Alternative interactions between the Tn7 transposase and the Tn7 target DNA binding protein regulate target immunity and transposition
2003; Springer Nature; Volume: 22; Issue: 21 Linguagem: Inglês
10.1093/emboj/cdg551
ISSN1460-2075
Autores Tópico(s)Bacteriophages and microbial interactions
ResumoArticle3 November 2003free access Alternative interactions between the Tn7 transposase and the Tn7 target DNA binding protein regulate target immunity and transposition Zachary Skelding Zachary Skelding Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD, 21205 USA Search for more papers by this author Jennie Queen-Baker Jennie Queen-Baker Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD, 21205 USA Search for more papers by this author Nancy L. Craig Corresponding Author Nancy L. Craig Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD, 21205 USA Search for more papers by this author Zachary Skelding Zachary Skelding Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD, 21205 USA Search for more papers by this author Jennie Queen-Baker Jennie Queen-Baker Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD, 21205 USA Search for more papers by this author Nancy L. Craig Corresponding Author Nancy L. Craig Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD, 21205 USA Search for more papers by this author Author Information Zachary Skelding1, Jennie Queen-Baker1 and Nancy L. Craig 1 1Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD, 21205 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:5904-5917https://doi.org/10.1093/emboj/cdg551 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Tn7 transposon avoids inserting into a target DNA that contains a pre-existing copy of Tn7. This phenomenon, known as 'target immunity', is established when TnsB, a Tn7 transposase subunit, binds to Tn7 sequences in the target DNA and mediates displacement of TnsC, a critical transposase activator, from the DNA. Paradoxically, TnsB–TnsC interactions are also required to promote transposon insertion. We have probed Tn7 target immunity by isolating TnsB mutants that mediate more frequent insertions into a potentially immune target DNA because they fail to provoke dissociation of TnsC from the DNA. We show that a single region of TnsB mediates the TnsB–TnsC interaction that underlies both target immunity and transposition, but that TnsA, the other transposase subunit, channels the TnsB–TnsC interaction toward transposition. Introduction Transposable elements use many different strategies to select their target DNAs (Craig, 1997; Craig et al., 2002). For example, insertion into nearby DNA is common for IS10/Tn10 elements. Insertion into DNA flanking the transposon can lead to the evolution of new composite IS10/Tn10 elements; however, insertion within the transposon itself can lead to element destruction (Kleckner et al., 1996). Other elements, such as Tn7, Mu and Tn3, preferentially insert into target sites that are distally located and do not already contain a copy of the transposable element (Robinson et al., 1977; Hauer and Shapiro, 1984; Reyes et al., 1987; Arciszewska et al., 1989). This bias in selecting a distal target site is known as 'target immunity' because the presence of a transposable element in a target DNA renders the DNA 'immune' to future insertions. The immunity signal is provided by the sequences at the ends of the transposon DNA that are bound by the transposase. In the case of Tn7 (Stellwagen and Craig, 1997a,b) and Mu (Maxwell et al., 1987; Adzuma and Mizuuchi, 1988), target immunity has been shown to depend on the interaction of the transposase with an element-encoded target DNA binding transposase activator protein; the interaction of these proteins results in the dissociation of the transposase activator from the target DNA. In this report, we explore the molecular mechanism by which TnsB, the component of the Tn7 transposase that binds to the ends of the transposon, interacts with TnsC, the target DNA binding transposase activator, to establish target immunity. Tn7 target immunity is effective over large distances in vivo (Arciszewska et al., 1989; DeBoy and Craig, 1996). For example, the presence of Tn7 transposon end sequences in the Escherischia coli chromosome decreases insertions into a target site 190 kb away; however, insertion into a target site 1900 kb away is not affected (DeBoy and Craig, 1996). Thus Tn7 target immunity is a distance-dependent cis-acting phenomenon. While Tn7 avoids inserting into DNA containing a 'negative' signal, i.e. Tn7 transposon end sequences, it can also be attracted to a target DNA by 'positive' signals. The Tn7 target selection proteins TnsD or TnsE are responsible for recognizing 'positive' target signals (Peters and Craig, 2001; Craig, 2002). The target selection proteins collaborate with the TnsAB transposase and the TnsC transposase activator to promote transposition into two types of target DNAs: (i) TnsABC + D direct insertions into DNA containing attTn7, a unique site in the E.coli chromosome (Bainton et al., 1993), and (ii) TnsABC + E direct insertions into DNA that is undergoing lagging strand DNA synthesis, such as conjugating DNA (Wolkow et al., 1996; Peters and Craig, 2001). However, both the TnsABC + D and the TnsABC + E transposition pathways are inhibited by target immunity, i.e. the presence of a 'negative' target signal is dominant to a 'positive' signal present on the same DNA (Arciszewska et al., 1989; DeBoy and Craig, 1996). Insight into the molecular interactions that promote Tn7 insertion into target DNA containing a 'positive' signal and inhibit insertion into DNA containing a 'negative' signal has come from the in vitro reconstituted TnsABC + D transposition system (Bainton et al., 1993; Craig, 2002). The attTn7 target DNA is recognized in a sequence-specific manner by TnsD, which then recruits the ATP-dependent sequence-independent DNA binding protein TnsC (Gamas and Craig, 1992; Bainton et al., 1993; Kuduvalli et al., 2001). TnsC, in turn, interacts with the TnsAB transposase and the transposon DNA resulting in the assembly of an essential transposition intermediate, the TnsABC + D transposon–target nucleoprotein complex (Skelding et al., 2002). Thus, the presence of TnsC on a target DNA is key to attracting Tn7. What feature of Tn7 provides a negative signal? That is, how does a pre-existing Tn7 element block insertions into the DNA in which it resides? The presence of TnsB binding sites on an attTn7 target DNA render it immune to insertions by increasing the local concentration of TnsB on the target DNA, which enables a TnsB–TnsC interaction that provokes displacement of TnsC from the DNA via a mechanism dependent on ATP hydrolysis by TnsC (Stellwagen and Craig, 1997a). Therefore 'positive' and 'negative' target signals determine whether or not TnsC binds stably to a DNA, which, in turn, determines whether that target DNA will receive a Tn7 insertion. Tn7 transposition and target immunity both depend on interactions between TnsB and TnsC. These interactions are essential for the assembly of a stable transposon–target complex (Skelding et al., 2002), and for the displacement of TnsC from DNA during target immunity (Stellwagen and Craig, 1997a). Are the TnsB–TnsC interactions that mediate the assembly and disassembly processes the same or different? To examine this question, we have probed the role of TnsB in establishing target immunity by isolating and analyzing TnsB mutants that are impaired in their ability to impose immunity on a target DNA that contains Tn7 ends close to attTn7. The mutations map to the carboxyl terminus of TnsB, a region shown previously to be essential for the TnsB–TnsC interaction that promotes transposition (Skelding et al., 2002). We show that these TnsB mutants are defective for imposing target immunity owing to a decrease in their ability to interact with TnsC and promote displacement of TnsC from target DNA. We have also found that TnsA, the other component of the Tn7 transposase, plays a critical role in influencing this TnsB–TnsC interaction: TnsA decreases the TnsB-mediated displacement of TnsC from target DNA, and thus promotes transposition. We discuss how a similar TnsB–TnsC interaction is able both to build nucleoprotein complexes for transposition in conjunction with TnsA and to displace TnsC from target DNA containing transposon ends to establish target immunity. Results Isolation of TnsB mutants that bypass target immunity Central to Tn7 target immunity is the interaction of TnsB, the component of the Tn7 transposase that binds specifically to the ends of Tn7, with TnsC, the target binding protein (Stellwagen and Craig, 1997a). A DNA containing a copy of Tn7 is made immune to further insertions because the TnsB–TnsC interaction results in the displacement of TnsC from the target DNA (Stellwagen and Craig, 1997a). To understand better the role of TnsB in Tn7 target immunity, we isolated TnsB mutants that are defective in establishing immunity but still maintain their ability to promote transposition. To isolate these mutants, we exploited the ability of Tn7 to insert at high frequency into attTn7, a specific chromosomal site 3′ of the bacterial glmS gene. Due to target immunity, insertion of a second element into attTn7 is much reduced in a strain containing a non-mobilizable mini-Tn7 (mTn7) element 8 kb from attTn7 (attTn7 + mTn78kb) (DeBoy and Craig, 1996). We used a visual assay to screen for TnsB mutants that promote transposition at a higher frequency into the attTn7 + mTn78kb site than TnsBwt (see Materials and methods) (Figure 1A). Figure 1.Isolation of TnsB immunity bypass mutants (A) A mini-Tn7lac (mTn7lac) element, consisting of a promoterless lacZY gene cassette flanked by the Tn7 left (Tn7L) and right (Tn7R) sequences required for its mobilization (triangles), resides in a transcriptionally silent location on a plasmid such that, in the absence of transposition, cells containing the plasmid have a Lac− phenotype. However, when the mTn7lac element inserts into attTn7, it is transcribed by the glmUS promoter upstream of attTn7 (Bainton et al., 1993), generating a cell with a Lac+ phenotype, i.e. formation of Lac+ papillae on a MacConkey lactose indicator plate. Thus, in a strain containing a non-mobilizable mTn7 element 8 kb from attTn7 (attTn7 + mTn78kb) (DeBoy and Craig, 1996), the frequency of mTn7lac insertion into attTn7 is very low, and this strain produces very few Lac+ papillae when grown on a MacConkey lactose indicator plate (second column). TnsB mutants that bypass immunity will promote higher-frequency transposition to attTn7 + mTn78kb, which increases the number of Lac+ papillae per colony (third column). (B) TnsB mutations that affect target immunity and transposition overlap. The TnsB immunity bypass mutations (top) were localized to the C-terminus of TnsB (middle). Alanine scanning mutagenesis was shown previously to block TnsB–TnsC interaction and transposition (bottom) (Skelding et al., 2002). Download figure Download PowerPoint Four TnsB 'immunity bypass' mutants were recovered by screening ∼42 000 colonies that were transformed with mutagenized TnsB plasmids. Although the entire TnsB gene was subjected to mutagenesis, DNA sequencing revealed that the mutations were tightly clustered at the carboxyl terminus of the TnsB 702 aa protein: TnsBP686S, TnsBV689M (two isolates) and TnsBP690L (Figure 1B). The location of the mutations is particularly striking because, as reported elsewhere (Skelding et al., 2002), other mutations within the same region prevent TnsB from interacting with TnsC-bound target DNA, which profoundly decreases TnsABC + D transposition (Figure 1B). Therefore mutation of a single region within TnsB can affect two processes that have been shown to depend upon contact between TnsB and TnsC: target immunity and TnsABC + D transposition. Indeed, the experiments presented in this work provide strong support for the idea that the same TnsB–TnsC interaction is being utilized for both processes. The ability of TnsBwt and the TnsB immunity bypass mutants to catalyze TnsABC + D transposition in vivo was examined using a quantitative assay that measures the translocation of a mTn7 element from a lambda phage to the bacterial chromosome (Materials and methods). Consistent with previous work (DeBoy and Craig, 1996), the presence of transposon ends 8 kb from attTn7 decreased TnsABC + D transposition into this site >60-fold (Table I, column 5), reflecting the target immunity effect. Table 1. The TnsB mutants promote increased insertion into an attTn7 + mTn78kb chromosome TnsB allele Transposition frequency into attTn7 + mTn78kba TnsBmut attTn7 + mTn78kbb Transposition frequency into attTn7 Tpn. attTn7c TnsBwt attTn7 + mTn78kb Tpn. attTn7 + mTn78kb TnsABwtC + D 0.4 (± 0.13) 1d 25 (± 5.4) 62.5 TnsABP686SC + D 1.7 (± 0.50) 4.2 5.0 (± 1.2) 2.9 TnsABV689MC + D 1.9 (± 0.53) 4.8 7.8 (± 0.71) 4.1 TnsABP690LC + D 1.1 (± 0.22) 2.8 3.5 (± 0.29) 3.2 a Transposition frequency is expressed as the ratio of chloramphenicol-resistant colonies to particle forming units of lambda phage (see Materials and methods). Each value represents the average of ≥3 trials where the final value is multiplied by 10. b TnsABC + D transposition frequency in a strain containing attTn7 + mTn78kb divided by the TnsABC + D transposition frequency in a strain containing attTn7 + mTn78kb. c TnsABC + D transposition frequency in a strain containing attTn7 divided by the TnsABC + D transposition frequency in a strain containing attTn7 + mTn78kb. d The numerator and denominator are equivalent: TnsABC + D transposition frequency in a strain containing attTn7 + mTn78kb. The TnsB immunity bypass mutants promoted 2.8–4.8 times more frequent transposition into attTn7 + mTn78kb than TnsBwt (Table I, column 3). Sequence analysis verified that 25/25 TnsABV689MC + D reactions contained a mTn7 element inserted at the appropriate position and with the appropriate orientation within attTn7. Thus the TnsB immunity bypass mutants do indeed promote increased transposition into attTn7 + mTn78kb, a site normally refractory to insertion because of target immunity. When the TnsB immunity bypass mutants were examined for their ability to carry out transposition into an attTn7 site not flanked by Tn7 ends, they all promoted less transposition than TnsABwtC + D (Table I, column 4). Thus, while able to promote increased transposition into a target site that lies close to 'immunizing' TnsB binding sites, these TnsB mutants were also slightly defective for transposition in general. The TnsBV689M mutant was further characterized because it increases transposition to attTn7 + mTn78kb more than the other mutants, while also promoting the most transposition into an attTn7 site lacking nearby Tn7 ends. We purified TnsBV689M and analyzed its properties in vitro. TnsBV689M is defective in establishing transposition immunity in vitro as evaluated by a redistribution assay In a previous study, target immunity was reconstituted in an in vitro system in which TnsB was able to provoke the 'redistribution' of TnsC from a target DNA containing the right end of Tn7 (attTn7 + Tn7R) to a target DNA lacking TnsB binding sites (attTn7). This reaction depends upon ATP hydrolysis (Stellwagen and Craig, 1997a). We examined the ability of TnsBV689M to provoke TnsC redistribution with a similar assay (Figure 2A). TnsD and a limited quantity of TnsC were bound to an attTn7 + Tn7R target DNA (Figure 2A, Tgt. #1). In a separate reaction, TnsD was bound to an attTn7 target DNA, which was present in equimolar concentration to the attTn7 + Tn7R target DNA (Figure 2A, Tgt. #2). The protein-bound target DNAs were combined in one tube and then incubated with TnsB for various times. After the TnsB incubation period, the distribution of TnsC among the target DNAs was evaluated by adding mTn7 donor DNA, TnsA, additional TnsB and Mg2+ to determine which target DNAs received Tn7 insertions. Figure 2.TnsBV689M is defective for promoting TnsC 'redistribution' (A) Reaction scheme. (B) In lanes 1–6, half of the TnsBwt was added during the 'TnsB incubation' and the remainder was added during the 'transposition' phase. In lanes 7–11, all the TnsBwt was added during the 'transposition' phase. The DNA products of the reaction were linearized and detected with a mTn7-specific probe. The slowest mobility species is the unreacted mTn7 donor DNA (Donor). The next two bands arise from simple insertion (SI) of the mTn7 element into either the attTn7 + Tn7R target DNA or the attTn7 target DNA. A double-strand break (DSB) at the Tn7R or the Tn7L end in the mTn7 donor DNA produces DSB-R and DSB-L, respectively. The band above DSB-L is due to cross-hybridization of the mTn7-specific probe to the unreacted attTn7 + Tn7R target DNA. (C) Reactions in lanes 1–11 were identical to the reactions in (B) except that TnsBV689M was substituted for TnsBwt. Download figure Download PowerPoint When the TnsBwt incubation period was short, the attTn7 + Tn7R target received all the insertions and target immunity was not observed, i.e. TnsC was not redistributed to the attTn7 target plasmid that lacked TnsB binding sites (Figure 2B, lane 1). However, after a 30 min TnsBwt incubation period, the majority of insertions were directed to the attTn7 target DNA (Figure 2B, lane 4), reflecting a redistribution of TnsC from the attTn7 + Tn7R target to the attTn7 target. When the combined target DNAs were incubated for 2 h in the absence of TnsB, the attTn7 + Tn7R target DNA still received most of the insertions, indicating that the spontaneous redistribution of TnsC is a slow process (Figure 2B, lane 11). Thus TnsBwt incubation is required to impose immunity on the attTn7 + Tn7R target DNA. In contrast, when we examined the ability of TnsBV689M to promote TnsC redistribution, we found that, even after a 60 min incubation, insertions occurred equally into both target DNAs (Figure 2C, lane 5), revealing that TnsBV689M does not effectively displace TnsC from the attTn7 + Tn7R target DNA. Thus, as was true in vivo, TnsBV689M is less able than TnsBwt to discourage transposition into a target DNA containing TnsB binding sites. Incubation with TnsBwt 'inactivates' TnsCD–attTn7 target DNA In the 'redistribution' experiment described above, the attTn7 + Tn7R target DNA became immune to insertion after incubation with TnsBwt because TnsC was displaced from attTn7 + Tn7R, leading to an accumulation of TnsC on the attTn7 target DNA. However, the mechanism by which TnsB interacts with TnsC in vitro to promote its displacement from a target DNA does not strictly depend upon TnsB and TnsC being bound to the same target DNA. For example, when Tn7R and attTn7 are on separate, but catenated plasmids, incubation with TnsB still decreases transposition into the tethered attTn7 plasmid (Stellwagen and Craig, 1997a). This finding suggests that binding of TnsB to the same DNA as TnsC is important only to increase the local concentration of TnsB around TnsC, as opposed to a model where the DNA itself plays an essential role in enabling TnsB to interact with TnsC (Adzuma and Mizuuchi, 1989). Consistent with this view, incubation of a TnsCD-bound attTn7 target DNA with TnsBwt can promote moderate redistribution of TnsC from its original attTn7 target DNA to another attTn7 target DNA, even when the original attTn7 target DNA lacks TnsB binding sites (Stellwagen and Craig, 1997a). Building on these observations, we evaluated the effect of TnsB incubation on TnsC DNA binding using an assay that does not depend upon the redistribution of TnsC from one target DNA to another. We bound TnsC and TnsD to an attTn7 target DNA, and then added TnsB, mTn7 donor DNA and Mg2+ and incubated for various times (Figure 3). The effect of TnsB incubation on the TnsCD-bound attTn7 target DNA was assessed by determining how much transposition occurred into the target DNA upon addition of TnsA. Figure 3.Incubation of TnsCD-bound attTn7 target DNA with TnsB decreases subsequent transposition. TnsBwt was added to the reactions in lanes 1–5 and TnsBV689M was added to the reactions in lanes 6–10. Bands are labeled as in Figure 2B. The bands above the donor result from a double-strand break at either Tn7R or Tn7L that was 'single end joined' to the target DNA (DSB-R/SEJ and DSB-L/SEJ) (Bainton et al., 1993). Download figure Download PowerPoint In the absence of TnsBwt incubation, transposition was robust; most of the mTn7 donor DNA was converted to the simple insertion product (Figure 3, lane 1). However, as the TnsBwt incubation period was increased, the amount of simple insertion product decreased (Figure 3, lanes 2–5). Thus incubation with TnsBwt can decrease the ability of the TnsCD-bound attTn7 target DNA to participate in transposition, even when that target DNA lacks specific TnsB binding sites. We found that TnsBV689M, like TnsBwt, promoted substantial transposition into the TnsCD–attTn7 target (Figure 3, lane 6 versus lane 1). In striking contrast, TnsBV689M incubation did not significantly reduce the amount of simple insertion product (Figure 3, lanes 6–10). Thus, while TnsBV689M can promote efficient transposition, it has lost considerable ability to 'inactivate' the TnsCD-bound attTn7 target DNA for transposition. The finding that incubation of the target complex with TnsBwt results in a decrease in target activity, whereas incubation with TnsBV689M does not, supports the view that 'inactivation' of the attTn7 target DNA occurs by a mechanism similar to the mechanism used to establish immunity on a target DNA that contains TnsB binding sites. TnsBV689M interacts poorly with TnsC The TnsC 'redistribution' experiment and the target 'inactivation' experiment described above demonstrate that the V689M mutation alters the ability of TnsB to impose target immunity. How might TnsBV689M fail to 'inactivate' the transposition potential of a TnsC-bound target DNA? One hypothesis is that TnsBV689M interacts poorly with TnsC and thus is incapable of displacing TnsC from a target DNA. Alternatively, TnsBV689M may interact with TnsC but be unable to provoke some downstream event required for displacement of TnsC from the target DNA, for example TnsC-ATP hydrolysis (Stellwagen and Craig, 1997a, 1998). To explore these possibilities, we directly examined TnsB–TnsC interaction by using a glutaraldehyde cross-linking method to assay for the assembly of a Tns-mediated transposon–target DNA nucleoprotein complex that depends upon TnsB–TnsC interaction (Figure 4)(Skelding et al., 2002). Figure 4.TnsBwt and TnsBV689M interact differently with TnsCD-bound attTn7 target DNA. The reactions were similar to Figure 3 except that a cross-linker was added instead of TnsA. The cross-linked products were digested with PflMI which cuts the mTn7donor DNA into two fragments containing either Tn7L or Tn7R. In lanes 1–10 TnsC and TnsD were bound to attTn7 target DNA in the presence of ATP, while in lanes 11–14 AMP–PNP was substituted for ATP. Download figure Download PowerPoint All the reactions produced an abundant amount of complex in which the Tn7 left and right ends were paired, designated (L,R) (Skelding et al., 2002); thus TnsBV689M is normal for Tn7 end binding and pairing (Figure 4, lanes 1–10). However, TnsBwt-bound transposon ends and TnsBV689M-bound transposon ends interact very differently with TnsCD-bound target DNA. Brief incubation of TnsBwt and the mTn7 donor DNA with the TnsCD-bound target DNA produced an abundant amount of nucleoprotein complex containing the Tn7 left and right ends and the target DNA designated (L,R)-Tgt (Figure 4, lane 1). In marked contrast, reactions containing TnsBV689M produced only a small amount of the (L,R)-Tgt complex (Figure 4, lane 6 versus lane 1). Thus TnsBV689M bound to the transposon ends interacts poorly with TnsC-bound target DNA. We also examined the effect of extended TnsB incubation on (L,R)-Tgt complex formation. As the TnsBwt incubation period was increased, the amount of (L,R)-Tgt complex decreased in an ATP-dependent manner (Figure 4, lanes 1 and 5 versus lanes 11 and 12). Taken together, the ATP dependence of the (L,R)-Tgt complex instability and the TnsB-provoked displacement of TnsC from DNA during target immunity suggests that (L,R)-Tgt complex dissociation is a consequence of TnsB-provoked TnsC displacement. The amount of TnsBV689M (L,R)-Tgt complex appears to decrease over time as well, but because such a small amount of complex is formed, it is difficult to compare dissociation of this complex with the TnsBwt (L,R)-Tgt complex (Figure 4, lanes 6–10). This analysis of donor–target nucleoprotein complex formation reveals directly that the ability of TnsBV689M-bound transposon ends to interact with TnsC-bound target DNA is much reduced compared with the interaction of TnsBwt with TnsC. The decreased ability of TnsBV689M to interact with TnsC likely prevents TnsBV689M from provoking displacement of TnsC from DNA, allowing the TnsC-bound target DNA to attract Tn7 insertions. TnsBV689M-bound transposon DNA forms a stable complex with TnsCD-bound target DNA in the presence of TnsA In the experiment described above, TnsBV689M bound to the transposon ends interacted poorly with TnsC-bound target DNA. This TnsB–TnsC interaction is also required for the assembly of a critical transposition intermediate, the TnsABC + D (L,R)-Tgt complex (Skelding et al., 2002). We tested for TnsABV689MC + D (L,R)-Tgt complex formation by setting up a reaction just like our 'standard' in vitro transposition reaction except that we substituted Ca2+ for Mg2+, a change which supports complex assembly but not the cleavage activity of the TnsAB transposase. We found both TnsABwtC + D and TnsABV689MC + D formed abundant (L,R)-Tgt complex (Supplementary figure 1, lanes 1–4, available at The EMBO Journal Online). We also found that TnsABV689MC + D and TnsABwtC + D formed equal amounts of (L,R)-Tgtsc complex in the presence of Mg2+ (Supplementary figure 1, lanes 5–8). Thus TnsA enables TnsBV689M-bound transposon DNA to interact effectively with the TnsC-bound target DNA, consistent with the ability of TnsBV689M to promote high-frequency TnsABC + D transposition. TnsB binding sites adjacent to attTn7 impose target immunity on attTn7 We have argued above and elsewhere (Stellwagen and Craig, 1997a) that the greater the local concentration of TnsB around a TnsC-bound target DNA, the greater the possibility that TnsB will impose immunity on that target DNA. One potential target DNA which contains TnsB binding sites positioned very close to DNA bound by TnsC is the simple insertion product of the TnsABC + D transposition reaction (McKown et al., 1988; Kuduvalli et al., 2001). Thus we created a DNA fragment containing Tn7R immediately adjacent to attTn7 (attTn7::Tn7R), as occurs following TnsABC + D transposition into attTn7 (Figure 5A). We evaluated the binding of the TnsC and TnsD proteins to this fragment with a band-shift assay (Figure 5B). We found that just as much TnsCD complex formed on the attTn7::Tn7R DNA fragment as on the attTn7 DNA fragment (compare lanes 1 in Figure 5C and D); thus the TnsB binding sites do not affect assembly of the TnsCD complex on attTn7::Tn7R. It should be noted that TnsD–attTn7 complex cannot be detected using this assay because TnsD alone cannot remain bound to attTn7 in the presence of 15 mM Mg2+. Figure 5.A direct assay for TnsB provoked dissociation of a TnsCD–attTn7::Tn7R complex. (A) The hatched bar beneath the attTn7 target DNA represents the hydroxyl radical footprint of the TnsD–attTn7 complex, while the solid bar represents the hydroxyl radical footprint of the TnsCD–attTn7 complex which extends beyond the position of Tn7 insertion centered around bp 0 (Kuduvalli et al., 2001). Insertion of Tn7 into attTn7 places the four contiguous TnsB binding sites of Tn7R adjacent to the region of DNA bound by the TnsCD complex. As the concentration of TnsB is increased, the sites in Tn7R are bound in the following order: χ; χ + ψ and χ + ω; χ + ψ + ω; χ + ψ + ω + Φ (Arciszewska and Craig, 1991). The outermost 8 bp of Tn7R is conserved with the outermost 8 bp of Tn7L; this sequence is referred to as the 'terminal inverted repeat' (TIR). (B) Reaction scheme. (C) Reactions in which different TnsBs were incubated with TnsCD–attTn7::Tn7R. The various TnsB–attTn7::Tn7R complexes are indicated using the χ, ψ and ω symbols. (D) Reactions in which different TnsBs were incubated with TnsCD–attTn7. Download figure Download PowerPoint Incubation of the assembled TnsCD–attTn7::Tn7R complex with a low concentration of TnsBwt (12 nM), dramatically decreased the amount of TnsCD–attTn7 complex and produced several new bands (Figure 5C, lane 1 versus lane 2). These new bands are the result of TnsB binding to different combinations of its binding sites in Tn7R (Figure 5A) (Arciszewska and Craig, 1991). In contrast, a TnsCD complex bound to an attTn7 fragment lacking TnsB binding sites was not affected by incubation with TnsB
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