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• W2110168944 abstract "Article27 January 2012free access Soj/ParA stalls DNA replication by inhibiting helix formation of the initiator protein DnaA Graham Scholefield Graham Scholefield Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle Upon Tyne, UK Search for more papers by this author Jeff Errington Jeff Errington Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle Upon Tyne, UK Search for more papers by this author Heath Murray Corresponding Author Heath Murray Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle Upon Tyne, UK Search for more papers by this author Graham Scholefield Graham Scholefield Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle Upon Tyne, UK Search for more papers by this author Jeff Errington Jeff Errington Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle Upon Tyne, UK Search for more papers by this author Heath Murray Corresponding Author Heath Murray Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle Upon Tyne, UK Search for more papers by this author Author Information Graham Scholefield1, Jeff Errington1 and Heath Murray 1 1Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle Upon Tyne, UK *Corresponding author. Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle Upon Tyne NE2 4AX, UK. Tel.: +44 191 208 3233; Fax: +44 191 208 3205; E-mail: [email protected] The EMBO Journal (2012)31:1542-1555https://doi.org/10.1038/emboj.2012.6 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Control of DNA replication initiation is essential for normal cell growth. A unifying characteristic of DNA replication initiator proteins across the kingdoms of life is their distinctive AAA+ nucleotide-binding domains. The bacterial initiator DnaA assembles into a right-handed helical oligomer built upon interactions between neighbouring AAA+ domains, that in vitro stretches DNA to promote replication origin opening. The Bacillus subtilis protein Soj/ParA has previously been shown to regulate DnaA-dependent DNA replication initiation; however, the mechanism underlying this control was unknown. Here, we report that Soj directly interacts with the AAA+ domain of DnaA and specifically regulates DnaA helix assembly. We also provide critical biochemical evidence indicating that DnaA assembles into a helical oligomer in vivo and that the frequency of replication initiation correlates with the extent of DnaA oligomer formation. This work defines a significant new regulatory mechanism for the control of DNA replication initiation in bacteria. Introduction Successful replication and segregation of genetic information prior to cell division is essential for all living organisms. Loss of replication control can dramatically reduce an organism's competiveness in its environment, and in extreme cases can lead to unchecked cell proliferation or cell death. Throughout the kingdoms of life, chromosome duplication is instigated by DNA replication initiator protein complexes (Mott and Berger, 2007; Wigley, 2009; Kawakami and Katayama, 2010). A unifying characteristic of initiator proteins is their AAA+ nucleotide-binding domain, which is critical for their structure and function (Tucker and Sallai, 2007; Kawakami and Katayama, 2010). Bacterial chromosomes are typically replicated bi-directionally from a single origin (oriC); an event orchestrated by the multi-domain initiator protein DnaA (Supplementary Figure S1; for review see Mott and Berger, 2007; Leonard and Grimwade, 2010). At the C-terminus, domain IV contains the helix-turn-helix and basic loop motifs required for specific double-stranded DNA-binding activity (Erzberger et al, 2002; Fujikawa et al, 2003). Domain III contains the AAA+ motif involved in ATP binding and ATP hydrolysis, as well as residues required for coordinating single-stranded DNA (Erzberger et al, 2002; Ozaki et al, 2008; Duderstadt et al, 2011). Domain II is a poorly conserved flexible linker (Abe et al, 2007; Molt et al, 2009) connecting domains III–IV to domain I, which acts as a hub for additional protein interactions and directs loading of the replicative helicase (Sutton et al, 1998). Initiation of DNA replication in bacteria requires stepwise structural transitions, resulting in the assembly of DnaA into an active initiation complex (for reviews see Ozaki and Katayama, 2009; Leonard and Grimwade, 2010). Through domain IV, DnaA is thought to stably bind conserved nine basepair sequences (DnaA-boxes) in the oriC region throughout the cell cycle (Cassler et al, 1995). These founding DnaA proteins recruit further DnaA molecules onto neighbouring low-affinity binding sites via dimerization of domain I (Simmons et al, 2003; Miller et al, 2009). Additional ATP-bound DnaA proteins then assemble onto this platform to form a large nucleoprotein complex observable by electron microscopy as a particle wrapped in DNA (Funnell et al, 1987). This oligomeric structure may correspond to the right-handed helix, built via interactions between neighbouring AAA+ domains, which has been observed by X-ray crystallography (Carr and Kaguni, 2001; Erzberger et al, 2006). Amino-acid substitutions in DnaA that perturb helix formation in vitro inhibit replication origin unwinding in vitro and functionality in vivo (Duderstadt et al, 2010), and it has recently been proposed that the DnaA helix destabilizes an AT-rich sequence within the origin (the DNA unwinding element; DUE) by stretching one strand of the DNA duplex to promote origin opening (Duderstadt et al, 2011). This activity appears to be accompanied by a transition in DNA-binding modes from double-stranded to single-stranded; a result of domain IV engaging the AAA+ motif of a neighbouring monomer within the helical oligomer (Erzberger et al, 2006; Duderstadt et al, 2010). This compact helix is thought to continue onto the upper strand of the now single-stranded DUE via residues in domain III, stabilizing the DUE in its unwound state (Speck and Messer, 2001; Ozaki et al, 2008). Following open complex formation, DnaA directly recruits the replicative helicase onto the single-stranded DNA via interactions with domains I and III (Sutton et al, 1998; Abe et al, 2007). The remaining replisomal components are then recruited in a stepwise manner, which culminates in an active DNA replication complex. There are several steps during initiation at which regulatory systems have been found to control bacterial DNA replication (for review see Katayama et al, 2010). DnaA binding to oriC can be inhibited either by protein occlusion (SeqA in Escherichia coli, Spo0A in Bacillus subtilis, and CtrA in Caulobacter crescentus), by spatial sequestration (YabA in B. subtilis), or by titration (datA in E. coli and DBCs in B. subtilis). DnaA assembly at oriC can be either stimulated (DiaA in E. coli and HobA in Helicobacter pylori) or repressed (SirA in B. subtilis) by the binding of regulatory proteins to domain I. Lastly, DnaA is inactivated following replisome formation through the stimulation of its ATP hydrolysis activity (Hda in E. coli and C. crescentus). In a previous study we identified the highly conserved ParA protein (Soj) as a novel regulator of DNA replication in B. subtilis (Murray and Errington, 2008). Soj is a Walker-type ATPase that forms an ATP-dependent sandwich dimer that can bind DNA (Leonard et al, 2005). We have shown that the monomeric Soj protein inhibits DnaA, while dimerization of Soj switches the protein into an activator of DnaA (Scholefield et al, 2011). These results indicate that Soj acts as a molecular switch to control DnaA activity, with its opposing regulatory activities being dictated by its quaternary state. Detailed biochemical characterization of Soj proteins has identified amino-acid substitutions that arrest Soj quaternary changes at different steps (Figure 1A; Leonard and Grimwade, 2005; Hester and Lutkenhaus, 2007; Scholefield et al, 2011). Two separate substitutions inhibit Soj dimerization: SojK16A is unable to bind ATP, while SojG12V can bind ATP but cannot dimerize due to a steric clash in the dimerization interface; both of these proteins inhibit DnaA activity. The SojR189A substitution allows ATP-dependent dimerization but disrupts DNA-binding activity: this mutant protein is relatively inert, presumably because DNA-binding activity is required for Soj to efficiently activate DnaA (Scholefield et al, 2011). Figure 1.Specific mutations in dnaA either bypass or suppress the inhibition of DNA replication initiation by SojG12V. (A) Pathway of the Soj activity cycle. (B) Point mutations in DnaA introduced by error-prone PCR were found to overcome the small colony phenotype characteristic of SojG12V overexpression. Strains were grown on NA plates in the presence or absence of 1% xylose to induce sojG12V expression. Wild-type (HM524), DnaAL294R (HM527), DnaAV323D (HM528), DnaAL337P (HM529), DnaAA341V (HM530). (C) The oriC-to-terminus ratios of dnaA point mutations generated using PCR mutagenesis were determined using MFA in the presence and absence of SojG12V overexpression (1% xylose). Suppressor mutations (red) were found to be recalcitrant to SojG12V activity. Cells were grown in LB medium at 30°C. Values were normalized to the ori:ter ratio of the wild-type strain grown in the absence of xylose. DnaAV121A (HM713), DnaAA131T (HM714), DnaAA132T (HM710), DnaAG151R (HM705), DnaAG154S (HM706), DnaAH162Y (HM707), DnaAR281G (HM708), DnaAN311D (HM712), DnaAN311T (HM709), DnaAE314G (HM711). (D) The SojG12V suppressor mutations in dnaA perturb the formation of a Soj:DnaA–His12 complex in vivo. Cells were grown in LB medium at 30°C, crosslinked with formaldehyde, and the DnaA–His12 complexes were purified before the crosslinks were reversed and proteins were separated by SDS–PAGE. Soj and DnaA–His12 were detected by western blot analysis. The top panel shows the amount of Soj protein in the cell lysate (Input) and the bottom panel shows the amount of Soj found in a complex with DnaA–His12 following purification (Complex). DnaA–His12 (HM657), DnaAL294R–His12 (HM716), DnaAV323D–His12 (HM555), DnaAL337P–His12 (HM658), DnaAA341V–His12 (HM725). (E) The amount of each DnaASup–His12 protein was determined by western blot analysis. DivIVA was used as a loading control. Download figure Download PowerPoint Here, we have investigated the negative regulation of DnaA by monomeric Soj. We have identified amino-acid substitutions in DnaA that render the protein insensitive to inhibition by monomeric Soj and that do not form a complex with Soj in vivo. Using these proteins we show that Soj directly interacts with DnaA in vitro. Importantly, we have developed a site-specific crosslinking assay that detects DnaA oligomers assembling on single-stranded and double-stranded DNA substrates, both of which appear to represent a helical conformation built upon the AAA+ domains. Using this assay we show that monomeric Soj specifically inhibits DnaA helix formation in vitro. Furthermore, we adapted our site-specific crosslinking assay to demonstrate that (i) DnaA forms oligomers in vivo, (ii) monomeric Soj inhibits DnaA oligomerization in vivo, and (iii) the extent of DnaA oligomerization in vivo correlates with the rate of DNA replication initiation. Together, these results establish the DnaA helix as an important target for regulation, as well as providing critical biochemical evidence supporting the physiological relevance of DnaA helix formation during DNA replication initiation. Results Specific point mutations in dnaA disrupt Soj inhibition in vivo Previously, we have shown that monomeric Soj inhibits DnaA activity and forms a complex with DnaA in vivo (Murray and Errington, 2008). However, it remained unclear whether this regulation was mediated by a direct interaction between the proteins. To address this question, we screened for mutations in dnaA that suppress the growth inhibition caused by overexpressing monomeric SojG12V (Figure 1B). A chloramphenicol marker was integrated downstream of the dnaAN operon and genomic DNA from this strain was used as a substrate for error-prone PCR to generate point mutations in dnaA. PCR products were transformed into a strain harbouring an inducible sojG12V allele and plated under SojG12V overexpression conditions, resulting in slow growth of wild-type colonies. Genomic DNA from large colonies was backcrossed into the parent strain to confirm that the mutation conferring fast growth was linked to dnaA. DNA sequencing identified 14 distinct mutations that caused single amino-acid substitutions within DnaA. To characterize the mutations in dnaA, marker frequency analysis (MFA) was used to measure the relative levels of origin and terminus DNA, thereby generating a measure of DNA replication initiation frequency (Figure 1C). The mutations within dnaA fell into two classes: hypermorphs that bypassed SojG12V inhibition (DnaAHyp proteins) by having a high basal rate of initiation, and suppressors that had an approximately wild-type rate of initiation but were resistant to SojG12V inhibition (DnaASup proteins: DnaAL294R, DnaAV323D, DnaAL337P, DnaAA341V). The suppressor mutations were each independently cloned into dnaA and transformed into the SojG12V overexpression strain to demonstrate that they were responsible for the large colony phenotype. The resulting strains displayed rates of DNA replication initiation and DnaA expression levels similar to wild type in the presence or absence of SojG12V overexpression (Supplementary Figure S2). The ability of Soj to form a complex with DnaA–His12 and DnaASup–His12 proteins in vivo was investigated using nickel affinity purification following formaldehyde crosslinking. Compared with wild-type DnaA–His12, all four DnaASup–His12 proteins were defective in their ability to form a complex with Soj (Figure 1D). Western blot analysis confirmed that all DnaA–His12 proteins were expressed to a similar level as wild type (Figure 1E). Taken together, the data indicate that these amino-acid substitutions in DnaA suppress SojG12V inhibition by disrupting DnaA–Soj complex formation. Soj interacts directly with DnaA in vitro To test whether Soj and DnaA directly interact, we purified several DnaA and Soj proteins and measured binding in vitro using surface plasmon resonance (SPR). B. subtilis DnaA lacks cysteine residues, allowing for the introduction of a C-terminal cysteine following a His5-tag. Conjugation of these proteins to the sensor chip using a ligand thiol coupling technique produced a homo-orientated DnaA surface. Wild-type and mutant Soj proteins were then systematically injected over the wild-type and DnaASup surfaces. SPR analysis showed that wild-type Soj in the monomeric, ADP-bound form (Soj:ADP) binds to DnaA with an KD of ∼30 μM (Figure 2A). In contrast, the DnaAL294R and DnaAV323D proteins were severely defective in their interaction with Soj:ADP, and the DnaAA341V protein displayed an intermediate interaction profile (Figure 2B). Furthermore, all the DnaASup proteins failed to support complex formation with monomeric SojG12V (Figure 2C). Figure 2.Soj directly interacts with DnaA. (A–D) SPR sensorgrams. DnaA proteins were immobilized onto the SPR chip surface via a unique C-terminal cysteine residue to create a homogenous surface. Cartoon representations of Soj are shown to indicate the quaternary state of various proteins. (A) Two-fold serial dilution of wild-type Soj:ADP injected over DnaAH485C. (B–D) The indicated Soj proteins (15 μM) were injected over wild-type and mutant DnaA surfaces starting at time zero for 360 s. (E) In vitro crosslinking assay using the primary amine-specific crosslinker (BS3) in the presence of DNA (pBSoriC4; 3 nM) and ATP (2 mM). Protein complexes were separated by SDS–PAGE and the DnaA protein was visualized by western blotting. Pluses located above each lane indicate the presence of BS3 and/or Soj protein (32 μM). The identity of the DnaA proteins (3 μM) are indicated below. The identity of the Soj proteins is indicated to the left of each gel. Download figure Download PowerPoint To substantiate the results observed by SPR, DnaA proteins were subjected to primary amine-specific crosslinking (BS3) in solution with and without SojG12V. Protein complexes were separated by SDS–PAGE and DnaA was detected by western blot analysis (Figure 2E). The appearance of a signal at a molecular weight expected for a Soj:DnaA complex (27 kDa + 54 kDa=81 kDa) was observed in the presence of SojG12V. By contrast, complex formation was dramatically reduced when the DnaASup proteins were tested. In addition, the DnaA:DnaA complex (108 kDa) was reduced in the presence of SojG12V. Cytological analysis of GFP–SojG12V localization in B. subtilis cells suggests that it associates with origin bound DnaA (Murray and Errington, 2008). To ascertain if SojG12V is capable of interacting with a DnaA:DNA complex in vitro, a pull-down experiment was performed. His-tagged SojG12V was incubated with pBsoriC4 in the presence and absence of native DnaA (DnaAnat). Proteins and DNA were crosslinked using a concentration of formaldehyde that yielded a specific Soj:DnaA interaction (Supplementary Figure S3A). Complexes were then bound to nickel beads via the histidine tag on Soj, washed, and the crosslinks reversed. The amount of pBsoriC4 in these complexes was detected using qPCR. There was an ∼13-fold enrichment of pBsoriC4 bound to SojG12V in the presence of DnaA, indicating that Soj is capable of forming a complex with DnaA bound to DNA (Supplementary Figure S3B). Taken together, the SPR and crosslinking assays indicate that monomeric Soj directly interacts with DnaA both in solution and bound to DNA, and that the substitutions in the DnaASup proteins disrupt complex formation. Mapping of the DnaASup and DnaAHyp substitutions onto DnaA structures suggests a mechanism for Soj regulation We noted that although our PCR mutagenesis strategy targeted the entire dnaA region (∼5 kb flanking either side of dnaA, including the dnaA promoter and all of oriC), all of the isolated DnaAHyp substitutions were located within the AAA+ motif of DnaA, while all of the DnaASup substitutions were located in domain IIIb (or at the border between domains IIIb and IV, depending upon the assignment of domain boundaries; see Supplementary Figure S1). Strikingly, when these amino-acid substitutions were mapped onto the crystal structure of DnaA domain III from Thermotoga maritima (Figure 3A), all four of the suppressor substitutions were found to cluster in domain IIIb, strongly suggesting that this region is the binding site for Soj. Figure 3.The DnaA hypermorph and suppressor substitutions are located in domain III. (A) A cartoon representation of monomeric DnaA from the T. maritima crystal structure (PDB ID: 2Z4S) bound to ADP (stick). Domains IIIa and IIIb are separated by a dashed line. The SojG12V hypermorph (black) and suppressor (red) substitutions are shown as spacefill representations. The identity and positions of the B. subtilis amino-acid substitutions are indicated above the corresponding residue of the T. maritima protein. (B) The majority of the hypermorphic substitutions are located either adjacent to, or buried within, the DnaA:DnaA interface. A surface representation of the helical DnaA structure from A. aeolicus (PDB ID: 2HCB) bound to AMP-PCP. DnaA monomers are coloured independently (yellow, cyan, blue, and green). The amino-acid substitutions are coloured and annotated as in (A) above. The inset shows the location of the two residues changed to cysteines for the crosslinking assays, with the black dashed line indicating where BMOE acts. Download figure Download PowerPoint The DnaAHyp substitutions were found more widely distributed throughout the AAA+ motif. Three of the amino acids (G151, G154, and V121) were located around the nucleotide-binding pocket, with the backbone of the latter two residues mediating direct contacts with the terminal phosphate(s) and sugar, respectively. However, the remaining six DnaAHyp substitutions appear to be surface exposed and distal from the nucleotide-binding pocket; thus, it was unclear what effect these substitutions were having. To gain insight into how the DnaAHyp proteins might affect DnaA activity, the positions of these amino-acid substitutions were mapped onto the helical crystal structure of Aquifex aeolicus DnaA bound to AMP-PCP (Figure 3B, note A. aeolicus DnaA lacks a 14 amino-acid stretch present in all classically studied bacteria including B. subtilis, which harbours two of the four suppressor substitutions (V323D and L337P); see Supplementary Figures S1 and S4). Significantly, all of these hypermorphic substitutions were either buried inside (70%) or adjacent to the DnaA:DnaA interface and were generally solvent exposed only at each end of the helix. Since these substitutions lead to hyperactivity of DnaA, we speculate that they may promote AAA+ mediated oligomerization by increasing the affinity of the DnaA:DnaA interaction. Taken together with the observation that SojG12V disrupts DnaA:DnaA complex formation (Figure 2E), we hypothesized that monomeric Soj regulates DnaA ATP-dependent oligomerization. B. subtilis DnaA assembles into an ATP-dependent oligomer in vitro To test this model we designed a crosslinking assay to specifically detect ATP-dependent oligomerization of DnaA (Chen, 1991). Guided by the A. aeolicus structure, a pair of cysteine residues were introduced into domain IIIa at N191 and A198 (Figure 3B, inset). Within the oligomer, the N191 residue from one DnaA monomer is in close proximity (∼9 Å) to the A198 residue of the adjacent monomer. DnaAN191C,A198C (hereafter referred to as DnaAcc) was incubated with the cysteine-specific crosslinker bis(maleimido)ethane (BMOE; spacer arm 8.0 Å), protein complexes were separated by SDS–PAGE, and DnaA was detected by western blot analysis. Figure 4A shows that crosslinking of DnaACC captures multiple high molecular weight complexes that run as a ladder on the gel. Formation of these DnaA oligomers was dependent on ATP and dramatically stimulated by DNA. Critically, mutation of the arginine finger residue (R264A) that coordinates the γ-phosphate of the ATP from the neighbouring DnaA molecule (Erzberger et al, 2006) greatly diminished DnaA oligomerization (lanes 7 and 8). The stimulation by DNA appeared to be non-specific as the formation of DnaA oligomers was indistinguishable when comparing plasmids with or without the B. subtilis origin of replication (pBsoriC4 versus pUC18, respectively), comparing supercoiled DNA with linear DNA, and comparing double-stranded DNA with single-stranded DNA (Figure 4B and data not shown). Figure 4.Monomeric Soj inhibits DnaA oligomerization in vitro. (A) In vitro oligomer formation assays using the cysteine-specific crosslinker BMOE. DnaA proteins were incubated in oligomer formation buffer for 15 min prior to the addition of BMOE. Pluses located above each lane indicate the presence of BMOE (2 mM), nucleotide (2 mM), and/or DNA (pBsoriC4; 3 nM). The identity of the DnaA proteins (3 μM) are indicated below. DnaA proteins were separated by SDS–PAGE and visualized by western blotting. (B) DnaA oligomers form on both single-stranded and double-stranded DNA. DnaA proteins (3 μM) were incubated in oligomer formation buffer with NaCl (400 mM) in the presence of ATP (2 mM), with or without DNA, for 15 min prior to the addition of BMOE. The identity of each DNA substrate is indicated above the respective lanes, and the amount of DNA is equal in each reaction (120 fmol). Both plasmids are supercoiled and the single-stranded DNA (ssDNA) is oligonucleotide oGJS159. DnaA proteins were separated by SDS–PAGE and visualized by western blotting. (C) Monomeric Soj disrupts DnaA oligomer formation in vitro. Triangles above the lanes represent an increasing concentration of various Soj proteins (12, 24, and 36 μM). DnaA proteins (3 μM) were incubated in oligomer formation buffer in the presence of ATP (2 mM) and DNA (pBsoriC4; 3 nM) for 15 min prior to the addition of BMOE. DnaA proteins were separated by SDS–PAGE and visualized by western blotting. Download figure Download PowerPoint Previous work has suggested that the crystalized DnaA oligomer cannot accommodate binding to double-stranded DNA through the helix-turn-helix in domain IV due to substantial steric clashes (Duderstadt et al, 2010). Because we readily observed that double-stranded DNA stimulates DnaA oligomer formation, we wondered whether this stimulation was dependent upon residues required for the DNA-binding activities of domains III and IV. An amino-acid substitution was introduced into domain III (I190A) that previously was shown to disrupt single-stranded DNA-binding activity of DnaA in vitro and DNA replication in vivo (Ozaki et al, 2008). As expected, oligomerization of DnaACC,I190A was not stimulated by single-stranded DNA, although it was stimulated by double-stranded plasmids (Figure 4B). Next, to investigate the role played by domain IV in oligomer formation, arginine 379 was mutated to an alanine. In E. coli DnaA, the equivalent residue has been shown to interact with both specific basepairs and backbone phosphates in the minor groove of double-stranded DNA (Fujikawa et al, 2003). In contrast to the results obtained with DnaACC,I190A, oligomerization of DnaACC,R379A was stimulated by single-stranded DNA, but not by the double-stranded pUC18 plasmid. Interestingly, oligomerization of DnaACC,R379A was stimulated by a plasmid that harboured oriC (Figure 4B). These results correlated with the ability of DnaACC,R379A to bind to the respective plasmids as judged by an electrophoretic mobility shift assay (Supplementary Figure S5). We note that single-stranded DNA stimulated helix formation of DnaACC,R379A to a greater degree than DnaACC. This observation suggests that the arginine residue may interact with the phosphate backbone of the single-stranded substrate and inhibit the docking of domain IV into the AAA+ domain, a transition proposed to be critical for single-stranded DNA-binding activity (Duderstadt et al, 2010). Taken together, these experiments indicate that DnaA is capable of forming ATP-dependent oligomers on both single-stranded and double-stranded DNA substrates, in contrast to the apparent constraints placed upon domain IV within the structure of the DnaA oligomer (see Discussion). Monomeric Soj specifically inhibits DnaA oligomer formation in vitro The DnaA oligomer formation assay was then used to investigate the effect of Soj on DnaA activity. The presence of SojG12V significantly reduced both the length and abundance of the DnaA oligomers formed in the presence of plasmid DNA (Figure 4C). To determine whether the observed inhibition was specific to monomeric Soj, the effect of SojK16A (monomeric) and SojR189A (dimeric) proteins was also investigated. While SojK16A clearly disrupted DnaA oligomer formation, SojR189A had little or no effect (Figure 4C) even though it was capable of interacting with DnaA (Figure 2D and E). Thus, despite sharing apparent interaction determinants, disruption of DnaA oligomerization is a specific property of the monomeric Soj protein. These results are consistent with our previous findings that monomeric Soj specifically inhibits DnaA initiation activity in vivo (Scholefield et al, 2011). Oligomers formed by DnaAL294R and DnaAV323D were highly resistant to SojG12V activity (Figure 5A). In comparison, oligomers formed by DnaAA341V were partially susceptible to SojG12V, consistent with the intermediate response observed between the two proteins by SPR analysis (Figures 2B and 5A). Importantly, all of the DnaASup proteins were found to form oligomers under the same conditions as wild type (Supplementary Figure S6A). These results indicate that the effect of SojG12V on DnaA oligomerization is dependent on the same residues required for the formation of a Soj:DnaA complex. Figure 5.Monomeric Soj specifically prevents DnaA oligomerization in vitro. (A) In vitro oligomer formation assay with DnaASup proteins. DnaA proteins (3 μM) and SojG12V (12, 24, and 36 μM) were incubated in oligomer formation buffer in the presence of ATP (2 mM) and DNA (pBsoriC4; 3 nM) for 15 min prior to the addition of BMOE. DnaA proteins were separated by SDS–PAGE and visualized by western blotting. (B) Monomeric Soj is unable to disassemble pre-formed DnaA oligmers. Reaction conditions are the same as in (A). DnaA was crosslinked at T=15 min. Lane 1, DnaA was added to the reaction buffer at T=13. Lane 2, SojG12V was added at T=0 and DnaA added at T=13. Lane 3, DnaA was added at T=0 and SojG12V added at T=13. (C) Monomeric Soj does not affect DnaA ATP binding. DnaAR313A (3 μM) and/or SojK16A (36 μM) were incubated with α-P32 ATP before being isolated from the reaction using magnetic nickel beads and denatured with methanol. The released nucleotides were separated on a PEI cellulose TLC plate and visualized by autoradiography. Density values were measured using Imag" @default.
• W2110168944 created "2016-06-24" @default.
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• W2110168944 date "2012-01-27" @default.
• W2110168944 modified "2023-09-25" @default.
• W2110168944 title "Soj/ParA stalls DNA replication by inhibiting helix formation of the initiator protein DnaA" @default.
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• W2110168944 doi "https://doi.org/10.1038/emboj.2012.6" @default.
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