Spo0A, the key transcriptional regulator for entrance into sporulation, is an inhibitor of DNA replication
2006; Springer Nature; Volume: 25; Issue: 16 Linguagem: Inglês
10.1038/sj.emboj.7601266
ISSN1460-2075
AutoresVirginia Castilla‐Llorente, Daniel Muñoz‐Espín, Laurentino Villar, Margarita Salas, Wilfried J. J. Meijer,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle3 August 2006free access Spo0A, the key transcriptional regulator for entrance into sporulation, is an inhibitor of DNA replication Virginia Castilla-Llorente Virginia Castilla-Llorente Search for more papers by this author Daniel Muñoz-Espín Daniel Muñoz-Espín Search for more papers by this author Laurentino Villar Laurentino Villar Search for more papers by this author Margarita Salas Margarita Salas Search for more papers by this author Wilfried J J Meijer Corresponding Author Wilfried J J Meijer Instituto de Biología Molecular 'Eladio Viñuela' (CSIC), Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain Search for more papers by this author Virginia Castilla-Llorente Virginia Castilla-Llorente Search for more papers by this author Daniel Muñoz-Espín Daniel Muñoz-Espín Search for more papers by this author Laurentino Villar Laurentino Villar Search for more papers by this author Margarita Salas Margarita Salas Search for more papers by this author Wilfried J J Meijer Corresponding Author Wilfried J J Meijer Instituto de Biología Molecular 'Eladio Viñuela' (CSIC), Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain Search for more papers by this author Author Information Virginia Castilla-Llorente, Daniel Muñoz-Espín, Laurentino Villar, Margarita Salas and Wilfried J J Meijer 1 1Instituto de Biología Molecular 'Eladio Viñuela' (CSIC), Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain *Corresponding author. Facultad de Ciencias, Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain. Tel.: (+34) 91 497 8434; Fax: (+34) 91 497 8490; E-mail: [email protected] The EMBO Journal (2006)25:3890-3899https://doi.org/10.1038/sj.emboj.7601266 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The transcription factor Spo0A is a master regulator for entry into sporulation in Bacillus subtilis and also regulates expression of the virulent B. subtilis phage ϕ29. Here, we describe a novel function for Spo0A, being an inhibitor of DNA replication of both, the ϕ29 genome and the B. subtilis chromosome. Binding of Spo0A near the ϕ29 DNA ends, constituting the two origins of replication of the linear ϕ29 genome, prevents formation of ϕ29 protein p6-nucleoprotein initiation complex resulting in inhibition of ϕ29 DNA replication. At the B. subtilis oriC, binding of Spo0A to specific sequences, which mostly coincide with DnaA-binding sites, prevents open complex formation. Thus, by binding to the origins of replication, Spo0A prevents the initiation step of DNA replication of either genome. The implications of this novel role of Spo0A for phage ϕ29 development and the bacterial chromosome replication during the onset of sporulation are discussed. Introduction The Gram-positive bacterium Bacillus subtilis belongs to a large family of bacteria that respond to nutritional stress by forming highly resistant endospores that can remain dormant for huge periods of time before germinating to resume growth. The multistage sporulation process has been and is widely used as a model system to study cell development. These studies have made of sporulation one of the best understood examples of cellular development and differentiation (for a review see, Piggot and Losick, 2002; Errington, 2003). The master regulator for entry into sporulation is the response regulator Spo0A (Hoch, 1993). Once activated by phosphorylation, Spo0A binds to a DNA sequence containing a so-called '0A-box' (Strauch et al, 1990), where it exerts its role by acting as a transcriptional activator or repressor. Besides being required for the onset of sporulation, Spo0A is also involved in the transcriptional regulation of various other stationary phase processes. Spo0A influences the expression of 520 B. subtilis genes showing that it has indeed a profound effect on the global gene expression pattern of B. subtilis (Fawcett et al, 2000; Liu et al, 2003). Of these 520 genes, 121 are under the direct control of Spo0A. Several of these encode proteins that themselves are directly or indirectly involved in transcriptional regulation, explaining the global effect of Spo0A on transcription (Molle et al, 2003, and references therein). The levels of Spo0A protein and activity increase gradually during the early stages of sporulation (Fujita and Losick, 2005) and the progressive increase of activated Spo0A explains the temporal fashion by which the low- and high-threshold Spo0A-regulated genes (Fujita et al, 2005) are activated or repressed. Spo0A has been subject to extensive mutational analysis, which, together with the recently resolved 3D structures of its phospho-receptor (Spo0AN) (Lewis et al, 1999) and effector (Spo0AC) domain (Lewis et al, 2000; Zhao et al, 2002), have led to major advances in the understanding of its DNA-binding and gene activation properties at the molecular level. Thus, a combination of genetic, biochemical, functional and structural studies has made of Spo0A one of the best-studied response regulators. The genome of ϕ29 consists of a linear double-stranded DNA (dsDNA) with a terminal protein (TP) covalently linked at each 5′ end (see Figure 1A for a genetic and transcriptional map). Phage ϕ29 transcription is divided into an early and a late stage (for a review, see Rojo et al, 1998; Meijer et al, 2001). All late genes are clustered in a single, centrally located, operon that is transcribed from the late promoter A3. The early-expressed genes, encoding all proteins required for phage DNA replication as well as the transcriptional regulator protein p4, are present in two operons. One, located at the right side of the genome, is under the control of the C2 promoter, and the other, located at the left side, is expressed from two tandemly organized promoters named A2b and A2c. Figure 1.Physical, genetic and transcriptional maps of the ϕ29 genome and the B. subtilis oriC region. (A) Map of the ϕ29 genome. The direction of transcription and length of the transcripts are indicated by arrows. The positions of genes are indicated with numbers and those of the main early (A2c, A2b, C2) and late (A3) promoters are boxed. The positions and directionality of the perfect 0A-box sequences are indicated with red triangles. The divergently oriented 0A boxes 5 and 6 are separated by 55 bp. Note that imperfect 0A boxes are not indicated. The bidirectional transcriptional terminator TD1 is indicated with a hairpin structure. Black circles represent the terminal protein covalently attached to the 5′ DNA ends. A black box indicates the region spanning the early promoters A2b and A2c and the late A3 promoter. Adapted from Meijer et al (2005). (B) Map of the B. subtilis oriC region. Positions of perfect DnaA boxes (5′-TTATCCACA-3′; filled) and those having one (diagonal stripes) or two (horizontal stripes) mismatches are indicated by green rectangles and numbered 1–21. DnaA boxes on the upper and lower DNA strand are shown offset high or low, respectively. The dnaA upstream (incA and incB) and downstream (incC) clusters of DnaA boxes are indicated. The position of the single consensus 0A-box sequence is indicated with a red triangle. The white vertical ovals and the white horizontal oval represent the three AT-rich tandem repeats and the 27-mer AT cluster, respectively. The direction of transcription from the dnaA promoter (blue triangle) is indicated by a bent arrow. The divergently oriented rpmH and dnaN genes at the oriC borders are shown on the left and right side, respectively. Dashed bent arrows encompass the minimal oriC region. Download figure Download PowerPoint The ϕ29 genome contains six perfect 0A boxes (5′-TGTCGAA-3′). Whereas one of these (0A-4) is present within gene 8.5, the other five 0A boxes are located in the vicinity of ϕ29 promoters. Three 0A boxes (0A-1, 0A-2, and 0A-3) are present in the intergenic A2c–A3 promoter region and two (0A-5 and 0A-6) are located upstream of promoter C2 (Figure 1A). Binding of Spo0A to sequences containing the 0A boxes in the A2c–A3 promoter region causes repression of the early A2b and A2c promoters and prevents p4-mediated activation of the late A3 promoter. Spo0A also represses the early C2 promoter (Meijer et al, 2005). Here, we describe a novel function for Spo0A. We show that Spo0A functions in vitro as an inhibitor of DNA replication of both, the ϕ29 genome and the B. subtilis chromosome, by preventing the initiation step of DNA replication. Initiation of ϕ29 DNA replication occurs via a so-called protein-primed mechanism and starts at either side of the genome (reviewed by Salas, 1991, 1999). A schematic overview of the in vitro ϕ29 DNA replication mechanism is shown in Figure 2. DNA replication starts at either DNA end by recognition of the origins of replication, constituted by the TP-containing DNA ends, by a heterodimer formed by the ϕ29 DNA polymerase and TP. The essential ϕ29-encoded protein p6 functions as initiator of DNA replication by forming a nucleoprotein complex near the DNA ends required to activate in vivo ϕ29 DNA replication (Blanco et al, 1986; Serrano et al, 1994). Figure 2.Schematic overview of in vitro ϕ29 DNA replication mechanism. Replication starts by recognition of the p6–nucleoprotein complexed origins of replication by a TP/DNA polymerase heterodimer. The DNA polymerase then catalyzes the addition of the first dAMP to the TP present in the heterodimer complex. Next, after a transition step (not shown), these two proteins dissociate and the DNA polymerase continues processive elongation until replication of the nascent DNA strand is completed. Replication is coupled to strand displacement. The ϕ29-encoded SSB protein p5 binds to the displaced ssDNA strands and is removed by the DNA polymerase during later stages in the replication process. Continuous polymerization results in the generation of two fully replicated ϕ29 genomes. Circles, TP; triangles, DNA polymerase; ovals, replication initiator protein p6; diamonds, SSB protein p5; de novo synthesized DNA is shown as beads on a string. Download figure Download PowerPoint Initiation of the circular B. subtilis chromosome also requires the formation of an initiation complex. In this case, the host-encoded DNA-replication initiator protein DnaA binds to multiple (im)perfect DnaA boxes within the oriC region. The DnaA boxes are clustered in three regions, named incA, incB and incC (Moriya et al, 1988, see Figure 1B). All three DnaA box clusters are required for initiation of chromosomal replication (Moriya et al, 1992). DnaA bound to its cognate sites in the incA and incB regions interact with DnaA bound to the DnaA boxes in incC resulting in loop formation of the intervening region (reviewed by Moriya et al, 1999; Messer, 2002). This looped complex appears necessary for local unwinding of dsDNA close to the 27-mer AT cluster in incC, which provides entry for the DNA helicase and recruitment of additional factors to allow formation of a functional replisome. Here we show that Spo0A binds specifically near both phage ϕ29 DNA ends, thereby preventing the formation of the p6–nucleoprotein complex and hence activation of ϕ29 DNA replication. Similarly, we show that Spo0A binds to sequences in the incA and incB oriC region of the B. subtilis chromosome and that Spo0A prevents DnaA-mediated oriC unwinding. Together, these results show for the first time that the well-studied transcriptional regulator protein Spo0A of B. subtilis acts directly as an inhibitor of DNA replication. Results Spo0A inhibits ϕ29 DNA replication Phage ϕ29 0A box 6, which is divergently oriented with respect to 0A box 5, is located 50 bp from the right DNA end, which constitutes an origin of ϕ29 DNA replication. Binding of Spo0A to the 0A box 6 region might, therefore, interfere with phage ϕ29 DNA replication. Using two different approaches, we studied whether Spo0A affects ϕ29 DNA replication in vivo. In the first approach, ϕ29 DNA replication was studied in wild-type B. subtilis 168 (168) and isogenic spo0A-deleted (168∷Δspo0A) cells infected under sporulation-inducing conditions. Thus, cells grown in Schaeffer's sporulation medium were infected with ϕ29 one hour after the end of logarithmical growth. The kinetics of ϕ29 DNA replication was then quantified by real-time PCR by determining the amount of intracellular ϕ29 DNA at different times after infection. The results presented in Figure 3A show that the level of intracellular ϕ29 DNA started to increase about 20 min after infection in 168∷Δspo0A but not in wild-type cells. These results show that ϕ29 is able to replicate its DNA in stationary phase growing spo0A mutant cells, and indicate that ϕ29 DNA replication is inhibited in a spo0A-dependent way. In the second approach, ϕ29 DNA replication was analyzed in logarithmical phase cells with or without artificial induction of Spo0A. The activity of Spo0A is normally subject to complex regulatory pathways (for a review, see Hoch, 1993). To bypass the transcriptional circuits that control Spo0A expression and the requirement of the phosphorelay for its activation, we used for these experiments strain sad67∷Tc in which a mutant spo0A gene, spo0A–sad67, is under the control of an IPTG inducible promoter (Veening et al, 2005). Spo0A–Sad67 does not require phosphorylation to be active (Ireton et al, 1993). Thus, real-time PCR was used to study in vivo ϕ29 DNA replication as a function of time using infected B. subtilis sad67∷Tc cells grown in rich medium (LB) without or with Spo0A–Sad67 induction. As expected, ϕ29 DNA replication occurred in the absence of Spo0A–Sad67 induction. However, phage DNA replication was almost completely blocked in cells in which Spo0A–Sad67 was induced (Figure 3B). Figure 3.In vivo ϕ29 DNA replication is inhibited in a spo0A-depedent way. (A) Cultures of wild-type B. subtilis 168 (168) or the isogenic spo0A-deletion strain WM90 (Δ0A), grown in Schaeffer's sporulation medium, were infected with ϕ29 (multiplicity of 5) 1 h after entry into the stationary phase. At the indicated times after infection, the amount of viral DNA was measured by real-time PCR. Data are expressed as nanograms of ϕ29 DNA per ml of culture. (B) A culture of the B. subtilis sad67∷Tc strain containing the spo0A–sad67 gene under the control of an inducible IPTG promoter (Veening et al, 2005) was grown in LB medium. At the mid-log phase, the culture was split into two and in one of them Spo0A–Sad67 was induced. After 10 min, the cultures were infected with ϕ29 (multiplicity of 10). Next, at the indicated times after infection, the amount of viral DNA was measured by real-time PCR. Data are expressed as nanograms of ϕ29 DNA per ml of culture. Download figure Download PowerPoint Whereas these latter results confirm that ϕ29 DNA replication is inhibited in a Spo0A-dependent way, these approaches do not allow to distinguish whether Spo0A inhibits phage DNA replication directly or indirectly. This distinction can be made by in vitro approaches in which the input of all components is controlled. Thus, native Spo0A was purified from overexpressing E. coli cells as described before (Muchová et al, 2004). Spo0A forms dimers upon phosphorylation and Spo0A dimers constitute the active form of Spo0A (Asayama et al, 1995; Lewis et al, 2002; Ladds et al, 2003; Muchová et al, 2004). Ladds et al (2003) showed that a portion of wild-type Spo0A purified from E. coli is in its active phosphorylated dimeric form. Indeed, gel filtration experiments showed that 15–40% of our purified Spo0A were dimers (not shown). The functionality of our purified Spo0A is demonstrated by the facts that (i) it produces highly similar footprints on the B. subtilis promoter spoIIG-associated 0A boxes to those published (Satola et al, 1992, not shown) and (ii) it has in vitro promoter activating and repressing activity (Meijer et al, 2005). Thus, to study the possibility that Spo0A is directly responsible for the observed inhibition of ϕ29 DNA replication in vivo, we analyzed the effect of purified Spo0A in the in vitro ϕ29 DNA amplification system. This system, which allows the amplification of low amounts of ϕ29 DNA, requires four ϕ29-encoded proteins: DNA polymerase, TP, replication initiator protein p6, and the SSB protein p5 (Blanco et al, 1994, see Figure 2 for in vitro ϕ29 DNA replication scheme). Figure 4A shows that Spo0A directly inhibits ϕ29 DNA amplification; thus, more than 75% was inhibited in the presence of 5 μM Spo0A. As at most 40% of the Spo0A of the purified sample is in its active dimeric form, this inhibitory effect is exerted at a maximum concentration of 2 μM active Spo0A. Figure 4.Spo0A inhibits in vitro ϕ29 DNA replication. (A) In vitro ϕ29 DNA amplification. Reaction mixtures contained ϕ29 DNA polymerase (6 nM), TP (6.5 nM), ϕ29 TP-DNA (32 pM), initiation protein p6 (33.3 μM), SSB protein p5 (24.0 μM), and no or increasing amounts of Spo0A. After incubation for 45 min at 30°C, reactions were stopped and subjected to alkaline agarose gel electrophoresis. The migration position of full-length ϕ29 DNA (19 285 bp) is indicated. Spo0A concentrations ranged from 1.25 to 80 μM (two-fold dilution steps). (B) In vitro protein-primed initiation of ϕ29 DNA replication (ϕ29 TP-dAMP formation). Protein-primed, TP-DNA-dependent replication initiation activity was measured as a function of Spo0A concentration. Reaction mixtures contained ϕ29 DNA polymerase (12 nM), TP (12.9 nM), initiation protein p6 (33.3 μM), ϕ29 TP-DNA template (1.6 nM), 0.1 μM [α-32P]dATP (1 μCi), and no or increasing amounts of Spo0A. After incubation for 5 min at 30°C, the reactions were stopped, processed and analyzed by SDS–PAGE and autoradiography. The position of TP-dAMP initiation product is indicated. Spo0A concentrations ranged from 2 to 16 μM (two-fold dilution steps). Note that higher amounts of ϕ29 TP-DNA are used in initiation than in amplification assays. Download figure Download PowerPoint The observed Spo0A-mediated inhibition of ϕ29 DNA amplification is not due to non-specific binding of Spo0A to DNA because Spo0A did not affect the efficiency and/or velocity in M13 replication assays in which ϕ29 DNA polymerase drives continuous rounds of replication on primed circular M13 DNA (not shown). Spo0A inhibits ϕ29 DNA replication at the initiation step To study whether Spo0A exerts its inhibitory effect on ϕ29 DNA amplification at the initiation step, TP-DNA-directed in vitro ϕ29 DNA replication initiation assays were performed in the absence or presence of Spo0A. As efficient initiation of in vitro ϕ29 DNA replication requires the replication initiation protein p6, this protein was included in the reaction mixtures. Figure 4B shows that the initiation of TP-DNA replication is inhibited in a Spo0A-dependent way. Low levels of TP-primed initiation activity can be obtained in the absence of protein p6 (Blanco et al, 1986). Under these conditions, Spo0A did not have a significant effect (not shown) indicating that Spo0A affects specifically the protein p6-mediated initiation of ϕ29 DNA replication (see also below). Spo0A binds to the ϕ29 origins of replication in vitro, thereby preventing formation of a p6–nucleoprotein initiation complex Phage ϕ29 DNA replication starts at either side of the genome. Spo0A almost fully prevented p6-stimulated initiation of DNA replication in reactions containing full-length ϕ29 DNA indicating that Spo0A affects the initiation reaction at either genome end. The possibility that Spo0A inhibits ϕ29 DNA replication by binding to the origins of replication was studied by DNase I footprinting using DNA fragments (202 bp) corresponding to the left or right side DNA sequences of the ϕ29 genome. Figure 5A and B show that Spo0A protects a specific region of about 45 bp near both DNA ends. Full protection of this region was observed using 1.8 μM of purified Spo0A. Thus, Spo0A binds at very similar positions near the left and right DNA ends. Interestingly, these Spo0A-protected DNA regions contain four adjacent 0A-box (like) sequences, each separated by a 3 bp spacer. Moreover, the positions of these boxes are located at exact equidistant positions from the extreme DNA ends (Figure 6). Figure 5.Spo0A prevents formation of p6–nucleoprotein initiation complexes at the ϕ29 origins of replication. Binding of Spo0A without (left panels) or with (right panels) the initiator protein p6 to the left (A) and right (B) ϕ29 origins of replication was analyzed by DNase I footprinting. DNA fragments corresponding to the 202 bp DNA ends of ϕ29, labeled at their 5′ ends, were incubated with the proteins as shown above the footprints. When indicated, 1 μg of initiator protein p6 was added 10 min after Spo0A addition. Spo0A concentrations ranged from 116 nM to 29.7 μM and 464 nM to 29.7 μM (four-fold dilution steps) in the left and right panels, respectively. Hatched boxes indicate the positions of imperfect 0A boxes. The single filled box indicates the position of the perfect 0A box sequence at the right ϕ29 origin (corresponding to 0A-6 in Figure 1A). Protein p6-induced hypersensitive sites are indicated with arrows. The top of the footprints correspond to the ϕ29 DNA ends. Download figure Download PowerPoint Figure 6.Organization of 0A boxes at the origins of replication of ϕ29 and the B. subtilis chromosome. The positions of 0A and DnaA boxes are indicated with red and green rectangles above and below the sequence, respectively. Mismatches with respect to their consensus sequence are indicated with crosses. (A) Right and left DNA sequences of the ϕ29 genome are shown in the upper and lower panel, respectively. Black circles represent TP bound to the 5′ DNA ends. The perfect 0A box at the right ϕ29 genome corresponds to 0A-6 in Figure 1A. (B) Sequence of the incB region of B. subtilis oriC containing the perfect 0A box. Note (i) that the organization of the 0A boxes is similar to that at the ϕ29 origins, (ii) that three 0A boxes coincide with imperfect DnaA boxes, and (iii) that the mismatches in the DnaA boxes result in (im)perfect 0A boxes (see text for details). (C) Sequence of the incA region of B. subtilis oriC containing two imperfect 0A boxes that coincide with imperfect DnaA boxes. Note that the stippled DnaA box containing three mismatches is not indicated in Figure 1. Numbering in (B) and (C) are according to Moriya et al (1992). Download figure Download PowerPoint Protein p6 binds preferentially at the ϕ29 DNA end regions, forming a specific nucleoprotein complex that highly stimulates ϕ29 DNA replication (Blanco et al, 1986; Serrano et al, 1989). We therefore studied whether binding of Spo0A at the ϕ29 origins prevents the formation of the p6–nucleoprotein complex. Lanes 8 of Figure 5A and B show the typical footprint generated by binding of protein p6 at the DNA ends (Serrano et al, 1989). However, the p6-induced footprint became lost when Spo0A bound to the regions near the DNA ends (Figure 5A and B, lanes 9 and 10), indicating that Spo0A prevents formation of the p6–nucleoprotein complex. Note that protein p6 generates a footprint that extends all along the DNA fragment used (Figure 5A and B, lanes 8). Binding of Spo0A to its confined sequences not only prevents p6 from binding to this region but also to sequences outside the Spo0A-binding region. Very similar results were obtained when Spo0A was added to the reaction after formation of the p6–nucleoprotein complex was allowed to take place (not shown), demonstrating that Spo0A can also annihilate a pre-existing replication initiation complex. Together, these results show that specific binding of Spo0A near the ϕ29 DNA ends inhibits initiation of phage DNA replication by preventing the formation of the replication-initiation p6–nucleoprotein complex. Spo0A binds to sequences within the B. subtilis oriC region A perfect 0A box is located in the incB oriC region and, as assessed by gel mobility shift assays, the C-terminal DNA-binding domain of Spo0A, Spo0AC, binds to the incB region (Molle et al, 2003). In addition, the 0A box sequence (5′-TGTCGAA-3′) shares homology with the complementary strand of the perfect DnaA box sequence (5′-TGTGGATAA-3′). As Spo0A can bind to some imperfect 0A boxes, it might bind to DnaA boxes. If this were the case, Spo0A might affect initiation of replication of the B. subtilis chromosome. DNase I footprinting was used to study possible binding of Spo0A to the incA, incB, and incC regions of oriC. Spo0A did not bind to the incC region (not shown). However, it did bind to sequences in the incB and incA regions (Figure 7A and B, respectively). In the case of the incB region, ∼30 bp including the perfect 0A box was protected at low Spo0A concentrations (0.46 μM). At higher Spo0A concentrations, the footprint was enlarged by ∼20 bp towards the 5′ direction. Indeed, the extended Spo0A-protected region contains, besides the perfect 0A box, three imperfect 0A boxes. These four 0A boxes have the same orientation and are all separated by a 3 bp spacer (see Figure 6B). Thus, the organization of the 0A boxes in this region is highly similar to that found at the ϕ29 origins of replication. Figure 7.Spo0A binds to sequences in the B. subtilis oriC incB and incA regions. Binding of Spo0A to the incB (A) or incA (B) region of B. subtilis oriC was analyzed by DNase I footprinting. DNA fragments, containing oriC positions 314 till 626 (A) and 86 till 387 (B) according to Moriya et al (1992) and labeled at their 5′ upper strand, were incubated without or with increasing amounts of Spo0A before DNase I digestion. Filled and hatched red boxes indicate the positions of the perfect and imperfect 0A boxes, respectively. Green boxes indicate the positions of the DnaA boxes. The numbering and coloring scheme of Figure 1B is used to indicate perfect or imperfect DnaA boxes. Spo0A concentration ranged from 116 nM to 29.7 μM (four-fold dilution steps). Download figure Download PowerPoint As shown in Figure 6B, three of the four 0A boxes coincide with imperfect DnaA boxes, which constitute bona fide binding sites for DnaA (Fukuoka et al, 1990). The perfect 0A box (5′-TGTCGAA-3′) corresponds to an imperfect DnaA box having two mismatches (5′-TTtTCgACA-3′) with respect to its consensus sequence (5′-TTATCCACA-3′). Interestingly, these two deviations result in the perfect 0A box sequence. A similar situation occurs for the other two upstream DnaA boxes that are protected by Spo0A; that is, the single deviation in the first upstream DnaA box (5′-TTgTCCACA-3′) and the two deviations in the second upstream DnaA box (5′-TTcTaCACA-3′) create 0A boxes that have two and one mismatches with respect to the consensus 0A box sequence, respectively. Thus, binding of Spo0A to this extended region overlaps with three functional DnaA-binding sites. At incA, Spo0A protected two separate regions. The downstream protected region contains two imperfect 0A boxes (one and two mismatches) that are separated by a 4 bp spacer. Also in this case, the 0A boxes coincide with imperfect DnaA boxes (see Figure 6C). The upstream incA region on which Spo0A produced a footprint corresponds to the 45 bp spacer that separates the three 16-mer AT-rich tandem repeats from the incA DnaA cluster. Whereas Spo0A produced a clear footprint at the upstream half of this region, the downstream half became only partially protected even at high Spo0A concentrations. Inspection of this 45 bp sequence revealed that the well-protected upstream half contains two heptamers, separated by a 3 bp spacer, whose sequences have three mismatches with respect to the consensus 0A box sequence. In summary, Spo0A binds to sequences present in the incA and incB oriC regions and, except for those in the 45 bp sequence downstream of the three 16-mer AT-rich tandem repeats, the Spo0A-binding sites overlap with functional DnaA-binding sites. Spo0A prevents DnaA-mediated unwinding of oriC The following approach was used to analyze whether Spo0A affects the initiation of DNA replication at oriC. Binding of DnaA to its cognate binding sites is required for initiation of DNA replication of the B. subtilis chromosome (Moriya et al, 1990). This binding causes local unwinding at or near an AT-rich region, which is a crucial step in the initiation of DNA replication. Krause et al (1997) precisely mapped the DnaA-mediated open complex to a short region that includes the left part of the 27-mer AT cluster and its 15 upstream bp (see Figure 1B). We used the system developed by Krause et al (1997) to study whether Spo0A interferes with DnaA-mediated open complex formation (see Materials and methods). In brief, purified DnaA was added without or with increasing amounts of Spo0A to supercoiled plasmid pBsoriC4, which carries the entire B. subtilis oriC region. Samples were then treated with potassium permanganate to oxidize any unpaired pyrimidines. Next, after linearization and purification of the DNA, primer extension was used to probe for the level of open complex formation at incC. As expected, DnaA-dependent open complex formation at positions identical to those described before (Krause et al, 1997) was observed in the absence of Spo0A (lanes 3 of Figure 8A and B). Open complex formation was progressively inhibited, however, in the presence of increasing amounts of Spo0A (lanes 4–8 of Figure 8A and B); ∼5
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