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Nucleic Acids Research, 2003, Vol. 31, No. 20 5917-5929
© 2003 Oxford University Press

Hexameric RSF1010 helicase RepA: the structural and functional importance of single amino acid residues

Günter Ziegelin1,2, Timo Niedenzu2, Rudi Lurz1, Wolfram Saenger2 and Erich Lanka*,1

1 Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, Dahlem, D-14195 Berlin, Germany and 2 Institut für Kristallographie, Freie Universität Berlin, D-14195 Berlin, Germany

*To whom correspondence should be addressed. Tel: +49 30 8413 1696; Fax: +49 30 8413 1130; Email: lanka{at}molgen.mpg.de

Received July 8, 2003; Revised August 15, 2003; Accepted August 26, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the known monoclinic crystals the 3-dimensional structure of the hexameric, replicative helicase RepA encoded by plasmid RSF1010 shows 6-fold rotational symmetry. In contrast, in the cubic crystal form at 2.55 Å resolution described here RepA has 3-fold symmetry and consists of a trimer of dimers. To study structure–function relationships, a series of repA deletion mutants and mutations yielding single amino acid exchanges were constructed and the respective gene products were analyzed in vivo and in vitro. Hexamerization of RepA occurs via the N-terminus and is required for NTP hydrolysis. The C-terminus is essential both for the interaction with the replication machinery and for the helicase activity. Functional analyses of RepA variants with single amino acid exchanges confirmed most of the predictions that were based on the published 3-dimensional structure. Of the five motifs conserved in family 4 helicases, all residues conserved in RepA and T7 gp4 helicases participate in DNA unwinding. Residues K42, E76, D77, D139 and H178, proposed to play key roles in catalyzing the hydrolysis of NTPs, are essential for RepA activity. Residue H178 of motif H3 couples nucleotide consumption to DNA strand separation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA helicases are ubiquitous motor proteins that utilize the energy obtained by the hydrolysis of nucleoside triphosphates (NTPs) to unwind double-stranded nucleic acids. The proteins play key roles in a variety of biological processes like DNA replication, recombination, repair and transcription. The enzymes have received increasing attention since it became known that at least six hereditary diseases, like xeroderma pigmentosum and Cockayne syndrome, are caused by variants of DNA helicases or of putative helicases (1). There are two groups of structurally known DNA helicases, one forming hexameric rings that operate at the DNA replication fork to separate both strands of the duplex DNA ahead of the DNA polymerase complexes, whereas the other group includes monomeric or dimeric enzymes.

Plasmid RSF1010 encodes its own replication initiation system, making its replication independent of the host initiation machinery. Proteins RepC and RepB are required for origin recognition and primer synthesis, respectively, whereas RepA is the hexameric replicative helicase essential for RSF1010 replication (2,3). RepA has 5'->3' polarity and requires a forked DNA substrate for optimal activity. Among ribonucleoside triphosphates ATP is the preferred low molecular weight substrate. The pH optimum of the helicase activity is at pH 5.5–6, which corresponds with optimal binding to single-stranded (ss)DNA (3). In contrast to other hexameric helicases RepA assembles into stable hexamers in the absence of any nucleotide or metal cofactor, as demonstrated by chemical cross-linking, gel filtration and electron microscopy (3).

In monoclinic RepA crystals grown at pH 6.0, dimers of hexamers in head-to-head orientation are observed (5). Both image reconstruction of electron microscopy data and the high resolution 3-dimensional crystal structures (2.4 and 1.95 Å) revealed a 6-fold rotational symmetry (3,5,6). The five helicase motifs H1, H1a and H2–H4 conserved in DnaB-like enzymes (7) are present in RepA and spatially clustered around the NTP binding pocket. The proposed catalytic site for ATP hydrolysis is located at the interface of neighboring monomers, with the adenine base being sandwiched between R85 of the NTP binding monomer and Y242 of the adjacent subunit.

Besides RepA, the 3-dimensional structures of several other helicases have been determined, e.g. the monomeric homologs Bacillus stearothermophilus PcrA and Escherichia coli Rep helicases, (8,9), the T7 helicase domain [amino acids 272–566 (10) and 241–566 (11)] and the 130 amino acid RNA binding domain of the E.coli Rho RNA helicase (12). The latter two enzymes are hexamers. A low resolution structure for the hexameric prototype E.coli DnaB has been determined by electron microscopy and 3-dimensional image reconstruction (13), but high resolution data of hexameric replicative DnaB-type helicases are still not available except for a short stretch of an N-terminal domain (14).

For translocation of helicases along DNA a variety of models have been proposed (15; reviewed in 16). For the monomeric or dimeric helicases the ‘inchworm’ model, the principles of which were proposed by Gefter in 1979 (17), has emerged as the most plausible in the light of recent work (reviewed in 18). Although the information on hexameric helicases is lagging behind, a modified version of this model might be applicable to these ring-shaped DNA unwinding enzymes (5,10,11).

Here we report on the high resolution structure of RepA crystallized at pH 7.5 in a cubic space group. Predictions made on the function of defined residues based on this 3-dimensional structure were evaluated experimentally. The variant proteins used for this purpose were generated by single amino acid exchange and deletion mutagenesis. The influence of specific residues proposed to play key roles in ATP hydrolysis and DNA unwinding were analyzed in vivo and in vitro. We discuss the implications for enzyme function of conformational differences of residues that probably bind the ATP between adjacent monomers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial strains and plasmids
Escherichia coli SCS1 [recA1, endA1, gyrA96, thi-1, hsdR17(rK– mK+), supE44, relA1; Stratagene] was used as host for repA overexpression plasmids, for construction of the RSF1010K{Delta}repA plasmid and for complementation experiments. Recombinant plasmids used in this study are given in Table 1. Media were as described previously (19). When appropriate, antibiotics were added at the following concentrations: 100 µg/ml ampicillin (sodium salt) or 30 µg/ml kanamycin sulfate.


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  		Table 1. Plasmids and bacteriophage used in this study
 
Construction of plasmids
For DNA manipulation standard techniques were used (20). In RSF1010, repA is flanked by repB' and repC, encoding the primase and the origin binding protein, respectively. repA was deleted leaving a few 5' and 3' codons in frame to avoid any polar effects. To construct RSF1010K{Delta}repA, the kanamycin-resistant derivative RSF1010K (Table 1) was used. The 2924 bp AflIII–BstEII fragment carrying most of repB', repA and the 5' portion of repC was replaced by two fragments generated by PCR using RSF1010K as template to restore repB' and repC. An AflII site that served to join the PCR fragments was created by changing the leucine codon repA L259 from CTC to CTT. Small 5' and 3' repA remnants were connected in frame encoding a 29 amino acid peptide that consists of RepA residues A1–L9 and L259–A278. The nucleotide sequence of this arrangement was verified by sequencing.

To construct pGZ18-20{Delta}1–5, pGZ18-20{Delta}1–7 and pGZ18-20{Delta}1–9, which lack RepA amino acid residues 1–5, 1–7 and 1–9, respectively, NdeI–HindIII fragments of 970, 964 and 958 bp were generated by PCR using pGZ18-20 as template and inserted into pMS470{Delta}8 prepared with NdeI and HindIII. To construct pGZ18-20{Delta}276–278, pGZ18-20{Delta}274–278 and pGZ18-20{Delta}269–278, which lack the very C-terminal RepA amino acid residues 276–278, 274–278 and 269–278, respectively, EcoRI–HindIII fragments of 901, 895 and 880 bp were generated by PCR using pGZ18-20 as template and inserted into pMS119EH prepared with EcoRI and HindIII. The nucleotide sequence of each amplified DNA fragment was determined and verified according to the published sequence (GenBank accession no. M28829 [GenBank] ; 21).

Point mutations and a single codon deletion (RepA {Delta}F12) were introduced into repA directly using a PCR-based method (22) with pGZ18-20 as template. The primers used were designed by changing as few bases as possible (Table 2). Following mutagenesis the nucleotide sequence of each repA allele was determined. Since formylmethionine is cleaved off post-transcriptionally, alanine occupies RepA amino acid position 1 (21). Therefore, our numbering differs from that used by Niedenzu et al. (5) and Xu et al. (6), who assigned methionine as position 1.


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  		Table 2. Oligonucleotides used for the molecular cloning of RSF1010K{Delta}repA and repA mutagenesis
 
In vivo complementation and interference assays
Complementation studies were done by transforming SCS1 harboring a pGZ18-20 derivative that carried a repA mutant allele with RSF1010{Delta}repA. An overnight culture was diluted with medium containing ampicillin (see above) and IPTG (5 µM) to an A600 of 0.1 and grown at 37°C with shaking. At an A600 of 0.4, the cells were harvested and made competent using the CaCl2 method (23). Aliquots of 20 ml of culture were centrifuged and resuspended to result in 1 ml of competent cells. To 100 µl of these cells was added 0.1 µg RSF1010K{Delta}repA (Table 1). Following incubation for 3 h on ice and 2 min at 40°C, 1 ml of medium was added. After incubation for 1 h at 37°C, the cells were diluted appropriately, plated on selective medium containing 5 µM IPTG and incubated overnight at 37°C. The colonies obtained were counted. Interference tests were done accordingly, except that RSF1010K was used instead of RSF1010K{Delta}repA.

Overexpression and purification of proteins
RepA mutant proteins were overproduced and purified using the procedure described previously (3) with the following modification. Chromatography on ATP–agarose as the last purification step was replaced by phenyl Sepharose. A column of phenyl Sepharose was equilibrated with 20 mM Tris–HCl (pH 8.0), 20 mM NaCl and 1 mM DTT (buffer A) containing 1 M ammonium sulfate. The DEAE Sephacel RepA fraction (3) was dialyzed against buffer A, adjusted to 1 M ammonium sulfate and loaded onto the column. The column was washed with buffer A containing 1 M ammonium sulfate and then with buffer A. Proteins were eluted with a gradient of 0–70% ethylene glycol in buffer A. RepA eluted at ~60% ethylene glycol.

Helicase and ATPase assays
A forked helicase substrate based on that of Crute et al. (24) was used. The unpaired 3'-portion of the 53mer oligonucleotide comprised 22 nt, whereas 31 nt were paired to viral M13mp18 DNA. The 5'-end was 32P-labeled. RepA-catalyzed unwinding of double-stranded (ds)DNA was assayed essentially as described (3) at 30°C for 15 min in 20 µl of buffer B [40 mM MES/NaOH pH 5.6, 10 mM MgCl2, 1 mM DTT and 50 µg/ml bovine serum albumin (BSA)] containing 1 mM ATP. The products were separated by electrophoresis on 10% polyacrylamide gels. The radioactivity of the substrate and the displaced oligonucleotide was visualized using phosphor storage technology and quantified by the use of ImageQuant software version 5.0 (Amersham/Pharmacia).

RepA-catalyzed ATP hydrolysis reactions were run for 15 min at 30°C as described (3) in 20 µl of buffer B containing 0.5 mM ATP, 100 nCi [{gamma}-32P]ATP and 1 µg viral M13mp18 DNA. Products were separated by thin layer chromatography and quantified as described above.

Crystallization and X-ray data collection
For crystallization, RSF1010 RepA was purified using a modification of the protocol described in Röleke et al. (4). To obtain crystals of the highest quality, the purity of the final product was further improved by inserting an additional anion exchange chromatography (Q-Sepharose) before the final gel filtration. For crystallization using the sitting drop vapor diffusion method equal volumes of 5 µl of RepA stock solution (26 mg/ml RepA, 10 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.1 mM EDTA) and reservoir solution (100 mM Tris–HCl pH 7.5, 26% PEG400, 100 mM MgSO4) were mixed and equilibrated against reservoir solution. Diffraction data from a native crystal were collected at 100 K using a mar345 Image Plate detector mounted on a Nonius FR571 rotating anode X-ray generator equipped with Osmic focusing mirrors. A mercury derivative was prepared by soaking crystals in reservoir solution with 1 mM o-chloromercurinitrophenol as described (5). MAD data were collected up to 3.0 Å resolution at beamline BW7A, EMBL outstation, DESY/Hamburg, using a mar CCD detector.

Phasing and refinement
All diffraction data were processed with DENZO/SCALEPACK (25). Programs from the CCP4 suite (26) were used for subsequent calculations and model refinement. The single mercury site in the derivative was located by inspection of an anomalous difference Patterson map. A first electron density map was calculated, the starting model taken from the previously published RepA crystal structure (5) was placed manually into the map and the orientation of the individual monomers was fitted. Further model building using the program O (27) and refinement employed the higher resolution native data set (Table 3). The coordinates of the cubic RepA form have been deposited in the RCSB Protein Data Bank under the accession code 1olo [PDB] .


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  		Table 3. Diffraction data collection and refinement statistics
 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A new crystal form of RepA
The improvement in the purification scheme for RepA for crystallization was visible in the PAGE analysis, where even in heavily overloaded lanes nothing but RepA was detectable (not shown). In addition to the described monoclinic RepA crystals (space group P21) grown at pH 6.0 (4,5), previously unobserved crystals of perfect octahedral shape appeared in one of the crystallization screen trials. Following optimization of crystallization conditions, crystals suitable for X-ray diffraction data collection were grown as described in Materials and Methods. The crystals belong to the cubic space group P4332, with two of the RepA subunits in the asymmetric unit (Matthews coefficient 4.8 Å3/Da, 74.4% solvent). The structure was determined by MAD using a mercury derivative (see Materials and Methods) and refined against a native data set at 2.55 Å resolution. The final model consists of 499 residues (249 and 250 for monomers A and B, respectively), 361 water molecules in the solvation shells of the protomers and 60 additional waters. A sulfate ion was bound to the p-loop of each monomer as described (6).

In contrast to the published RepA crystal structures (5,6) comprising pairs of hexameric rings with 6-fold rotation symmetry in head-to-head orientation in the asymmetric unit, the RepA oligomers in the cubic crystal system occur as single hexameric rings. Since the crystallographic C3 axis passes right through the molecule, the hexamers have 3-fold rotation symmetry and the crystallographic asymmetric unit contains only two monomers. Although a few more of the C-terminal amino acids (A262–S270) could be modeled here that were not seen in the monoclinic RepA (5), the loop formed by residues 180–199 containing half of the helicase motif H4 still had a poorly defined electron density and could not be modeled (6). A view from the top of the hexamer is shown in Figure 1a. In cubic RepA crystals, two different conformations of the monomer–monomer interface are found that feature different orientations of the side chain of Y242 (Fig. 2). A closed conformation is observed for the A–B interface, whereas the B–A interface shows a more open conformation (Fig. 2a and b). ATP was modeled manually into the RepA structure with a similar procedure to that described in Niedenzu et al. (5). The overlay of the open and the closed conformation shows that both conformations can be converted into each other by a rotation of the left protomer (monomer A at the A–B interface; see Fig. 2a) around the axis of helix F (Fig. 2c). This movement opens and closes the cleft between the monomers, which is the proposed binding site for the purine moiety of ATP. The position of the N-terminal hook (see below and Fig. 3) to the neighboring protomer (monomer B at the A–B interface; see Fig. 2a) is not affected by this movement.



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  		Figure 1. Localization of mutations in the RepA structure. (a) View of the RepA hexamer from the C-terminal side (top) of the ring. Monomer A is shown in green, monomer B in orange. (b) 3-Dimensional structure of the RepA monomer viewed from inside the ring. Positions of mutated amino acids are highlighted, N-terminal deletions are marked. The conserved family 4 helicase motifs are colored: H1, green; H1a, orange; H2, blue; H3, red; H4, violet. Dotted lines represent regions of poorly defined electron density of segment 180–199. (c) The bar represents RepA consisting of 278 amino acid residues. The positions of exchanged residues are given. Single dots (monomer A) and double dots (monomer B) mark the proposed ATPase catalytic residues at the monomer–monomer interface. Single and double colons highlight the residues of monomers A and B, respectively, that may sandwich the adenine in the closed conformation (see also Fig. 2a). Within the bar, regions of poorly defined electron densities are marked in black. (d) Sequence comparison of residues structurally equivalent according to the output of MAPS (http://bioinfo1.mbfys.lu.se/TOP/maps.html) in RepA, T7 gp4, E.coli RecA, F1 ATPase subunit B, E.coli Rep and B.stearothermophilus PcrA. Above the RepA sequence the corresponding {alpha}-helices A–G and ß-strands 1–9 are shown by boxes and arrows, respectively. Dotted lines represent the disordered segments. The conserved motifs H1, H1a, H2, H3 and H4 are boxed and colored according to (a). Amino acids exchanged are highlighted in cyan. Deleted N-terminal and C-terminal residues are in cyan boxes.

 


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  		Figure 2. Subunit interface: the catalytic center of the NTPase. Stereo views of sections of the two different interfaces of the monomer–monomer interface in the cubic crystal structure are shown. ATP was modeled into the structure as described (5). Monomer A is shown in green, monomer B in orange. The side chains of amino acids mutated for this study are shown as stick models. The dotted line indicates the disordered segment 180–199. (a) The closed conformation at the A–B interface. (b) The open conformation at the B–A interface. (c) Overlay of the closed conformation (dark colors) and the open conformation (light colors). Monomer A of the open interface was superposed on monomer B of the closed interface. Helix F is marked.

 


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  		Figure 3. Linkage between adjacent monomers that results in stable interaction. The proposed dimerization regions of two adjacent monomers are given in green and orange (monomers A and B, respectively). The dotted lines represent hydrogen bonds. The interactions are supported/enhanced by van der Waals contacts (not indicated). Oxygen and nitrogen atoms are in red and blue, respectively. The red dot represents a water molecule that fits in between the I6 backbone oxygen of monomer A and the I107 backbone nitrogen of monomer B.

 
The RepA N-terminus is essential for hexamer formation
The remarkable stability of the RepA hexamers might result from interactions of the hook-like N-terminus of one monomer with a central portion of the neighboring molecule (5) (Fig. 3). To test this hypothesis, deletion derivatives were constructed lacking five, seven or nine N-terminal amino acid residues (Figs 1 and 3). Since F12 is situated at the end of helix A, which is the central part of the N-terminal hook (Fig. 3), we predict that the angle would widen if this residue was deleted. Then the stability of the hexamer might be altered. Therefore, the derivative RepA {Delta}F12 lacking only residue F12 was made. To study the oligomeric state of these RepA variants, they were purified and subjected to velocity sedimentation in a glycerol gradient. Subsequently aliquots of the collected fractions were analyzed by gel electrophoresis (Fig. 4). Both wild-type RepA and RepA {Delta}F12 behaved as hexamers of ~180 kDa. However, compared to the wild-type protein, RepA {Delta}F12 was less stable, since protein sedimenting with reduced velocity was observed in significant amounts. Obviously the presence of F12 stabilizes the RepA hexamer significantly through hydrophobic interactions (Fig. 3).



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  		Figure 4. Glycerol gradient centrifugation of RepA proteins. A volume of 150 µl of purified protein (RepA wild-type and RepA {Delta}F12, 0.4 mg each; RepA {Delta}1–7, 0.12 mg) was layered onto a 3.7 ml, 15–35% linear glycerol gradient in buffer containing 20 mM Tris–HCl pH 7.6, 20 mM NaCl, 2 mM DTT, 0.1% Brij-58 and 0.1 mM EDTA and centrifuged at 270 000 g for 15 h at 4°C. After the run, the bottoms of the tubes were pricked with a needle and fractions were collected. Aliquots were taken and analyzed for protein content by gel electrophoresis and for ATPase activity in the presence and absence of ssDNA as described in Materials and Methods. Open and filled circles, wild-type RepA without and with ssDNA; filled squares, RepA {Delta}F12 both with and without ssDNA; filled triangles, RepA {Delta}1–7, both with and without ssDNA. Note the logarithmic scale of the ordinate. Aliquots of the fractions were electrophoresed in 15% polyacrylamide gels containing 0.1% SDS. The gels were stained with Serva Blue R and scanned. Fraction numbers of the gradient correspond to slot numbers of the gel. The direction of sedimentation is from right to left. 1, catalase (1250 kDa, s20,w = 11.3 S), 2, aldolase (2158 kDa, s20,w = 7.8 S) and 3, BSA (368 kDa, s20,w = 4.4 S) were run in parallel as standards, the peak positions are marked by arrows.

 
RepA {Delta}1–7 sedimented with a coefficient below that of BSA (68 kDa), suggesting the presence of monomeric or dimeric RepA subunits (Fig. 4). In the electron microscope, RepA {Delta}F12 and RepA {Delta}1–5 had a hexameric ring-like structure comparable to wild-type RepA, whereas for RepA {Delta}1–7 and for RepA {Delta}1–9 no hexamers were observed (Table 4 and data not shown). These data show that hexamerization occurs via the very N-terminus of each monomer with position I6 and/or N7 being essential, whereas residues A1–P5 are dispensable. Consequently, at least part of the oligomerization domain is assigned to the N-terminal end of the molecule A. Its N-terminal segment A1–N7 interacts with the neighboring molecule B through {alpha}-helices C and D and ß-strand 3, which form four hydrogen bonds, one water-mediated (Ile107N-H···W···OIle6) and the other three direct (Ile6N-H···OIle107; Gln108N{epsilon}-H···OHis3; His3N{epsilon}-H···OIle111). In addition, I6 and I8 of monomer A form hydrophobic interactions with I82, I107, L86 and L105 of monomer B, respectively (Fig. 3).


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  		Table 4. In vivo and in vitro properties of RepA mutant proteins
 
The replication-deficient plasmid RSF1010K{Delta}repA served as a tool for the phenotypic evaluation of repA mutations in vivo
The repA deletion derivative RSF1010K{Delta}repA was constructed as described in Materials and Methods. Since repA is essential for RSF1010 replication, such a construct is only viable in the presence of repA in trans. Consequently the ability of the repA variants to support RSF1010 replication in vivo could be tested by complementation. Introducing RSF1010K{Delta}repA DNA into E.coli strain SCS1(pGZ18-20) which provides repA in trans restores the replication ability of RSF1010K{Delta}repA. Accordingly, RSF1010K{Delta}repA was introduced into each strain that carried a repA allele. This in vivo system (Fig. 5) was used to analyze the replication properties of all repA mutations generated and served as a tool to select replication-deficient repA variants. The mutated repA genes were overexpressed and the encoded products were purified for in vitro studies.



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  		Figure 5. Scheme of the in vivo repA complementation assay. SCS1 cells harboring a plasmid that carries a repA allele were transformed with RSF1010K{Delta}repA. Then the cells were plated onto solid medium containing ampicillin and kanamycin. If the mutation leaves RepA protein active, RSF1010K{Delta}repA replicates, confers resistance to kanamycin and the cells form colonies. If the mutation renders RepA inactive, RSF1010K{Delta}repA cannot replicate and no colonies are observed.

 
A stable hexamer is a prerequisite for RepA activity
Purified RepA {Delta}F12 hydrolyzed ATP in the presence of M18mp18 ssDNA less efficiently than wild-type protein, whereas RepA {Delta}1–7 showed rather minor activity, although clearly above background. To separate minor impurities from these preparations, the ATPase activity of different fractions of the glycerol gradient centrifugation (Fig. 4) was measured. For RepA {Delta}F12 the peak of maximum activity corresponded to the protein peak, whereas for the monomeric/dimeric RepA {Delta}1–7 (see above), the peak of the residual activity did not coincide with the protein peak, indicating that the observed minor ATP hydrolysis was mainly due to an impurity (Fig. 4). RepA {Delta}F12 is less stable than the wild-type protein. The ATPase activity of this derivative was not stimulated by ssDNA. In the absence of ssDNA, the hydrolyzing activity of RepA {Delta}F12 was reduced 3-fold compared to the wild-type protein (Fig. 4). Since for the RepA {Delta}F12 variant no helicase activity was detectable (not shown), a stable RepA hexamer is required for stimulation of the ATPase activity by ssDNA and for unwinding of dsDNA.

Of the 5'-terminal deletions in repA (Fig. 1b and d), only the {Delta}1–5 derivative was able to complement RSF1010K{Delta}repA, whereas the {Delta}1–7 and {Delta}1–9 deletions were not (Table 4). Also, repA{Delta}F12 failed to restore replication of RSF1010K{Delta}repA. Hence, interactions with the replisome did not stabilize the labile hexamers of RepA {Delta}F12 in vivo to fulfill its role in replication, although this protein still possesses ATPase activity (Fig. 4). These data coincide with those for the helicase activity of the respective purified protein (Table 4) and demonstrate that a stable hexamer is not only essential in vitro for helicase activity but also in vivo for RSF1010 replication.

Alanine scan of single amino acid residues proposed to play key roles
Nine residues proposed to be essential for RepA function (5) were substituted by alanine (Fig. 1c). These substitutions include amino acids within all five motifs conserved in helicase family 4 (Fig. 1d). Y242 of one monomer and R85 of the neighboring monomer were proposed to bind the purine moiety of ATP at the interface between RepA monomers (Fig. 2a and b). For steric reasons R253 might serve as an alternative for R85 (Fig. 2a). R206 points towards the {gamma}-phosphate of the nucleotide and forms an ‘arginine finger’ analogous to that observed for GTPase-activating proteins and also discussed for T7 gp4 helicase (10). K42, E76 and D139 are conserved in RepA, RecA and T7 gp4 and proposed to function as essential residues of the catalytic center. H178 is located in the vicinity of the {gamma}-phosphate of ATP and conserved in RepA and T7 gp4. D77 was changed to alanine since its carboxyl group was likely to interact with N6 of ATP (Fig. 2b).

All these nine repA mutations failed to complement replication of RSF1010K{Delta}repA (Table 4). Accordingly, all nine purified RepA variants were inactive in hydrolyzing ATP except for RepA H178A (see below and Table 4). RepA Y242T, which in contrast to RepA Y242A still provides a hydroxyl residue, was inactive, as was the Y242A variant. Therefore the aromatic side chain of tyrosine proposed to bind the purine residue of ATP is essential for enzymatic function.

Decoupling ATPase and helicase activity
Residue H178 is part of the conserved helicase motif H3 and is located at the end of ß-strand 5 (Fig. 1b and d; 5). H178 was suggested to be part of the catalytic centers of RepA helicase. Since it is in close proximity to the {gamma}-phosphate of ATP (Fig. 2a and b) it might act as a sensor for this phosphate to distinguish ATP from ADP (10). While the ATPase activity of RepA H178A was similar to that of the wild-type protein, its helicase activity was only marginally above background levels: at a monomer concentration of 50 pM, less than 5% of the wild-type activity was observed (Fig. 6). Helicase activity of RepA H178A was neither increased notably by larger amounts of protein nor by higher ATP concentrations in the reaction mixture. Since the ATPase activity of RepA H178A was stimulated by the addition of ssDNA, the RepA H178A hexamer is suggested to still be able to move along ssDNA comparable to the wild-type protein. Hence, the results demonstrate that H178 is dispensable for catalyzing the hydrolysis of ATP, but plays an essential role in DNA strand displacement.



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  		Figure 6. Helicase activity of wild-type and mutant RepA proteins. Purified RepA proteins were assayed for the ability to displace a radioactively labeled oligonucleotide bound to viral M13mp18 DNA as described in Materials and Methods. Circles, RepA wt; squares, RepA H178A; triangles, RepA Q192A; dots, RepA {Delta}269–278. The percentage of the oligonucleotide displaced was determined. For each protein, the values plotted were averaged from at least two independent experiments.

 
Hexamerization is independent of proposed catalytic amino acids
Hexamerization is required for RepA to function as a motor protein. Since each of the six ATPase catalytic centers of the RepA hexamer is constituted by two adjacent monomers, local alteration of the 3-dimensional structure at the contact surface may influence oligomerization and the orientation of subunits, besides inhibiting catalysis. Therefore the oligomerization state of the replication-deficient RepA variants was studied by electron microscopy. They all maintained a hexameric ring-like structure comparable to wild-type RepA irrespective of the position or the proposed task of the residue exchanged (summarized in Table 4). Consequently, none of the amino acids suggested to participate in catalysis are essential to maintain the hexameric state.

One of the negative charges of the glutamate triplet in loop L2 is essential for RepA enzymatic activity
The triplet E147–E148–E149, located in the loop L2 formed between ß-strand ß4 and {alpha}-helix {alpha}F, confines the narrowest part of the central channel of the hexamer. To answer the question as to whether the negative charge of the side chain is important, each glutamate was changed to the uncharged glutamine. Using the in vivo complementation of RSF1010K{Delta}repA assay outlined in Figure 5, only repA E149Q resulted in a negative phenotype, whereas E147Q and E148Q behaved similarly to wild-type repA (see above). Since E149Q still forms hexamers but lacks ATPase and helicase activity, the carboxyl group exerts an important function in the protein. In the cubic structure, this carboxyl group chain forms a hydrogen bridge with H177, thereby fixing the position of helix F relative to the central ß-sheet (Fig. 1b).

Do mutant RepA monomers form heterooligomers with wild-type protein?
Transdominance was found for hexameric replicative helicases like DnaB, T7 gene 4 protein or SV40 large T-antigen (28–30). If mutant and wild-type protein monomers assembled into an inactive heterohexamer that inhibits the replication process, a dominant lethal phenotype was observed. The system used to study the behavior of the mutations was identical to that outlined in Figure 6 except that RSF1010K was used. Each strain containing a repA allele was transformed with the replication-proficient and selectable RSF1010K as described in Materials and Methods. None of the repA mutations that were functional in vivo showed any interference with the wild-type (Table 4). Even for RepA E148Q and RepA Q193E, which restored replication of RSF1010K{Delta}repA less efficiently than the wild-type gene, no transdominant effect was observed. Therefore, in the case that mixed complexes of mutant and wild-type RepA exist, these heterooligomers are both enzymatically active and able to functionally interact with the replication machinery. For the majority of the replication-deficient repA mutations, including all N-terminal and C-terminal deletion derivatives, no or only slight transdominance was observed (Table 4). However, the mutations repA K42A, repA D77A, repA D139A and repA R206A in helicase motifs H1, H1a, H2 and H4, respectively (Fig. 1d), strongly interfered with wild-type repA. Compared to the wild-type, the number of colonies obtained with repA K42A, repA D77A and repA R206A was reduced 100-fold, with the D139A mutation even 1000-fold (Table 4). Hence, in vivo the enzymatically inactive variants RepA K42A, RepA D77A, RepA D139A and RepA R206A assemble into complexes with wild-type protein, which are unsuitable to promote RSF1010K replication.

The amide of glutamine Q192 is essential for RepA function in vivo, but irrelevant for enzymatic activity
To explore the possible roles of the glutamines of the triplet Q191–Q192–Q193 located in the disordered segment of 20 residues (S180–S199) between ß5 and ß6 each of the residues was changed to glutamic acid. Only the Q192E mutation showed a negative phenotype in repA complementation. repA Q191E behaved like the wild-type, but repA Q193E was 10-fold less efficient in restoring the replication ability of RSF1010K{Delta}repA (see below). Since the in vitro activities of purified RepA Q192E proved to be comparable to wild-type protein (Fig. 6), we speculate that this residue might be involved in the interaction with other replication proteins.

The C-terminus is required for RepA enzymatic activity and interacts with the replication machinery
To test whether the flexibility of the RepA C-terminus is of functional importance for motoring, derivatives lacking three, five and ten C-terminal residues were constructed. Since all three C-terminally truncated RepA proteins assemble into hexamers, the C-terminal 10 residues are dispensable for hexamerization (Table 4). RepA {Delta}276–278 and RepA {Delta}274–278 lacking three and five amino acids, respectively, behaved similarly to the wild-type protein concerning ATP hydrolysis and DNA unwinding (data not shown). Although RepA {Delta}269–278 lacking 10 residues still hydrolyzes ATP, the DNA unwinding capability was only marginally above background (Fig. 6). Addition of higher amounts of this protein did not result in a significantly higher helicase activity. Therefore, the RepA helicase domain extends into the C-terminus except for the last five C-terminal residues that do not contribute to helicase activity. In contrast to the enzymatic activities, none of the three C-terminal variants was functional in vivo in restoring the replication ability of RSF1010K{Delta}repA (Table 4). This demonstrates that even the three very C-terminal residues are essential for RSF1010 replication and suggests that the C-terminus interacts with RSF1010-encoded and/or host-encoded replication proteins.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Domain structure of RepA
The combination of the data of this study yielded a partial domain structure of RepA that includes the residues for hexamerization, for interaction with the low molecular weight substrate ATP and regions possibly involved in the interaction with replication proteins of RSF1010 and the host (Fig. 7). We consider the unstructured flexible 20 residue region (180–199) of RepA as the most interesting portion of the molecule since it is likely to constitute the moving part(s) of the motor protein. This stretch of the protein points to the central hole of the hexamer, which is proposed to enclose or embrace the DNA single strand on which the relative movement takes place, driving the strand separation process (31). To determine the structure of the flexible region in RepA additional work is required that would arrest the residues in one conformation by means of mutations or by the addition of tightly binding effector molecules. A comparison with the corresponding region of the T7 gp4 structure shows that in the monomer a similarly unstructured region is present at an almost identical position in motifs H3 and H4 (10), whereas in the hexameric structure the same region is clearly defined (11). Since in RecA the equivalent loop proposed to be involved in DNA binding is also unstructured (32), we speculate that this segment in RepA may only be arrested and of defined structure in the presence of a suitable ssDNA substrate. Since the distance between the C{alpha} atoms of the flanking amino acids positions 179 and 200 is 11.2 Å, there is no space for a single {alpha}-helical connection of ~30 Å corresponding to these 20 disordered residues. Otherwise, in this region an extensive conformational change of RepA would have to take place.



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  		Figure 7. RepA domains for protein–protein interaction. Above and below the bar, which represents RepA, boxes mark the domains for hexamerization and interaction with host and/or plasmid-encoded replication proteins. The N-terminal hexamerization domain around positions 1–12 of one monomer interacts with the counterpart of the adjacent monomer around positions 107–111 given below the bar (for details see Fig. 3). K42 marks the position of the Walker A motif.

 
Stability of the hexameric ring structure of RepA
Since no cofactors like divalent cations or nucleotides are needed to sustain the hexameric ring structure of RepA, it proves to be robust under our experimental conditions in vitro, contrasting with other hexameric helicases that require cofactors to stabilize the ring structures. The high stability of the ring architecture of RepA is obviously associated with hooking up the N-terminus of one subunit to an eye of the adjacent subunit by hydrogen bonding and several hydrophobic interactions. If the inter-linkage of subunits is abolished by deletion of the N-terminal seven amino acids, the ring structure and in parallel the enzymatic activity are lost. This demonstrates that the ring structure is essential for RepA function in vivo and in vitro. In other systems similar observations were made: hexamerization is also a prerequisite for DNA unwinding activity of the T7 helicase, since deletions at the N-terminus of the helicase domain prevent hexamerization and abolish helicase activity (33).

Mechanistic implications of the two conformations observed at the subunit interfaces
There are additional interaction surfaces between adjacent subunits that contribute to ring stability and/or enzymatic activity. The hook–eye interaction to hold the ring structure together appears to be identical in the A–B and B–A interfaces, whereas differences are obvious in the proposed binding site for the purine moiety of ATP (Fig. 2). Although the major part of the ATP binding site resides in one subunit, the adjacent subunit seems to contribute considerably to ATP interaction mainly by residues Y242 and R206 at the interaction interface. An open and a more closed conformation are arranged in alternating positions in the cubic structure of RepA, demonstrating the existence of three equivalent dimers in the hexameric ring structure associated with two types of ATP binding sites. This finding is also in agreement with biochemical studies which showed that the ATP binding sites in RepA are not identical but three are of low and three of high affinity for ATP (34). Mechanistically this is probably important because the conversion of the open into the closed type adenine binding site may lead to some kind of a pulsation of the molecule that could periodically change the diameter or the shape of the central hole. Such conformational changes may induce the relative movement between RepA and DNA.

Motif H4 was proven to be part of the ssDNA binding domain of the T7 gp4 helicase (35). In RepA, R206, which is located in a position suitable to serve as an ‘arginine finger’, is part of H4. This suggests that binding of ssDNA alters the conformation of H4, pushing R206 towards the ATPase active site, thereby stimulating ATP hydrolysis. Since RepA R206A is ATPase-deficient, the ‘arginine finger’ appears to be required for ATP hydrolysis.

Coupling of ATPase and translocation via conformational switching
Of particular importance seems to be residue H178 as part of the ATP binding pocket that may act as a sensor for the {gamma}-phosphate. Replacement of H178 by alanine renders RepA completely inactive in vivo. In addition, the in vitro properties are interesting because RepA H178A is the only variant that still hydrolyses ATP in an ssDNA-dependent manner but lacks helicase activity. This variant still binds to viral M13 DNA as does the wild-type protein (data not shown). In helicases the hydrolysis of NTPs is generally thought to be coupled to the movement along a single DNA strand (reviewed in, for example, 16,18,36). One intriguing explanation for the effect observed for RepA H178A is that the released energy cannot be utilized for DNA unwinding although the translocation on ssDNA apparently takes place, as indicated by the stimulation of ATPase activity. However, it is conceivable that the fork mimicked by the non-base paired tail of the substrate acts as an insurmountable obstacle for the H178 variant.

Alternatively, the coupling between the ATPase and the concerted binding/release of ssDNA required for translocation of the single strand is lost. The histidyl residue at the end of motif H3 (H178) is positionally conserved in RepA and T7 gp4 and in close proximity to the bound {gamma}-phosphate of the NTP (Fig. 1d; 5,10,11). In analogy to the suggestion made for T7 gp4 (10), H178 may act as a conformational switch or sensor for the {gamma}-phosphate of NTP. Such conformational switching has already been proposed for the strand exchange activity of RecA (37) and the DNA unwinding activity of the monomeric helicase PcrA (8) and the RNA helicase NS3 (38). In DnaB proteins, PcrA and RecA, the positional equivalent to H178, is a conserved glutamine. For Bacillus subtilis DnaB, Soultanas and Wigley demonstrated by site-directed mutagenesis that substitution of this glutamine results in a protein proficient in ATPase activity but mostly deficient in DNA unwinding (39). Depending on the presence or absence of a {gamma}-phosphate, the ssDNA binding site, part of which is located in loop L2 that connects motifs H3 and H4, and motif H4 (Fig. 1d) are altered from high to low affinity and vice versa. If this switch is lost in RepA H178A, the affinity for ssDNA cannot be modulated. The enzyme is left in a non-functional static state with at least one catalytic ATPase active site activated by eventually bound ssDNA.

Interaction of RepA with other proteins
Two segments of RepA play vital roles in the interaction with the replication machinery: (i) the very C-terminus, since deletion of three C-terminal residues abolishes RSF1010 replication without affecting the enzymatic activities (Table 4 and Fig. 7); (ii) the flexible loop (positions 180–199) containing one half of motif H4. Q192 is hypothesized to be an essential residue in protein–protein interaction, since the in vitro activities of RepA Q192E are comparable to wild-type protein, however, in vivo the variant is unable to complement repA (Table 4). SSB protein and the polymerase III holoenzyme have to be taken into account as interaction partners. Since in other replication systems primase and helicase are interacting (E.coli DnaG–DnaB, phage T4 gp61–gp41) or even located on a single polypeptide chain (phages T7 gp4, P4 gp{alpha}), the most likely interaction partner for RepA helicase is the RSF1010-encoded RepB primase, which could not be substituted by the host primase DnaG. Another promising candidate is the RSF1010-specific RepC origin binding protein. We will purify RepB and RepC to experimentally prove these predictions.


   ACKNOWLEDGEMENTS
 
We thank Hans Lehrach for generous support. The expert technical assistance of Marianne Schlicht is greatly appreciated. This work was supported by European Commission grant QLRT-1999-30634 to E.L. and W.S.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. van Brabant,A.J., Stan,R. and Ellis,N.A. (2000) DNA helicases, genomic instability and human genetic disease. Annu. Rev. Genomics Hum. Genet., 1, 409–459.

  2. Scherzinger,E., Haring,V., Lurz,R. and Otto,S. (1991) Plasmid RSF1010 DNA replication in vitro promoted by purified RSF1010 RepA, RepB and RepC proteins. Nucleic Acids Res., 19, 1203–1211.[Abstract/Free Full Text]

  3. Scherzinger,E., Ziegelin,G., Bárcena,M., Carazo,J.M., Lurz,R. and Lanka,E. (1997) The RepA protein of plasmid RSF1010 is a replicative DNA helicase. J. Biol. Chem., 272, 30228–30236.[Abstract/Free Full Text]

  4. Röleke,D., Hoier,H., Bartsch,C., Umbach,P., Scherzinger,E., Lurz,R. and Saenger,W. (1997) Crystallization and preliminary X-ray crystallographic and electron microscopic study of a bacterial DNA helicase (RSF1010 RepA). Acta Crystallogr., D53, 213–216.

  5. Niedenzu,T., Röleke,D., Bains,G., Scherzinger,E. and Saenger,W. (2001) Crystal structure of the hexameric replicative helicase RepA of plasmid RSF1010. J. Mol. Biol., 306, 479–487.[CrossRef][Web of Science][Medline]

  6. Xu,H., Sträter,N., Schröder,W., Böttcher,C., Ludwig,K. and Saenger,W. (2003) Structure of DNA helicase RepA in complex with sulfate at 1.95 Å resolution implicates structural changes to an ‘open’ form. Acta Crystallogr., D59, 815–822.

  7. Ilyina,T.V., Gorbalenya,A.E. and Koonin,E.V. (1992) Organization and evolution of bacterial and bacteriophage primase-helicase systems. J. Mol. Evol., 34, 351–357.[CrossRef][Web of Science][Medline]

  8. Subramanya,H.S., Bird,L.E., Brannigan,J.A. and Wigley,D.B. (1996) Crystal structure of a DExx box DNA helicase. Nature, 384, 379–383.[CrossRef][Medline]

  9. Korolev,S., Hsieh,J., Gauss,G.H., Lohman,T.M. and Waksman,G. (1997) Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP. Cell, 90, 635–647.[CrossRef][Web of Science][Medline]

  10. Sawaya,M.R., Guo,S., Tabor,S., Richardson,C.C. and Ellenberger,T. (1999) Crystal structure of the helicase domain from the replicative helicase-primase of bacteriophage T7. Cell, 99, 167–177.[CrossRef][Web of Science][Medline]

  11. Singleton,M.R., Sawaya,M.R., Ellenberger,T. and Wigley,D.B. (2000) Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides. Cell, 101, 589–600.[CrossRef][Web of Science][Medline]

  12. Allison,T.J., Wood,T.C., Briercheck,D.M., Rastinejad,F., Richardson,J.P. and Rule,G.S. (1998) Crystal structure of the RNA-binding domain from transcription termination factor rho. Nature Struct. Biol., 5, 352–356.[CrossRef][Web of Science][Medline]

  13. San Martin,M.C., Stamford,N.P., Dammerova,N., Dixon,N.E. and Carazo,J.M. (1995) A structural model for the Escherichia coli DnaB helicase based on electron microscopy data. J. Struct. Biol., 114, 167–176.[CrossRef][Web of Science][Medline]

  14. Fass,D., Bogden,C.E. and Berger,J.M. (1999) Crystal structure of the N-terminal domain of the DnaB hexameric helicase. Structure, 7, 691–698.[Medline]

  15. Ali,J.A. and Lohman,T.M. (1997) Kinetic measurement of the step size of DNA unwinding by Escherichia coli UvrD helicase. Science, 275, 377–380.[Abstract/Free Full Text]

  16. Patel,S.S. and Picha,K.M. (2000) Structure and function of hexameric helicases. Annu. Rev. Biochem., 69, 651–697.[CrossRef][Web of Science][Medline]

  17. Yarranton,G.T. and Gefter,M.L. (1979) Enzyme-catalyzed DNA unwinding: studies on Escherichia coli rep protein. Proc. Natl Acad. Sci. USA, 76, 1658–1662.[Abstract/Free Full Text]

  18. Marians,K.J. (2000) Crawling and wiggling on DNA: structural insights to the mechanism of DNA unwinding by helicases. Structure, 8, R227–R235.[Medline]

  19. Ziegelin,G., Linderoth,N.A., Calendar,R. and Lanka,E. (1995) Domain structure of phage P4 {alpha} protein deduced by mutational analysis. J. Bacteriol., 177, 4333–4341.[Abstract/Free Full Text]

  20. Sambrook,J., Fritsch,E.T. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

  21. Scholz,P., Haring,V., Wittmann-Liebold,B., Ashman,K., Bagdasarian,M. and Scherzinger,E. (1989) Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010. Gene, 75, 271–288.[CrossRef][Web of Science][Medline]

  22. Weiner,M.P., Costa,L.G., Schoettlin,W., Cline,J., Mathur,E. and Bauer,J.T. (1994) Site-directed mutagenesis of double-stranded DNA by polymerase chain reaction. Gene, 151, 119–123.[CrossRef][Web of Science][Medline]

  23. Hanahan,D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol., 166, 557–580.[Web of Science][Medline]

  24. Crute,J.J., Mocarski,E.S. and Lehman,I.R. (1988) A DNA helicase induced by herpes simplex virus type 1. Nucleic Acids Res., 16, 6585–6596.[Abstract/Free Full Text]

  25. Otwinowski,Z. and Minor,W. (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol., 276, 461–472.[CrossRef]

  26. Bailey,S. (1994) The CCP4 suite-programs for protein crystallography. Acta Crystallogr., D50, 760–763.

  27. Jones,T.A., Zou,J.Y., Cowan,S.W. and Kjeldgaard,M. (1991) Improved methods for binding protein models in electron density maps and the location of errors in these models. Acta Crystallogr., D47, 110–119.

  28. Maurer,R. and Wong,A. (1988) Dominant lethal mutations in the dnaB helicase gene of Salmonella typhimurium. J. Bacteriol., 170, 3682–3688.[Abstract/Free Full Text]

  29. Notarnicola,S.M. and Richardson,C.C. (1993) The nucleotide-binding site of the helicase-primase of bacteriophage T7: interaction of mutant and wild-type proteins. J. Biol. Chem., 268, 27198–27207.[Abstract/Free Full Text]

  30. Farber,J.M., Peden,K.W. and Nathans,D. (1987) Trans-dominant defective mutants of simian virus 40 T antigen. J. Virol., 61, 436–445.[Abstract/Free Full Text]

  31. Egelman,E.H., Yu,X., Wild,R., Hingorani,M.M. and Patel,S.S. (1995) Bacteriophage T7 primase/helicase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases. Proc. Natl Acad. Sci. USA, 92, 3869–3873.[Abstract/Free Full Text]

  32. Story,R.M., Weber,I.T. and Steitz,T.A. (1992) The structure of the E. coli RecA protein monomer and polymer. Nature, 355, 318–325.[CrossRef][Medline]

  33. Guo,S., Tabor,S. and Richardson,C.C. (1999) The linker region between the helicase and primase domains of the bacteriophage T7 gene 4 protein is critical for hexamer formation. J. Biol. Chem., 274, 30303–30309.[Abstract/Free Full Text]

  34. Xu,H., Frank,J., Niedenzu,T. and Saenger,W. (2000) DNA helicase RepA: cooperative ATPase activity and binding of nucleotides. Biochemistry, 39, 12225–12233.[CrossRef][Medline]

  35. Washington,M.T., Rosenberg,A.H., Griffin,K., Studier,F.W. and Patel,S.S. (1996) Biochemical analysis of mutant T7 primase/helicase proteins defective in DNA binding, nucleotide hydrolysis and the coupling of hydrolysis with DNA unwinding. J. Biol. Chem., 271, 26825–26834.[Abstract/Free Full Text]

  36. Singleton,M.R. and Wigley,D.B. (2002) Modularity and specialization in superfamily 1 and 2 helicases. J. Bacteriol., 184, 1819–1826.[Free Full Text]

  37. Story,R.M. and Steitz,T.A. (1992) Structure of the recA protein–ADP complex. Nature, 355, 374–376.[CrossRef][Medline]

  38. Yao,N., Hesson,T., Cable,M., Hong,Z., Kwong,A.D., Le,H.V. and Weber,P.C. (1997) Structure of the hepatitis C virus RNA helicase domain. Nature Struct. Biol., 4, 463–467.[CrossRef][Web of Science][Medline]

  39. Soultanas,P. and Wigley,D.B. (2002) Site-directed mutagenesis reveals roles for conserved amino acid residues in the hexameric DNA helicase DnaB from Bacillus stearothermophilus. Nucleic Acids Res., 30, 4051–4060.[Abstract/Free Full Text]

  40. Balzer,D., Ziegelin,G., Pansegrau,W., Kruft,V. and Lanka,E. (1992) KorB protein of promiscuous plasmid RP4 recognizes inverted sequence repetitions in regions essential for conjugative plasmid transfer. Nucleic Acids Res., 20, 1851–1858.[Abstract/Free Full Text]

  41. Guerry,P., van Embden,J. and Falkow,S. (1974) Molecular nature of two nonconjugative plasmids carrying drug resistance genes. J. Bacteriol., 117, 619–639.[Abstract/Free Full Text]

  42. Yanisch-Perron,C., Vieira,J. and Messing,J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene, 33, 103–119.[CrossRef][Web of Science][Medline]

  43. Krause,S., Bárcena,M., Pansegrau,W., Lurz,R., Carazo,J.M. and Lanka,E. (2000) Sequence-related protein export NTPases encoded by the conjugative transfer region of RP4 and by the cag pathogenicity island of Helicobacter pylori share similar hexameric ring structures. Proc. Natl Acad. Sci. USA, 97, 3067–3072.[Abstract/Free Full Text]


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