand in the double-stranded substrate) and kept on ice. The quenched samples were run on non-denaturing polyacrylamide gels (29:1, acrylamide/bis-acrylamide) in 1x TBE buffer (90 mM Tris, 89 mM boric acid, 1.98 mM EDTA, pH 8.3) at 150 volts constant power. The gels were then dried and analyzed using the Storm PhosphorImager. The percent DNA unwound was determined by quantitating the relative amount of 32P-labeled ds and ssDNA in each lane, and then calculated as % unwound = ss/(ss + ds), where ss and ds are the integrated amounts of radioactivity in the ss and ds bands in that lane. The background of ssDNA present in the zero time point (usually <5% of the total DNA) was subtracted out. DNA unwinding rates were calculated from the slope of the initial, nearly linear, part of the reaction time course. Reannealing of the single-stranded products was found to be insignificant at the DNA concentration in the reactions (1 nM) and could be ignored when analyzing the unwinding reactions.

DNA Binding Measurements—The hairpin oligonucleotides used in these experiments are listed in Table I. These molecules have 20 bp of double-stranded DNA, a three-nucleotide loop, and either a 6-nt or 12-nt 5'-single-stranded tail, or a 12-nt forked end. These constructs were used to avoid the presence of any single-stranded DNA in mixtures of separate, complementary, oligonucleotides. The hairpin oligonucleotides were 5'-end-labeled with [32P] as above.

Most binding mixtures contained, in 5 µl, 25 mM Tris acetate, pH 7.5, 10 mM magnesium acetate, 1 nM [5'-32P]hairpin DNA and 1.25 µl of appropriately diluted RecD. The mixtures were kept on ice for 30 min before 1.5 µl of 30% glycerol was added. The samples were then loaded quickly onto a 10% polyacrylamide gel (37.5:1, acrylamide/bis-acrylamide) in 1x TBE buffer. The gels were chilled in a cold room for 30 min and then pre-run at 10 mA current before use. High voltage (~500 volts) was applied after the samples were loaded to run the gels for a short time (15~20 min) in the cold room. The gels were dried and analyzed using the Storm PhosphorImager.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
ATP Hydrolysis by D. radiodurans RecD—We first tested the purified RecD protein for ATP hydrolysis activity. The protein exhibits efficient ATP hydrolysis activity in the presence of a single-stranded DNA oligomer, with no detectable activity in the absence of DNA (Fig. 2). A low level of ATP hydrolysis was detected with linear dsDNA, and even less with circular dsDNA. The latter ATPase activity may result from a small amount of linear DNA in the circular plasmid preparation.



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  		FIG. 2.
ATP hydrolysis by D. radiodurans RecD. Reaction mixtures contained 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT, 5% (v:v) glycerol, 0.1 mg/ml BSA, 10 µM [{gamma}-32P]ATP, 5 nM RecD enzyme, and a 17-nt single-stranded oligonucleotide (closed circles), plasmid pTZ19R linearized by cleavage with EcoRI (open triangles), circular plasmid pTZ19R DNA (closed triangles), or no DNA (open circles). DNA concentration (when present) was 10 µM nt. Production of [32P]phosphate was determined by TLC as described under "Experimental Procedures."

 
DNA Unwinding Activity—We then tested for DNA unwinding activity using substrates shown in Table I. RecD unwound a 20-bp duplex with a 12-nt 5'-single-stranded extension (oligonucleotides I + II; see Table I), and the same duplex with a forked end (I + IV: 12-nt non-complementary extensions on both the 3'- and 5'-ends) (Fig. 3). There was no detectable unwinding of a 20-bp duplex with blunt ends nor of the duplex with the 3'-single-stranded tail (Fig. 3). Thus the enzyme requires a DNA duplex with a 5'-single-stranded extension for helicase activity. While the forked substrate is unwound more rapidly than the tailed substrate, the enzyme does not require the fork, in contrast to some other helicases (26). There was no unwinding in the absence of either ATP or magnesium ion (data not shown).



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  		FIG. 3.
DNA unwinding by D. radiodurans RecD. Reaction mixtures contained 25 mM Tris acetate, pH 7.5, 10 mM magnesium acetate, 1 mM ATP, 1 mM DTT, 0.1 mg/ml BSA, 1 nM (DNA molecules) 32P-labeled double-stranded DNA substrate, and 5 nM RecD protein. The DNA substrates had 20 base pairs and 12-nt single-stranded tails where shown. Reactions were run at 30 °C, and samples were analyzed on non-denaturing 15% polyacrylamide gels as described under "Experimental Procedures." Samples in lanes M were taken before enzyme was added and were heated to 100 °C before loading, to provide a marker for the labeled single strand.

 
The RecD protein was in excess over the DNA in the experiment shown in Fig. 3, but unwinding of the 5'-tailed and forked substrates was efficient even when the DNA was in 10–1000-fold excess over the protein (Fig. 4, A and B). The fact that a substantial fraction of the DNA was unwound under these conditions shows that the enzyme acts catalytically, with a single enzyme molecule unwinding several DNA molecules (20–100, depending on the RecD concentration and the type of DNA substrate). The initial rates of the reactions, determined from the slopes of the linear part of the time courses, were 0.10 (±0.025) DNA molecules unwound/RecD/sec, or 2 (±0.5) bp/RecD/sec, for the 5'-tailed substrate (Fig. 4A), and 0.8 (±0.1) DNA molecules unwound/RecD/sec, or 16 (±2) bp/RecD/sec, for the forked substrate (Fig. 4B).



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  		FIG. 4.
Unwinding at various RecD concentrations. A, DNA substrate (1 nM) was 20 bp with a 12-nt 5'-tail (Table I), with 0.01 nM RecD (closed circles), 0.03 nM RecD (open circles), 0.06 nM RecD (closed triangles), and 0.12 nM RecD (open triangles). Other conditions were as in Fig. 3. B, DNA substrate (1 nM) was 20 bp with a 12-nt single-stranded fork on one end (Table I), with 0.001 nM RecD (closed circles), 0.003 nM RecD (open circles), 0.006 nM RecD (closed triangles), and 0.012 nM RecD (open triangles). Samples were run on 15% non-denaturing polyacrylamide gels and analyzed using a Phosphorimager as described under "Experimental Procedures."

 
We tested next whether the enzyme can unwind duplex substrates with 5'-terminal extensions shorter than 12 nt. We detected no unwinding of a 28-bp duplex with a 4-nucleotide 5'-single-stranded tail (oligomers IV + V, Table I), with 1 nM DNA and 10 nM RecD (data not shown). Twenty-base pair duplexes with 6- or 8-nucleotide 5'-tails (III + VI or VII, respectively) were very poor substrates under conditions where the substrates with a 10- or 12-nt overhang were unwound efficiently (Fig. 5). Greater unwinding of the 8-nt tailed substrate, and slight unwinding of the 6-nt tailed substrate, could be detected at higher enzyme concentration (10 nM; data not shown). These results suggest that the enzyme requires at least 10 nt of single-stranded DNA for tight binding to the DNA substrates.



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  		FIG. 5.
Effect of 5'-single strand extension length on DNA unwinding. Reaction mixtures were as in Fig. 3, with 0.25 nM RecD and 1nM [5'-32P]DNA substrates consisting of 20 bp with 5'-single-stranded extensions of 6 nt (closed circles), 8 nt (open circles), 10 nt (closed triangles), or 12 nt (open triangles). Samples were taken and analyzed on a 15% polyacrylamide gel as under "Experimental Procedures."

 
DNA Binding Measurements—We measured binding of RecD to DNA molecules with various end structures directly in a gel shift assay (Fig. 6), using hairpin oligonucleotides (Table I). The hairpin with the 12-nt forked end was bound tightly and two shifted complexes were observed. We presume that these complexes are DNA molecules with RecD bound to one or both ss tails. The enzyme bound less tightly to the 5'-tailed DNA than to the fork (Fig. 6B). The hairpin with the 6-nt extension was bound weakest of all, with a large excess of protein required before a significant amount of the DNA was shifted in the gel. Weak binding to this hairpin DNA is consistent with the fact that the duplex with the 6-nt tail is a very poor unwinding substrate (Fig. 5). This hairpin (at 20 nM) also did not inhibit the unwinding reaction of RecD (0.1 nM) with the 5'-tailed 20-bp substrate (1 nM substrate; data not shown). Interestingly, two shifted bands appeared at the high protein concentrations used for the 6-nt tailed hairpin. The slower mobility complex could result from additional enzyme molecules binding to the DNA (perhaps to the loop or to the double-stranded region in the hairpin), or from dimerization/oligomerization of the protein on the DNA. We do not know whether DNA unwinding requires the combined action of more than one RecD monomer on the DNA substrate, although the fact that the 5'-tailed substrate is unwound efficiently but only a single complex is observed in the binding experiment with the tailed hairpin (Fig. 6) suggests that the RecD enzyme may unwind these molecules as a monomer. Some Superfamily I helicases unwind DNA as dimers (27, 28) whereas others apparently function as monomers (29, 30). Further experiments must be done to resolve this point definitively for D. radiodurans RecD.



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  		FIG. 6.
RecD binding to dsDNA hairpins with different 5'-tail structures. A, mixtures containing the indicated RecD concentrations and [5'-32P] DNA (1 nM) were analyzed on non-denaturing 10% polyacrylamide gels as under "Experimental Procedures." B, percent DNA bound was analyzed by integrating the amount of radioactivity in the unbound dsDNA band, the DNA-protein complexes, and the radioactive material migrating intermediate between the two. The amount of radioactivity in the two shifted DNA bands in the 12-nt fork and the 6-nt tail experiments was summed for this plot. Plots are for the hairpin DNA with a 12-nt 5'-tail (closed circles) and 12-nt forked end (open circles). Inset, hairpin DNA with a 6-nt 5'-tail (closed circles).

 
Unwinding of Longer DNA Substrates—We next asked whether the enzyme is able to unwind substrates longer than the 20-bp substrates tested so far, using 52 and 76 bp substrates with a 12-nt 5'-single-stranded overhang (Table I). In striking contrast to the results with the 20-bp substrates, a large molar excess of protein over DNA (10–100-fold) was required for significant unwinding of the 52-bp substrate (Fig. 7, A and B). There was little or no unwinding of the 52-bp substrate with enzyme concentrations equal to or less than the DNA (data not shown). The 76-bp substrate was unwound even less efficiently than the 52-bp substrate (Fig. 7B).



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  		FIG. 7.
Unwinding of 52 and 76 bp duplexes. A, reaction mixture contained 1 nM of the 52-bp substrate with a 12-nt 5'-tail (see Table I), 100 nM RecD, and other conditions as in Fig. 3. Samples were analyzed on a non-denaturing 10% polyacrylamide gel as described under "Experimental Procedures." B, percent DNA unwound in the reaction shown in A was determined as in Fig. 4, for the 52-bp substrate, 100 nM RecD (closed circles). Other reactions (gels not shown) contained 52-bp substrate (1 nM), 10 nM RecD (closed triangles); and 76-bp substrate (1 nM; see Table I), 10 nM RecD (open triangles). C, reaction mixture contained 1 nM of the 52-bp substrate with a 12-nt 5'-tail, 5 nM RecD, and other conditions as in the legend to Fig. 3. D, reaction was as in C, with 390 nM single-stranded DNA-binding protein from D. radiodurans (DrSSB). E, percent DNA unwound in the reactions shown in C and D. No DrSSB (open circles); +DrSSB (closed circles).

 
There are at least two possible explanations for the inefficient unwinding of the longer substrates compared with the 20-bp substrates. One is that RecD is a helicase with low processivity, such that the enzyme is able to unwind a short duplex (i.e. 20 bp) but it falls off before it can unwind completely the longer dsDNA molecules. A second possibility is that the individual DNA strands might rewind behind the RecD as it travels along the DNA duplex. A way to prevent this rewinding is to include a single-stranded DNA-binding protein in the unwinding reaction mixtures, to grab the unwound strands before they can reanneal. The rate and extent of unwinding of the 52-bp substrate were increased significantly (about 4–5-fold) in the presence of the D. radiodurans SSB protein (Fig. 7, C–E), although the rate was still much lower than for the 20-bp substrates. Inclusion of SSB from E. coli (at 20 nM, an amount sufficient to bind all of the DNA in the reaction mixture (31)) had little effect on the unwinding rate or extent for either the 52 bp or the 76 bp DNA substrates (data not shown).

We also tested whether unwinding of the longer substrates could be more efficient, in the absence of SSB protein, under other reaction conditions. The rate of unwinding the 52-bp DNA was about 2-fold greater at lower magnesium ion concentration (1–3 mM) than at 10 mM Mg2+ as was used in previous experiments (data not shown). The unwinding rate decreased slightly (<2-fold) at 5 or 10 mM ATP rather than 1 mM ATP, with Mg2+ concentration kept equal to the ATP (data not shown). Added salt (NaCl or KCl) inhibited at all concentrations tested (25–400 mM; data not shown) and was not included in further experiments.

The one change that did affect significantly the unwinding of the longer substrates was pH. Substantially more 52-bp DNA was unwound at pH 6–6.5 than at pH 7.5, the conditions used in previous experiments (Fig. 8A), or at higher pH (pH 8–9, data not shown). Thus, at pH 6.5 and 2 mM Mg2+, the 52 bp DNA was unwound at a rate of 0.168 (±0.015) bp/RecD/sec, as compared with about 0.001 bp/RecD/sec at pH 7.5 and 10 mM Mg2+. The rate and extent of unwinding the 76-bp DNA was also enhanced at pH 6.5 versus pH 7.5, but the 76-bp substrate was still unwound less efficiently than the 52-bp substrate under these conditions (data not shown). The DrSSB protein further enhanced the unwinding rate of the 52-bp substrate by about 2-fold at pH 6.5 (Fig. 8C). The unwinding rate also increased in the presence of E. coli SSB, but only by about 30–60% (data not shown).



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  		FIG. 8.
pH dependence of 52-bp substrate unwinding. A, reaction mixtures contained 25 mM sodium PIPES buffer, adjusted to the indicated pH with NaOH, 2 mM magnesium acetate, 2 mM ATP, 1 nM of the 52-bp substrate (Table I), and 10 nM RecD. B, reaction mixtures contained 25 mM sodium PIPES buffer, pH 6.5, and other conditions as in A, with 10 nM RecD (closed circles), 1 nM RecD (open circles), and 0.1 nM RecD (closed triangles). C, reaction mixtures were as in B with 0.1 nM RecD and 390 nM DrSSB protein (closed circles) or no DrSSB (open circles).

 
We then compared other enzymatic activities of RecD at pH 6.5 and 7.5. The dramatic increase in unwinding efficiency of the longer DNA at pH 6.5 versus 7.5 is not a result of enhanced ATP hydrolysis or intrinsic unwinding at the lower pH. Thus, ATP hydrolysis was slightly greater at pH 7.5 (9.0 (±0.3) ATP hydrolyzed/sec/RecD) than at pH 6.5 (6.7 (±0.3) ATP/sec/RecD), with 0.01–1 nM RecD and 10 µM of a 17-nt ssDNA cofactor. Unwinding of the 5'-tailed 20-bp substrate was only about 2-fold faster at pH 6.5, with 2 mM Mg2+ (4.7 (±0.7) bp/RecD/sec; data not shown) than at pH 7.5 and 10 mM Mg2+ (see above). Interestingly, the unwinding of the 20-bp DNA with a forked end was about 2-fold slower at pH 6.5 than at pH 7.5 (data not shown). The most striking observation is that binding to the 12-nt 5'-tailed hairpin DNA is even tighter at pH 6.5 than at 7.5 (Fig. 9). Stronger DNA binding at pH 6.5 is consistent with apparently higher processivity at pH 6.5 than at pH 7.5. The enzyme would be able to stay bound to the substrate until it is unwound completely and detected as separate single strands in the gel unwinding assay. Nevertheless, unwinding of the 52- and 76-bp substrates is significantly slower than for the 20-bp substrates under all conditions we have tested.



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  		FIG. 9.
DNA binding at pH 6.5 and 7.5. A, binding mixtures contained 25 mM sodium PIPES, pH 6.5, 2 mM magnesium acetate, 1 nM [5'-32P]hairpin DNA (12-nt 5'-overhang), and the indicated RecD concentrations. Samples were analyzed on a non-denaturing 10% polyacrylamide gel as under "Experimental Procedures," except that the gel buffer was 30 mM histidine/30 mM MOPS, pH 6.6. B, reaction mixtures contained 25 mM Tris acetate, pH 7.5, 10 mM magnesium acetate, 1 nM [5'-32P]hairpin DNA (12-nt 5'-overhang), and the indicated RecD concentrations. Samples were analyzed on a non-denaturing 10% polyacrylamide gel in 1x TBE buffer as described under "Experimental Procedures."

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our study of the D. radiodurans RecD protein is the first examination of any of the RecD-like proteins that are found in this organism and in some other Gram-positive bacteria (Fig. 1). The RecD protein is a DNA helicase whose substrate specificity indicates that it moves with 5'-3' polarity along single-stranded DNA. It requires at least a 5'-single-stranded tail, and does not unwind duplexes with blunt ends or a 3'-single-stranded tail. The best substrate that we have tested has a forked end. Greater unwinding of a duplex with a forked end versus one with only one single-stranded tail was also observed with the Pif1 protein from yeast, a helicase included in the COG0507 RecD helicase family. It was suggested that this preference may indicate that Pif1 works at replication forks ((32), and see below). Unwinding of the longer substrates (52 and 76 bp) is always much less efficient than the 20-bp substrates, indicating that the RecD unwinds DNA with low processivity. Low processivity could indicate that its function is to unwind DNA only over short distances, or there may be other proteins that enhance its processivity, such as we observe for DrSSB, and has been observed for the RecB helicase subunit in RecBCD (33) and the PcrA helicase (34).

The unwinding rates for RecD with the 20-bp substrates are comparable to rates reported for similar substrates with some other helicases, including the bacteriophage T4 Dda helicase (35), the bacteriophage T7 helicase (36), and the E. coli rep helicase (37). The rates that we have measured are initial reaction rates determined at low DNA substrate concentrations, and thus they may be less than the maximal unwinding rate of which the enzyme is capable. The DNA binding measurements showed that little RecD protein bound to the DNA under conditions similar to the unwinding reactions (except that ATP was not present in the binding mixtures). Greater unwinding rates might be observed either at higher DNA concentrations (Vmax conditions), or at higher protein concentrations under single turnover conditions. Some helicases do unwind DNA much more rapidly than does RecD, including RecBCD (38) and E. coli UvrD (39).

It should be noted that the D. radiodurans RecD protein is expressed with a C-terminal hexahistidine tag to facilitate its purification (see "Experimental Procedures"). The fact that we detect high unwinding activity under some conditions indicates that the His tag does not interfere substantially with the RecD function. However, we cannot rule out the possibility that the His tag might affect the rate or processivity of unwinding, or a putative interaction with DrSSB. Further study with the unmodified RecD would clarify these issues (the His tag encoded by the pET-21a vector does not include a protease recognition site that would allow it to be removed from the RecD protein).

The RecD subunit of the E. coli RecBCD enzyme is also a helicase with apparent 5'-3' polarity (19), and it is thought to act as a helicase during RecBCD enzyme activity (19, 40). However, RecD is dispensable for unwinding, since RecBC enzyme is also a helicase (41, 42), and the major effect of removing RecD from RecBCD is on the nuclease activity of RecBCD. Loss of the RecD subunit reduces the nuclease activity to very low levels that are not biologically relevant (43, 44). Thus, the phenotypes of E. coli recD-null mutants are quite different from those of recB or recC null mutants. Cells with null mutations in recB or recC, and recBCD deletion mutants, are deficient in homologous recombination, sensitive to various DNA-damaging agents, and susceptible to infection by certain mutant bacteriophages that are normally destroyed by the RecBCD nuclease activity (45, 46). The recD-null mutants are recombination proficient and resistant to DNA-damaging agents (43, 46), presumably due to activity of RecBC in these mutants (47). The recD mutants are however susceptible to phage infections because of the low level of nuclease activity in the mutants (43).

The E. coli RecD protein is largely insoluble when overexpressed and must be purified in denatured form and then renatured in order to detect its enzymatic activity (18, 19). The D. radiodurans RecD protein is soluble when overexpressed and purified, has high enzymatic activity, and retains activity during prolonged storage. Part of the motivation for the present study was to have a RecD protein that could be studied biochemically. The D. radiodurans RecD protein may be suitable for structure-function study of a RecD-like helicase.

Properties of Other RecD Family Members—Some examples of the hits from a BLAST search using either D. radiodurans RecD or E. coli RecD as the query, and the E-values that indicate the significance of the alignment, are shown in Table II. The list includes definite (E. coli, Haemophilus influenzae) or likely (Chlamydia muridarum RecD (2)) subunits of RecBCD enzymes, and proteins from B. subtilis and C. muridarum (RecD (1)) that group closely with D. radiodurans RecD in COG0507, and are unlikely to be RecBCD-enzyme subunits. Others (Rrm3 and its close relative Pif1 from yeast, and proteins related to the TraA conjugative transfer protein of the Ti plasmid in Agrobacterium tumefasciens (48)) are helicases that are more distantly related to the RecD proteins and are also included in COG0507. The weak similarity of bacteriophage T4 Dda helicase and of human helicase B to the RecD-like proteins has been noted by others (49, 50).


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  		TABLE II
Selected results of BLAST searches using D. radiodurans or E. coli RecD protein as the query

The non-redundant GenBankTM protein sequence database was searched using BLASTp 2.2.9 (64), at www.ncbi.nlm.nih.gov/BLAST/, with either D. radiodurans RecD (DrRecD) or E. coli RecD (EcRecD) as the query sequence.

 
Several of the proteins in Table II, including Rrm3 and Pif1 (51–53), helicase B (50), and Dda (54), have been found to be 5'-3' helicases, like the D. radiodurans and E. coli RecD proteins. These proteins are all thought to be involved in DNA replication. Pif1 and Rrm3 affect replication fork movement through certain parts of the chromosome, including ribosomal RNA genes, telomeres, and others (53, 55). Dda may enable replication fork movement past bound proteins (56) and may also be involved in initiation of bacteriophage T4 replication (57). Helicase B is also thought to be involved in DNA replication, although its precise role is unknown (50). The conjugative transfer proteins (e.g. TraA (48), TrwC from plasmid R388 (58)) are large (~1000 amino acid residues) and contain several functional domains. An N-terminal domain cleaves a specific phosphodiester bond in the oriT region of the plasmid and remains covalently bound via a tyrosine residue to the 5'-phosphoryl group of the cleaved DNA. The C-terminal helicase domain then unwinds the double-stranded plasmid, acting as a 5'-3' helicase, to provide the single-stranded DNA that is transferred to the recipient cell (58).

The function of RecD in D. radiodurans is not known. The recD mRNA was detected in an experiment that measured global gene expression in D. radiodurans using a DNA microarray (59). The recD mRNA expression level did not increase after gamma irradiation of a stationary phase culture (in fact, it decreased slightly). This may suggest that the D. radiodurans RecD protein is not involved in DNA repair. However, transcription of genes encoding proteins involved in DNA repair does not necessarily increase in response to DNA damage (60). For example, the recB, recC, and recD genes of E. coli are not induced after UV irradiation (61). The mRNAs from the two recD genes were also detected in the transcriptome of Chlamydia trachomatis (62). The RecD protein itself was detected in the D. radiodurans proteome by mass spectrometry, but only in cells grown under certain conditions (e.g. stationary phase cells (63)).


   FOOTNOTES
 
* This work was supported by Grant GM39777 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 301-405-1821; Fax: 301-314-9121; E-mail: dj13{at}umail.umd.edu.

1 The abbreviations used are: dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; DrSSB, D. radiodurans single-stranded DNA-binding protein; MOPS, 3-(N-morpholino)-propanesulfonic acid; PIPES, 1,4-piperazine bis-(2-ethanesulfonic acid); TLC, thin layer chromatography; nt, nucleotide; BSA, bovine serum albumin; TBE, Tris borate/EDTA. Back


   ACKNOWLEDGMENTS
 
We thank Prof. Mike Cox for the generous gift of the D. radiodurans SSB protein.



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