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The Bacillus subtilis Primosomal Protein DnaD Untwists Supercoiled DNA

Centre for Biomolecular Sciences School of Chemistry,¹ Laboratory of Biophysics and Surface Analysis, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom²

Received 9 March 2006/ Accepted 17 May 2006

	ABSTRACT

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The essential Bacillus subtilis DnaD and DnaB proteins have been implicated in the initiation of DNA replication. Recently, DNA remodeling activities associated with both proteins were discovered that could provide a link between global or local nucleoid remodeling and initiation of replication. DnaD forms scaffolds and opens up supercoiled plasmids without nicking to form open circular complexes, while DnaB acts as a lateral compaction protein. Here we show that DnaD-mediated opening of supercoiled plasmids is accompanied by significant untwisting of DNA. The net result is the conversion of writhe (Wr) into negative twist (Tw), thus maintaining the linking number (Lk) constant. These changes in supercoiling will reduce the considerable energy required to open up closed circular plectonemic DNA and may be significant in the priming of DNA replication. By comparison, DnaB does not affect significantly the supercoiling of plasmids. Binding of the DnaD C-terminal domain (Cd) to DNA is not sufficient to convert Wr into negative Tw, implying that the formation of scaffolds is essential for duplex untwisting. Overall, our data suggest that the topological effects of the two proteins on supercoiled DNA are different; DnaD opens up, untwists and converts plectonemic DNA to a more paranemic form, whereas DnaB does not affect supercoiling significantly and condenses DNA only via its lateral compaction activity. The significance of these findings in the initiation of DNA replication is discussed.

	INTRODUCTION

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DNA replication is the most fundamental function in all biology. It is divided in three main stages known as initiation, elongation, and termination. Initiation involves remodeling of a replication origin (oriC) through the action of the main initiator protein DnaA and primosomal multiprotein cascades that ultimately load two replicative ring helicases, one on each strand of the DNA duplex. The helicases in turn recruit the DNA primases, thus signaling the switch from initiation to elongation and the two replication forks migrate in opposite directions, one on each strand (11, 21). Progression to the elongation stage does not guarantee completion of DNA replication since replication forks could be challenged and arrested anywhere along the DNA. In Escherichia coli, reconstitution of an active replication fork at arrested sites is mediated by PriA and/or PriC primosomal pathways that include the PriA, PriB, PriC, and DnaT proteins (19, 25). Homologues of both DnaA and PriA are found in gram-positive bacteria but another primosomal cascade involving the DnaD, DnaB, and DnaI proteins is found only in some low G+C content gram-positive bacteria, including Bacillus subtilis. Although DnaI is the gram-positive functional homologue of the Escherichia coli helicase-loader DnaC (not to be confused with the Bacillus subtilis DnaC helicase), both DnaD and DnaB have no homologues in gram-negative bacteria. They are essential for viability and required for both DnaA and PriA-mediated initiation of DNA replication (3, 27). The molecular events that underpin the priming mechanism are unclear at present, but the data thus far indicate that DnaD interacts with DnaA, PriA, and DnaB (16, 20), whereas DnaI interacts with the DnaC helicase (15, 26, 32, 36). DnaD is believed to act early in the cascade setting the stage for helicase recruitment.

We have recently discovered that DnaD and DnaB have global DNA-remodeling activities. DnaD forms scaffolds and converts supercoiled DNA to an open circular form, whereas DnaB compacts laterally supercoiled and linear DNA (35, 40). The biological significance of these activities is not clear at present, but we have proposed a functional model with DnaD and DnaB being the link between global or local nucleoid remodeling and the initiation of DNA replication (35, 40). Atomic force microscopy (AFM) revealed that DnaD converts all of the writhe of supercoiled plasmids into twist and suggested that this DNA remodeling might be accompanied by significant untwisting of the duplex (40). Untwisting of the DNA may compensate for the considerable force required to open up supercoiled plasmids without nicking. Once opened up, the plasmid is held firmly in the perimeter of a circular protein scaffold made up of DnaD molecules.

The actual details of the molecular mechanism of the DnaD-induced topological effects are not known. In an effort to understand this mechanism, we examined the effects of DnaD binding to DNA on the activity of Escherichia coli topoisomerase I (topo I). Topo I relaxes negatively supercoiled DNA and changes in topology can be probed by effects on its activity. We demonstrate that DnaD untwists the DNA in a concentration-dependent manner. Untwisting requires the formation of scaffolds by intact DnaD, since a C-terminal domain (Cd) that binds to DNA and exhibits a DNA-dependent oligomerization activity (5) is unable to significantly untwist supercoiled plasmids. By comparison, DnaB does not affect the topology of DNA and its effect on DNA condensation is simply the result of lateral compaction. Our combined data suggest that the two proteins have different DNA remodeling effects. The significance of our findings and the putative roles of these proteins in DNA replication are discussed.

	MATERIALS AND METHODS

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Protein purifications. DnaD and DnaB proteins and the Cd domain of DnaD were purified as described before (5, 35, 40).

Topo I relaxation assays. pBluescript plasmid was purified from Escherichia coli XL1-Blue cells using a plasmid Midi-Prep kit (Sigma). The same batch of pBluescript was used for all of the topo I relaxation assays shown here. pBluescript (18 nM) was incubated in a total volume of 25 µl with various concentrations of protein(s) at 37°C for 20 min to allow complex formation. Subsequently, 3 µl of 10x NEB buffer 4 (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol), 1 µl of bovine serum albumin (30 mg/ml), and 1 µl of topo I (4 U/µl; New England Biolabs) were added, and the reaction mixture (total volume, 30 µl) was incubated at 37°C for 40 min. All proteins in the reaction mixture were removed by digestion with excess of PK at 37°C for 20 min. A total of 5 µl of 6x DNA gel loading buffer was added, and the mixture loaded onto 1% (wt/vol) agarose gel casted in TAE buffer. The gel was run at 40 V in TAE buffer in a cold room for 7 to 8 h, stained in 1 µg of ethidium bromide/ml for 40 min, and photographed with a digital camera.

Second-dimension agarose gel electrophoresis. Topo I relaxation assays were set up as described above. The reaction mixtures were subjected first to normal one-dimensional (1D) agarose gel electrophoresis in 1.2% (wt/vol) agarose gels, casted in TAE buffer, at 4°C (cold room). The samples were loaded into a well on the top left corner of the gels, run at 40 V for 7 h in one dimension, and then soaked in TAE buffer supplemented with 4.5 µg of chloroquine/ml for 2 h. The gels were then turned clockwise by 90° and run in the second dimension at 20 V for 12 h in TAE buffer containing 4.5 µg of chloroquine/ml. The gel was stained in 10 µg of ethidium bromide/ml for 35 min, destained in deionized water for 2 h, and photographed with a digital camera.

	RESULTS

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DnaD untwists supercoiled plasmids in a concentration-dependent manner. The effects of DnaD binding on the activity of E. coli topo I were investigated. We argued that if DnaD binding to supercoiled plasmids causes compensatory untwisting, treatment with topo I will relax this untwisting with a corresponding positive linking number change, and subsequent removal of the bound DnaD will yield more relaxed supercoiled DNA. Because purifications of low-copy number pBR322 plasmid (4,361 bp) yielded small amounts, to produce higher amounts of plasmid required for our experiments, we used the high-copy-number pBluescript SK(–) plasmid (2,961 bp) instead.

Incubation of pBluescript with increasing concentrations of DnaD, subsequent treatment with topo I, and removal of all proteins by digestion with proteinase K (PK) resulted in a stimulation of relaxation up to 16 µM DnaD, followed by apparent inhibition of relaxation at 32 µM DnaD (Fig. 1A). By comparison, free pBluescript was relaxed less efficiently, as can be clearly seen from the appearance of distinct topoisomers in the absence of DnaD compared to fully relaxed plasmid in the presence of DnaD (0.1 to 10 µM) under identical conditions (Fig. 1B, left panel). In the presence of 10 µM DnaD four to five times less topo I was sufficient to relax the bound plasmid relative to the amount required to relax unbound plasmid (Fig. 1B, right panel). The simplest interpretation of these data is that DnaD binding and remodeling of the DNA causes increasing compensatory untwisting as the DnaD concentration is increased. Treatment with topo I relaxes this compensatory untwisting with corresponding positive linking number changes, progressively increasing with increased untwisting. Subsequent removal of the bound DnaD yields supercoiled DNA with progressively higher positive linking number changes, resulting in the appearance of more relaxed negatively supercoiled DNA. However, at high concentrations of DnaD, inhibition of the topo I activity is observed which is likely to be due to excess bound DnaD molecules sterically preventing access to the DNA. These DnaD-induced superhelical changes were unequivocally confirmed by 2D gel electrophoresis (Fig. 1C).

View larger version (73K): [in this window] [in a new window]   FIG. 1. DnaD stimulates the topo I activity. (A) The effect of increasing concentrations of DnaD on the activity of topo I. pBluescript (18 nM) was incubated with increasing concentrations of DnaD (0.5, 1, 8, 16, and 32 µM; lanes 4 to 8, respectively) and then treated sequentially with topo I (4 U) and PK prior to electrophoresis. Controls are shown in lanes 1 to 3, as indicated. Relaxed, linear, and negatively supercoiled plasmid is indicated by R, L, and SC(–), respectively. Increased relaxation of the SC(–) plasmid is observed up to 8 µM DnaD (lanes 4 to 6), manifested by the increasing appearance of R. At higher concentrations of DnaD, topo I is inhibited as manifested by the appearance of supercoils with higher electrophoretic mobility in lanes 7 and 8. (B) The left panel shows that the initial stimulation of the topo I relaxation activity is evident at 1 and 10 µM DnaD (lanes 6 and 7, respectively), whereas at 0.1 and 0.01 µM DnaD there was no observable effect (lanes 4 and 5, respectively). Controls are shown in lanes 1 to 3, as indicated. The right panel compares the relaxation of pBluescript by increasing concentrations of topo I (4, 8, and 16 U) in the presence or absence of 10 µM DnaD, as indicated. A total of 16 U of topo I was required to relax pBluescript with approximately the same efficiency as 4 U in the presence of 10 µM DnaD (compare lanes 4 and 7). Controls are shown in lanes 1 to 3, as indicated. (C) 2D gel electrophoresis verifying that DnaD untwists duplex DNA. The gels from left to right show 2D gels of the products from the reactions in lanes 3, 4, 5, 7, and 8 of panel A, respectively. The positions of the R, L, and SC(–) plasmids are indicated. Topoisomers with progressively increasing negative Lk are apparent from top to bottom. Only topoisomers with Lk values from 0 to –9 are indicated for clarity.

DnaB does not cause significant changes in plasmid supercoiling. Previous observations with AFM revealed that tetrameric DnaB binds to, and laterally compacts, supercoiled plasmids forming bead-like structures with the DNA wrapped around the protein (40). An important question to answer is whether this condensing activity is accompanied by significant changes in the superhelical properties of the DNA, analogous to that observed for DnaD. We examined the effects of DnaB binding on the activity of topo I. DnaB up to 4 µM only slightly enhanced topo I relaxation (Fig. 2A, lane 4, and Fig. 2B) that can be attributed to minor effects by DnaB binding rather than specific significant untwisting, as was observed for DnaD. As the concentration was raised to above 8 µM inhibition of the topo I activity was observed (Fig. 2A, lanes 5 to 7), presumably because of excess bound DnaB preventing access to the DNA. These data were verified with 2D gel electrophoresis (Fig. 2B).

View larger version (35K): [in this window] [in a new window]   FIG. 2. DnaB does not alter the superhelical properties of DNA. (A) Effect of increasing concentrations of DnaB on the activity of topo I. pBluescript (18 nM) was incubated with increasing concentrations of DnaB (4, 8, 16, and 26.7 µM; lanes 4 to 7, respectively) and then treated with topo I (4 U). The reaction was terminated, and the mixture was treated with PK prior to electrophoresis. Controls are shown in lanes 1 to 3, as indicated. Relaxed, linear, and negatively supercoiled plasmid is indicated by R, L, and SC(–), respectively. Marginal nonspecific stimulation is barely visible at 4 µM DnaB (lane 4), and inhibition is observed at 8, 16, and 26.7 µM DnaB (lanes 5, 6, and 7). Overall, there is no significant effect on the topo I activity. (B) 2D gel electrophoresis verifying that DnaB does not untwist duplex DNA. 2D gels showing reactions in the absence (left) and presence of 4 and 8 µM DnaB (middle and right, respectively), equivalent to the products of the reactions in lanes 3, 4, and 5 from panel A. The positions of the R, L, and SC(–) plasmids are indicated together with the position of the Lk = 0 topoisomer. Topoisomers with progressively increasing negative Lk are apparent from top to bottom.

The DnaD scaffold is essential for untwisting the duplex. DnaD consists of two domains with distinct activities (5). Its N-terminal domain (Nd) has a DNA-independent oligomerization activity, while its C-terminal domain (Cd) binds to DNA and exhibits a separate DNA-dependent oligomerization activity (5). An important mechanistic detail to reveal is whether DNA binding per se is sufficient to untwist the duplex. To answer this question, the effect of Cd binding to pBluescript on the activity of topo I was investigated (Fig. 3A). Increasing concentrations of Cd 0.5 to 1 µM did not affect the activity of topo I (Fig. 3A, compare lanes 3, 4, and 5), but at 8 µM a slight stimulation of topo I was apparent (Fig. 3A, compare lanes 3 and 6). This was better than the minor effect observed for DnaB (Fig. 2A, lane 4, and Fig. 2B) but much less compared to the strong effect seen with the full-length DnaD protein (Fig. 1), indicating that simply Cd binding to pBluescript causes only minor untwisting of the duplex. At 18 and 64 µM Cd there was apparent steric inhibition of topo I (Fig. 3A, lanes 7 and 8). These observations were verified by 2D gel electrophoresis (Fig. 3B). The combined data indicate that the DNA binding and DNA-induced oligomerization activities of Cd were not sufficient to untwist the DNA significantly.

View larger version (33K): [in this window] [in a new window]   FIG. 3. The Cd of DnaD cannot untwist duplex DNA. (A) Effect of increasing concentrations of Cd on the activity of topo I. pBluescript (18 nM) was incubated with increasing concentrations of Cd (0.5, 1, 8, 16, and 64 µM; lanes 4 to 8, respectively) and then treated sequentially with topo I (4 U) and PK prior to electrophoresis. Controls are shown in lanes 1 to 3, as indicated. No effect on topo I activity was observed at 0.5 and 1 µM Cd (compare lanes 3, 4, and 5), but a slight stimulation of relaxation was observed at 16 µM Cd (compare lanes 3 and 6). At higher Cd concentrations (16 and 64 µM) inhibition of relaxation was observed. (B) 2D gel electrophoresis verifying that Cd inhibits the topo I at high concentration. 2D gels showing reactions in the absence (left) and presence of 8 and 64 µM Cd (middle and right), equivalent to the products of the reactions in lanes 3, 6, and 8 from panel A. The positions of the R, L, and SC(–) plasmids are indicated together with the position of the Lk = 0 topoisomer. Topoisomers with progressively increasing negative Lk are apparent from top to bottom.

View larger version (41K): [in this window] [in a new window]   FIG. 4. DnaB does not inhibit untwisting by DnaD. (A) Effect of DnaB (8 µM) on the twisting activity of increasing amounts of DnaD (0.5 to 33 µM), as indicated. Appropriate mixtures of proteins were incubated with pBluescript (18 nM), sequentially treated with topo I and PK before electrophoresis. The positions of fully relaxed, linear, and supercoiled plasmids are indicated by R, L, and SC, respectively. Increasing amounts of DnaD and treatment with topo I resulted in gradual positive increases of Lk by duplex untwisting, manifested as a gradual relaxation of the negatively supercoiled plasmid (lanes 4, 6, and 8), followed by steric inhibition of topo I activity at high concentrations of DnaD (lanes 10 and 12). Under identical conditions but in the presence of 8 µM DnaB, the untwisting activity of DnaD was totally inhibited, as manifested by the presence of mainly supercoiled plasmid throughout (lanes 5, 7, 9, 11, and 13). Controls are shown in lanes 1 to 3, as indicated. (B) The same experiment as the one shown in panel A was carried out with 4 µM DnaB at 0.5, 1, and 9.7 µM DnaD, as indicated. Increasing the concentrations of DnaD (0.5 to 1 µM) in the presence of 4 µM DnaB resulted in a slight enhancement of topo I relaxation the presence of equivalent concentrations of DnaD alone (lanes 3 to 6).

	DISCUSSION

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Binding of DnaD to supercoiled pBR322 plasmid results in the formation of circular protein scaffolds with the plasmids held firmly around the periphery of the scaffolds (40). The contour lengths of the bound plasmids were found to be consistently longer than unbound molecules and directly proportional to the sizes of the scaffolds. Larger scaffolds held plasmid molecules with longer contour lengths compared to smaller scaffolds (40). The net result was the conversion of all of the Wr into Tw. However, in the absence of a nicking activity there will be no net change in Lk, implying that the elimination of Wr must be compensated by negative twisting, i.e., untwisting of the double helix. From statistical measurements of 134 pBR322 molecules bound to DnaD, it has been established that untwisting results in an average increase of the double helical turn from 10.6 to 16.1 bp (40). Our data offer direct biochemical evidence that DnaD binding to a supercoiled plasmid causes significant duplex untwisting. Simply DnaD binding to DNA is not sufficient to untwist the duplex. The formation of the scaffold is essential and thus duplex untwisting is the result of the sum of both DNA binding and scaffold formation. Since the size of the scaffolds (40) and the extent of untwisting are both concentration dependent, the implication is that as the scaffold increases in size at higher DnaD concentrations so does the untwisting of the duplex.

DnaD binds efficiently to a 19-mer oligonucleotide (5) and assuming 19 bases as the binding size compared to the size of pBluescript (2,961 bp), this represents approximately 155 binding sites per plasmid for DnaD. Therefore, at 28 and 56 molar excess of DnaD, it is unlikely that the whole of the plasmid will be opened up by DnaD, and only at a 448 molar excess is it likely that the whole of the plasmid will have been forced to open up. In fact, untwisting increased progressively as the concentration of DnaD was raised and reached its maximum level at 8 µM (Fig. 1A), representing a 1:448 molar excess of DnaD over the plasmid. We hypothesize that initially at low concentrations, DnaD binds to few sites along the plasmid via its Cd domain and forms localized scaffolds that untwist the duplex only partially. Since the concentration of DnaD increases the sizes of the local scaffolds also increase to form a large circular scaffold holding the open plasmid in the periphery. As the scaffold grows duplex untwisting also increases progressively until the maximum possible open/paranemic plasmid conformation is achieved (Fig. 5). We estimate this to be the case at around 1:440 molar excess of DnaD over pBluescript. Untwisting of the duplex will compensate against the considerable force required to eliminate all of the Wr, open up supercoiled DNA without nicking, and increase its contour length in a paranemic conformation (Fig. 5).

View larger version (19K): [in this window] [in a new window]   FIG. 5. Schematic diagram illustrating conversion of DNA from plectonemic to paranemic by DnaD concentration-dependent untwisting. DNA remodeling by DnaD is the sum of separate DNA-independent and DNA-dependent oligomerization activities residing on its Nd and Cd domains, respectively, plus a DNA-binding activity on the Cd domain (15). Based upon a DNA-remodeling model proposed before (15), we suggest that initial binding of DnaD to DNA via its Cd domain causes only minor untwisting of the duplex but, as the scaffold forms at higher concentrations, via oligomerization interactions mediated by the Nd domain, gradual untwisting of the duplex becomes significant, and the duplex is eventually converted to a paranemic form. Only three helical turns are shown for simplicity and although untwisting is depicted to open up individual helical turns, it is likely that it will be distributed throughout all of the helical turns in the circular plasmid molecule as the DnaD scaffold grows.

DnaD is a potential modulator of global superhelical density. The precise role of DnaD in vivo may not be confined to the initiation of DNA replication. For example, could its DNA remodeling activity with the significant untwisting of the DNA and stimulation of topo I activity be part of the essential homeostatic control of global superhelical density in vivo? Bacterial nucleoids are negatively supercoiled closed circular compacted structures separated into distinct topological domains (6, 12, 27). Modulation of the superhelical density has been shown to be an important factor in bacterial physiology affecting global transcriptional regulation, replication, recombination, and response to environmental challenges (8, 34, 37, 39), and nucleoid architectural changes are apparent during growth (1, 17). Excess negative supercoiling is growth inhibitory (10) and, in E. coli, topA-null mutants exhibit a growth defect due to increased negative supercoiling and frequently acquire compensatory mutations that inactivate DNA gyrase or overproduce topo IV resulting in a reduction of negative supercoiling (7, 9, 28, 29). It is attractive to speculate that, in addition to other functions, DnaD may also act as a global regulator of superhelical density in B. subtilis. Indeed, its relative abundance in the cell (4) suggests functions in addition to initiation of DNA replication. Overproduction of DnaD in vivo may be an alternative mechanism for stimulating topo I activity and thus relieving excess negative supercoiling. A putative functional cooperation between DnaD and topo I does not imply a direct interaction between the two proteins (although this has not been excluded), since topo I activity can be modulated indirectly by DNA-binding proteins in the absence of a direct interaction (31).

Role of DnaB. The precise function(s) of DnaB in the initiation of DNA replication is rather ambiguous. It has been suggested to act in conjunction with DnaI, effectively forming a pair of helicase loaders that load DnaC onto DNA (36) or alternatively as a membrane attachment protein to regulate initiation of DNA replication by regulating the recruitment of DnaD from the cytoplasm to the membrane (13, 24, 30, 33). The latter is consistent with the localization of green fluorescent protein (GFP)-DnaD and GFP-DnaB fusions proximal to the cell membrane (22), suggesting that the two proteins are likely to interact at some stage during primosomal assembly. A mutation in dnaB (DnaBS371P) suppresses the temperature-sensitive phenotype caused by the dnaD23ts mutation and while DnaB does not interact with DnaD, the resulting DnaBS371P mutant interacts directly with DnaD (30). These data indicate that there is a genetic link between the two proteins and that DnaB can adopt a conformation (induced by the S371P mutation) that interacts directly with DnaD. Indeed, a DNA-dependent interaction between the two proteins has been detected (20), but it has not been established whether this interaction alters the mode of DnaD binding to DNA. AFM revealed that both proteins bind simultaneously to the same plasmid resulting in characteristic bipolar structures with the two proteins located at diametrically opposite ends of a rod-like supercoiled plasmid (40). Under the conditions of our experiment the two proteins are bound simultaneously to the plasmid and partial opening should occur (40). This is also supported by the observation that, as the concentration of DnaD is increased in the presence of DnaB, topo I is eventually sterically inhibited, suggesting that DnaD binds to DNA simultaneously with DnaB. These data suggest that DnaB binding does not inhibit untwisting by DnaD.

	ACKNOWLEDGMENTS

 
The Cd of DnaD was purified by Maria Carneiro.

This study was supported by a BBSRC grant to P.S., C.J.R., and S.A. (grant reference BB/C500579/1).

	FOOTNOTES

 
* Corresponding author. Mailing address: Centre for Biomolecular Sciences School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom. Phone: 44(0)115-9513525. Fax: 44-(0)115-8468002. E-mail: panos.soultanas{at}nottingham.ac.uk.

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