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• W2007332550 abstract "Mu DNA transposition from a negatively supercoiled DNA substrate requires interaction of an enhancer element with the left (attL) and right (attR) ends of Mu. The orientation of the L and R ends with respect to each other (inverted) and with respect to the enhancer is normally inviolate. We show that when the enhancer is provided in trans as a linear fragment, the head to head orientation of the L/R ends is still required. Each functional half of the linear enhancer maintains the same “cross-wise” interaction with the subsites L1 and R1, when present in cis or intrans. In reactions catalyzed by an enhancer-independent variant of the Mu transposase, the need for negative supercoiling of the substrate and the inverted orientation of L and R ends is not relaxed. These results show that the orientation specificity of the enhancer is not determined by its topological linkage to the Mu ends. There is a functional asymmetry inherent to the enhancer. Furthermore, the enhancer does not directly impose topological constraints on the transposition reaction or specify the reactive orientation of the Mu ends. Mu DNA transposition from a negatively supercoiled DNA substrate requires interaction of an enhancer element with the left (attL) and right (attR) ends of Mu. The orientation of the L and R ends with respect to each other (inverted) and with respect to the enhancer is normally inviolate. We show that when the enhancer is provided in trans as a linear fragment, the head to head orientation of the L/R ends is still required. Each functional half of the linear enhancer maintains the same “cross-wise” interaction with the subsites L1 and R1, when present in cis or intrans. In reactions catalyzed by an enhancer-independent variant of the Mu transposase, the need for negative supercoiling of the substrate and the inverted orientation of L and R ends is not relaxed. These results show that the orientation specificity of the enhancer is not determined by its topological linkage to the Mu ends. There is a functional asymmetry inherent to the enhancer. Furthermore, the enhancer does not directly impose topological constraints on the transposition reaction or specify the reactive orientation of the Mu ends. base pair nucleotide polymerase chain reaction open circle Site-specific recombination systems in both prokaryotes and eukaryotes require interaction of specific DNA sequences. Although some systems can carry out recombination with the interacting recombination sites in any orientation (e.g. phage λ integration/excision, phage P1 Cre/lox recombination, yeast 2-μm plasmid Flp/FRT recombination), other systems have a strict requirement for just one orientation (e.g. Mu transposition, Tn3/γδ resolvase reactions, Hin/Gin inversion reactions) (see Ref. 1Gellert M. Nash H. Nature. 1987; 325: 401-404Crossref PubMed Scopus (83) Google Scholar). A hallmark of the latter systems is the employment of enhancers (or accessory DNA sites) and the requirement for negative DNA supercoiling. Either one or both of these elements (enhancers and DNA supercoiling) could contribute to the orientation specificity of the recombination sites in these systems. For the Mu transposition system, the role of supercoiling was explored by Craigie and Mizuuchi (2Craigie R. Mizuuchi K. Cell. 1986; 45: 793-800Abstract Full Text PDF PubMed Scopus (98) Google Scholar) prior to the discovery of the enhancer. Transposition of the L and R ends of Mu present in two different orientations on catenated or knotted supercoiled DNA substrates was monitored. The pattern of reactivity of Mu ends on these novel substrates led the authors to suggest that the normally inverted L and R ends might juxtapose most easily in an “intertwined” parallel configuration on negatively supercoiled DNA; for directly oriented sites an energy barrier must be overcome to bring them together in this parallel configuration. Thus, DNA topology was responsible for sensing the relative orientation of the Mu ends. Experiments with Tn3 resolvase were consistent with the Mu results in suggesting the existence of a “topological filter” (defined as a combined effect of constraints derived from both the circularity of the DNA substrate and the requirement for a highly specific interwrapping of the recombining sites; see Ref. 1Gellert M. Nash H. Nature. 1987; 325: 401-404Crossref PubMed Scopus (83) Google Scholar) that limits productive interaction to a particular orientation of sites (3Boocock M.R. Brown J.L. Sherratt D.J. Biochem. Soc. Trans. 1986; 14: 214-216Crossref PubMed Scopus (38) Google Scholar). Recent experiments with Tn3 resolvase have implicated interwrapping at the enhancer in determining the topological selectivity of the reaction (4Arnold P.H. Blake D.G. Grindley N.D.F. Boocock M.R. Stark W.M. EMBO J. 1999; 18: 1407-1414Crossref PubMed Scopus (81) Google Scholar); enhancer-independent resolvase mutants simultaneously lost their normal specificity for directly oriented sites and for supercoiled substrates. Similar results have been reported with the Gin invertase family (5Klippel A. Cloppenborg K. Kahmann R. EMBO J. 1988; 7: 3983-3989Crossref PubMed Scopus (93) Google Scholar, 6Haffter P. Bickle T.A. EMBO J. 1988; 7: 3991-3996Crossref PubMed Scopus (51) Google Scholar). Since the Mu experiments implicating DNA topology in determining the orientation specificity of the ends (2Craigie R. Mizuuchi K. Cell. 1986; 45: 793-800Abstract Full Text PDF PubMed Scopus (98) Google Scholar) were done before the discovery of the enhancer, one objective of the present study was to dissect the contribution of the enhancer, if any, to the topological filter. These studies were facilitated by the existence of an enhancer-independent variant of the Mu transposase (7Yang J.-Y. Jayaram M. Harshey R.M. Genes Dev. 1995; 9: 2545-2555Crossref PubMed Scopus (22) Google Scholar). A second objective of this study was to understand the basis of the orientation specificity of the transposition enhancer itself (8Leung P.C. Teplow D.B. Harshey R.M. Nature. 1989; 338: 656-658Crossref PubMed Scopus (113) Google Scholar, 9Mizuuchi M. Mizuuchi K. Cell. 1989; 58: 399-408Abstract Full Text PDF PubMed Scopus (121) Google Scholar, 10Surette M.G. Lavoie B.D. Chaconas G. EMBO J. 1989; 8: 3483-3489Crossref PubMed Scopus (59) Google Scholar). Since the Mu ends are inverted with respect to each other, the transposase synapse (in which the enhancer interacts with the L and R ends to form an LER complex; see Ref. 11Watson M.A. Chaconas G. Cell. 1996; 85: 435-445Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar; see Fig. 1 A) is expected to be most closely related to the synapse made by the Hin/Gin invertases, whose enhancers do not display orientation specificity (13Heichman K.A. Johnson R.C. Science. 1990; 249: 511-517Crossref PubMed Scopus (117) Google Scholar,14Kanaar R. Klippel A. Shekhtman E. Dungan J.M. Kahmann R. Cozzarelli N.R. Cell. 1990; 62: 353-366Abstract Full Text PDF PubMed Scopus (107) Google Scholar). Does the orientation specificity of the Mu enhancer arise as a result of an inherent asymmetry in its sequence and/or from being topologically linked to the L and R ends? If the enhancer were free from the constraints of DNA topology, might it acquire the freedom to be active in either orientation? Two developments facilitated this investigation. The first was the demonstration that the enhancer is functional “in trans” on a linear DNA fragment (15Surette M.G. Chaconas G. Cell. 1992; 68: 1101-1108Abstract Full Text PDF PubMed Scopus (60) Google Scholar). The second was the availability of a convenient assay for testing the orientation specificity of the enhancer by exploiting similarities and differences between the transposition systems of Mu and the Mu-related phage D108; these two systems share the same att site specificity but have different enhancer specificities (16Jiang H. Yang J.-Y. Harshey R.M. EMBO J. 1999; 18: 3845-3855Crossref PubMed Scopus (21) Google Scholar). The latter studies found a highly specific “cross-wise” interaction between the left O1 site of the enhancer and the right R1 att site and between the right O2 site of the enhancer and the left L1 att site (Fig. 1 B). We have used these two assays to explore the orientation specificity of both the enhancer and the L/R ends in this study. DNA supercoiling plays a critical role in recombination systems that employ accessory enhancer sites to organize the recombination synapse (e.g. Mu transposition, Hin/Gin inversion, Tn3/γδ resolution; see Refs. 12Chaconas G. Lavoie B.D. Watson M.A. Curr. Biol. 1996; 7: 817-820Abstract Full Text Full Text PDF Scopus (71) Google Scholar, 17Stark W.M. Boocock M.R. Sherratt D.J. Trends Genet. 1992; 8: 432-439Crossref PubMed Google Scholar, and 18Grindley N.D.F. Nucleic Acids Mol. Biol. 1994; 8: 236-267Crossref Google Scholar). Varied roles for supercoiling have been deduced in these systems (see “Discussion”). In the Mu transposition system supercoiling is not essential at the enhancer, which can function in trans on a linear DNA fragment when the L/R ends are present on supercoiled DNA (15Surette M.G. Chaconas G. Cell. 1992; 68: 1101-1108Abstract Full Text PDF PubMed Scopus (60) Google Scholar). These results do not rule out a role for supercoiling at the enhancer when it is present incis. It is clear that a cis location increases the effective concentration of the enhancer, since a 50-fold molar excess is required for function when the enhancer is supplied intrans in the presence of Escherichia coli protein IHF (15Surette M.G. Chaconas G. Cell. 1992; 68: 1101-1108Abstract Full Text PDF PubMed Scopus (60) Google Scholar). A requirement for the DNA bending protein IHF (which binds between the O1 and O2 sites of the enhancer; see Ref. 19Krause H.M. Higgins N.P. J. Biol. Chem. 1986; 261: 3744-3752Abstract Full Text PDF PubMed Google Scholar) on linear enhancer DNA fragments, or on circular DNA with low superhelical densities (10Surette M.G. Lavoie B.D. Chaconas G. EMBO J. 1989; 8: 3483-3489Crossref PubMed Scopus (59) Google Scholar), suggests that supercoiling may also favor DNA bending at the enhancer. In addition, supercoiling has been shown to be important for binding HU protein at the L end (20Kobryn K. Lavoie B. Chaconas G. J. Mol. Biol. 1999; 289: 777-784Crossref PubMed Scopus (50) Google Scholar) as well as for a critical post-synaptic event (21Wang Z. Harshey R.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 699-703Crossref PubMed Scopus (33) Google Scholar, 22Wang Z. Namgoong S.-Y. Zhang X. Harshey R.M. J. Biol. Chem. 1996; 271: 9619-9626Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The enhancer interacts with the L and R ends early in the Mu transposition reaction to form an unstable nucleoprotein complex LER (see Fig. 1 A; not shown are three sets of individual transposase-binding sites L1–L3, O1–O3, and R1–R3 within L, enhancer, and R, respectively). Interactions within LER lead to formation of a stable type 0 complex in which the Mu transposase (MuA protein) assumes its active tetrameric form, catalyzing the cleavage (type I complex) and joining (type II complex) reactions of transposition (12Chaconas G. Lavoie B.D. Watson M.A. Curr. Biol. 1996; 7: 817-820Abstract Full Text Full Text PDF Scopus (71) Google Scholar). Only two subunits within the tetramer, those located on L1 and R1, have thus far been implicated in catalysis on supercoiled substrates (Fig. 1 B; see Ref. 23Namgoong S.-Y. Harshey R.M. EMBO J. 1998; 17: 3775-3785Crossref PubMed Scopus (61) Google Scholar). Similar results have been reported using R1–R2 oligonucleotide substrates under dimethyl sulfoxide (Me2SO) reaction conditions (24Williams T.L. Jackson E.L. Carritte A. Baker T.A. Genes Dev. 1999; 13: 2725-2737Crossref PubMed Scopus (64) Google Scholar). The catalytic “DDE” residues of these active subunits work intrans (25Aldaz H. Schuster E. Baker T.A. Cell. 1996; 85: 257-269Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 26Savilahti H. Mizuuchi K. Cell. 1996; 85: 271-280Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), i.e. DDE+ subunit at L1 is responsible for cleavage and strand transfer of the opposite R end whereas the DDE+ subunit at R1 is responsible for these reactions at the L end (23Namgoong S.-Y. Harshey R.M. EMBO J. 1998; 17: 3775-3785Crossref PubMed Scopus (61) Google Scholar). The specific function of the other two subunits is not known (although they also interact with the enhancer). We report that Mu ends maintain their orientation specificity as well as their requirement for a supercoiled substrate in reactions with a topologically unlinked enhancer as well as in those not dependent on the enhancer. We compare the role of the enhancer in imposing topological selectivity (defined as the specificity for a particular orientation of topologically linked reactive sites) in other recombination systems. Plasmids pJMM and pJDD have been described (16Jiang H. Yang J.-Y. Harshey R.M. EMBO J. 1999; 18: 3845-3855Crossref PubMed Scopus (21) Google Scholar). pJHN (enhancer−) was created by deleting aSal I-Eco RI fragment encoding the enhancer from pJMM. pJH25 (enhancer−, inverted attL) was created by inverting a 233-bp1 Xba I-Bgl II fragment encompassing attL in pJHN. pJH26 (enhancer+, inverted attL) was constructed in a manner similar to pJH25, except that pJMM was used as the substrate. pJH27 (O2−) was constructed by first incorporating anEco RI site at nt 964 (between IHF and O2 sites) in a PCR primer used to amplify the O1-IHF region on pJMM. ABgl II-Eco RI digest of the PCR product was exchanged into similar sites on pJMM, resulting in deletion of O2. pJH28 (O1−) was constructed in a manner similar to pJH27, except by introducing a Sal I site at nt 938 (between O1 and IHF sites) into the PCR primer used for amplifying the IHF-O2 region, followed by exchange of the Sal I-Eco RI-digested PCR product into these sites on pJMM. pJH29 (IHF−O2−) was constructed similarly, by introducing theEco RI site at nt 943 between O1 and IHF sites. These were prepared by PCR amplification of template DNA with appropriate primers. They are shown in Table I. O1L (long) was designed to be the same size as MM and contains nonenhancer DNA in place of O2. The numbers in the last column are the starting and ending nucleotide coordinates of the enhancer fragments encompassing the Mu and/or D108 genome.Table ILinear enhancer fragments derived by PCR amplificationEnhancerDescriptionTemplateSizeNuc on Mu/D108 genomebpMMO1-O2 MupJMM171872–1042DDO1-O2 D108pJDD140831–970MDO1 Mu-O2 D108pJMD140872, 970DMO1 D108-O2 MupJDM167831, 1042O1LO1 MupJH27171872–1042O1O1 MupJMM103872–974O2O2 MupJMM89954–1042 Open table in a new tab Purification of MuA, MuA(E392A), MuAΔ84, D108A, D108(E392A), and HU proteins has been described (7Yang J.-Y. Jayaram M. Harshey R.M. Genes Dev. 1995; 9: 2545-2555Crossref PubMed Scopus (22) Google Scholar). IHF was a generous gift from Steve Goodman, University of Southern California. Strand cleavage (type I complex) assays were done in 20-μl reaction volumes in 20 mm HEPES-KOH (pH 7.6), 140 mm NaCl, 10 mm MgCl2. Final Me2SO concentrations were 15% when included. In reactions employing linear enhancer DNA fragments the molar ratio of the donor plasmid DNA to enhancer DNA was 1:50 and that of enhancer DNA to IHF protein was 1:1.6 (15Surette M.G. Chaconas G. Cell. 1992; 68: 1101-1108Abstract Full Text PDF PubMed Scopus (60) Google Scholar). Besides the amounts of transposase proteins (0.1–0.4 μg), the reaction contained 0.8 μg of donor mini-Mu DNA and 0.2 μg ofE. coli HU protein. Reaction mixtures were incubated at 30 °C for 20 min and analyzed by agarose gel electrophoresis. DNA bands were excised from ethidium bromide-stained agarose gels as described (16Jiang H. Yang J.-Y. Harshey R.M. EMBO J. 1999; 18: 3845-3855Crossref PubMed Scopus (21) Google Scholar) and digested with either Bam HI plusXba I or with Bam HI plus Aat II. The DNA fragments were labeled with [α-32P]cordycepin phosphate using terminal nucleotidyltransferase, electrophoresed on 6% denaturing polyacrylamide gels, and detected by autoradiography as described (16Jiang H. Yang J.-Y. Harshey R.M. EMBO J. 1999; 18: 3845-3855Crossref PubMed Scopus (21) Google Scholar). The “cleavage-in-trans” rule (i.e. the action of a catalytically active MuA monomer away from the end to which it is bound) has provided the rationale for mapping the enhancer-att interactions, whose functional relationships were deduced by assaying how enhancers of Mu-D108 hybrid specificity respond to mixtures of MuA and D108A proteins and their catalytically inactive DDE−variants (see Ref. 16Jiang H. Yang J.-Y. Harshey R.M. EMBO J. 1999; 18: 3845-3855Crossref PubMed Scopus (21) Google Scholar; these two transposases bind to each other's att sites but are highly specific for their cognate enhancers). We have now employed the same system for testing the orientation specificity of an enhancer when supplied on a linear fragment (in trans) to attL and attR ends present on supercoiled DNA. These studies will be presented first, followed by those employing a variant transposase, which functions in the absence of the enhancer. Both Mu and D108 enhancers are composed of three transposase binding regions (O1-O3; see Ref. 16Jiang H. Yang J.-Y. Harshey R.M. EMBO J. 1999; 18: 3845-3855Crossref PubMed Scopus (21) Google Scholar). The polarity of these sites with respect to each other is not known (see “Discussion”). Although the O3 site participates in optimal enhancer function, the O1-O2 sites are sufficient for activity (10Surette M.G. Lavoie B.D. Chaconas G. EMBO J. 1989; 8: 3483-3489Crossref PubMed Scopus (59) Google Scholar,16Jiang H. Yang J.-Y. Harshey R.M. EMBO J. 1999; 18: 3845-3855Crossref PubMed Scopus (21) Google Scholar). We have therefore used O1-O2 as the enhancer in the present study. Linear DNA fragments encoding wild-type enhancers from Mu and D108 (MM and DD, respectively) as well as hybrid enhancers (MD and DM) were generated by PCR amplification as described under “Materials and Methods.” The first and second letters in the enhancer name denote the source of O1 and O2, respectively: M for Mu and D for D108. The logic for the design of the hybrid enhancer substrates has been described (16Jiang H. Yang J.-Y. Harshey R.M. EMBO J. 1999; 18: 3845-3855Crossref PubMed Scopus (21) Google Scholar). The E. coli IHF protein is required for function of linear Mu enhancer fragments (15Surette M.G. Chaconas G. Cell. 1992; 68: 1101-1108Abstract Full Text PDF PubMed Scopus (60) Google Scholar). When the enhancer and att sites are all present on the same plasmid, the O1 site specifies the occupancy of the transposase monomer at R1, and the O2 site promotes placement of its cognate transposase at L1 (Fig. 1 B). The O1-R1 rule is rigid; the O2-L1 rule is less so (16Jiang H. Yang J.-Y. Harshey R.M. EMBO J. 1999; 18: 3845-3855Crossref PubMed Scopus (21) Google Scholar). These conclusions were reached from strand cleavage assays using negatively supercoiled plasmid substrates containing the hybrid enhancers DM and MD incis (16Jiang H. Yang J.-Y. Harshey R.M. EMBO J. 1999; 18: 3845-3855Crossref PubMed Scopus (21) Google Scholar). Reactions of an enhancer-less plasmid pJHN with the DM enhancer provided in trans are shown in Fig.2. Except where indicated, reactions contained the IHF protein. Wild-type MuA did not yield the cleaved type I complex (lane b), and the catalytically inactive variant MuA(E392A) did not produce the uncleaved type 0 complex (lane d). We know that MuA and its variants are not active on a DM substrate in cis unless D108A is present (16Jiang H. Yang J.-Y. Harshey R.M. EMBO J. 1999; 18: 3845-3855Crossref PubMed Scopus (21) Google Scholar). Wild-type D108A was only weakly active (lane c; the type I band is barely visible). Interestingly, this activity was elevated when IHF was omitted from the reaction (lane a*). D108A(E392A) gave type 0 complex (lane h) and, as expected, no type I product. When wild-type D108A was paired with MuA(E392A), the type I product and, more prominently, the nicked circular product were formed (lanes e–g). From previous work (16Jiang H. Yang J.-Y. Harshey R.M. EMBO J. 1999; 18: 3845-3855Crossref PubMed Scopus (21) Google Scholar), we know that single cleavage at the left end of Mu results in an unstable form of the type I complex. This product migrates as the open circle (OC) during electrophoresis due to relaxation of a substrate domain that is normally held supercoiled in the stable complex. The diffused migration of the OC band suggests dissociation of MuA from the DNA, perhaps during electrophoresis. Further characterization of the cleavage product is described below. In reactions that paired MuA with D108A(E392A), stable type I complex was formed (lanes i–k). The strand cleavage position was mapped in this product as well. Unlike left end cleavage, single cleavage at the right end does not destabilize type I (16Jiang H. Yang J.-Y. Harshey R.M. EMBO J. 1999; 18: 3845-3855Crossref PubMed Scopus (21) Google Scholar). Note that these reactions also yielded the type 0 complex, its formation being favored at higher molar ratios of D108A(E392A). The reason for the higher activity of wild-type D108A in the absence of IHF, rather than its presence, is not known (Fig. 2, comparelanes a and c). All the other reactions in Fig.2, including those containing D108A as one of the binary protein partners, were strictly dependent on IHF (data not shown). In our previous work (16Jiang H. Yang J.-Y. Harshey R.M. EMBO J. 1999; 18: 3845-3855Crossref PubMed Scopus (21) Google Scholar), we observed that D108A acted efficiently on a negatively supercoiled substrate containing the DM enhancer incis under reaction conditions that did not require IHF. The hybrid enhancer may not have retained all the structural features of the native enhancer, causing the odd behavior of D108A noted here. For example, an intrinsic bend or flexibility within the DM enhancer could be unfavorably modulated by IHF. Subtle differences between wild-type and mutant proteins in their binding affinities for the enhancer and/or att sites and in their intersubunit cooperativities could also come into play. However, these factors do not diminish the significance of the clear-cut selectivity in strand cleavage observed with specific protein pairs (see below). The scheme for mapping the cleavage positions in the type I and OC products formed in Fig. 2 is diagrammed in Fig.3 A, and the results are shown in Fig. 3 B. The DNA samples extracted from the excised gel bands were digested with Bam HI and Xba I in one case and with Bam HI and Aat II in the other. The 3′-hydroxyl ends generated from the digestion as well as those produced by cleavage of Mu ends were radioactively labeled with 32P and fractionated by electrophoresis in denaturing polyacrylamide gels. The diagnostic bands for left end cleavage are LC1 and LC2; those for right end cleavage are RC1 and RC2 (see Ref. 16Jiang H. Yang J.-Y. Harshey R.M. EMBO J. 1999; 18: 3845-3855Crossref PubMed Scopus (21) Google Scholar for more details). The results of the cleavage analysis from reactions in Fig. 2,lanes f (containing equimolar D108A and MuA(E392A)) andlane j (containing equimolar MuA and D108A(E392A)), are displayed in Fig. 3 B. The OC product from the f reaction was cleaved almost exclusively at the left end (LC1 and LC2 in lane 2 and undetectable RC1 and RC2 in lane 6). The type I product from the f reaction also showed predominant cleavage at the left end (LC1 and LC2 in lane 3 and faint bands of RC1 and RC2 in lane 7). By contrast, the type I product from the j reaction shown in Fig. 2 (MuA/D108A(E392A)) showed nearly exclusive cleavage at the right end (RC1 with undetectable LC1 and LC2 inlane 4; RC1 and RC2 in lane 8). The conclusions from the data in Fig. 2 and Fig. 3 B are summarized in Fig. 3 C. The placement of the wild-type or mutant MuA or D108A monomers is dictated by the trans rule for DDE donation during MuA active site assembly; left end and right end cleavages require the DDE-containing monomer to be positioned at the R1 and L1 sites, respectively (23Namgoong S.-Y. Harshey R.M. EMBO J. 1998; 17: 3775-3785Crossref PubMed Scopus (61) Google Scholar). The DM enhancer strictly specifies the occupancy of D108A at R1, either wild-type (Fig.3 C, left) or the DDE mutant (Fig. 3 C, right). Left end cleavage is promoted in the former case, whereas it is blocked in the latter. The DM enhancer also promotes the occupancy of MuA (over D108A) at the L1 end, but the specificity is less rigorous. L1 is primarily occupied by MuA(E392A) when paired with D108A (Fig.3 B; the preponderance of cleavage at the left over the right end in lanes 2 and 3 and 6 and7 corresponding to reaction f in Fig. 2) and incorporates wild-type MuA when paired with D108A(E392A) (right end cleavage inlanes 4 and 8 in Fig. 3 B corresponding to reaction j in Fig. 2). However, the presence of D108A(E392A) at L1 in a fraction of the molecules is denoted by the formation of the type 0 complex (for example, reaction j in Fig. 2). Similarly, D108A is not totally excluded from L1, as indicated by the faint cleavage observed at the right end (lane 7 in Fig. 3 B corresponding to reaction f in Fig. 2). In summary, our results with the DM enhancer in trans are essentially identical to those obtained by Jiang and co-workers (16Jiang H. Yang J.-Y. Harshey R.M. EMBO J. 1999; 18: 3845-3855Crossref PubMed Scopus (21) Google Scholar) for the same enhancer in cis. Together, these findings ascertain that the distribution of the transposase subunits at the Mu ends is determined by their cross-wise specificities for the enhancer elements. The left O1 region of the enhancer specifies the right R1 site; and to a lesser degree, the right O2 region specifies L1. This rule for the distribution of transposase molecules is the same regardless of whether the enhancer is present in cis or intrans. We have carried out assays similar to those in Fig. 2 with the same substrate plasmid pJHN and the MD enhancer supplied intrans. The efficiency of the MD reactions was very poor. The action of the native enhancers MM or DD in trans on the plasmid pJHN was assayed in presence of IHF, and the points of strand cleavage were mapped (Fig.4). A control set of reactions was done with plasmid pJMM containing MM in cis (Fig. 4 A) and plasmid pJDD containing DD in cis (data not shown). Thecis reactions used standard conditions for supercoiled substrates that did not include IHF. Wild-type MuA was active and D108A was inactive in the type I reaction with the MM enhancer in cis (Fig. 4 A, lanes a andb) or in trans (lanes d ande). An equimolar mixture of D108A and MuA(E392A) yielded both type I and type 0 products (lanes c and f). The type 0 complex would be expected to contain MuA(E392A) monomers at R1 and L1. The results were similar for the DD enhancer intrans (lanes g–i). Type I reaction was obtained with D108A (lane g) but not MuA (lane h), and both type I and type 0 were obtained with a mixture of MuA and D108A(E392A) (lane i). The reaction profile was the same for the plasmid containing DD in cis (not shown). The type I complex from the wild-type reactions (Fig. 4 B, lanes a, d, and g) contained both left and right end cleavages as expected (LC1, LC2, and RC1 in lanes 2, 5, and8; RC1, RC2, and LC1 in lanes 11, 14, and17). On the other hand, type I from the mixed reactions (lanes c, f, and i) harbored only right end cleavages (RC1 alone in lanes 3, 6, and 9; RC1 and RC2 alone in lanes 12, 15, and 18). These results are in accordance with the strict O1-R1 rule and the less stringent O2-L1 rule. With the MM enhancer, MuA(E392A) would occupy R1, thereby negating left end cleavage. When MuA(E392A) occupies L1 as well, neither end can be cleaved, yielding the type 0 complex. When D108A occupies L1, right end cleavage can occur. These arguments apply to the DD enhancer as well. Table II summarizes the activities of various cis and trans arrangements of the Mu enhancer (see “Materials and Methods: for details). Some of these experiments have been previously reported (10Surette M.G. Lavoie B.D. Chaconas G. EMBO J. 1989; 8: 3483-3489Crossref PubMed Scopus (59) Google Scholar, 15Surette M.G. Chaconas G. Cell. 1992; 68: 1101-1108Abstract Full Text PDF PubMed Scopus (60) Google Scholar), but we present them here for the sake of completeness. The negatively supercoiled pJMM plasmid (harboring MM in cis) was active in the type I reaction with or without added IHF and provides a reference for the other reactions. The enhancer-less pJHN could be complemented intrans by MM in presence of IHF. No complementation was observed with O1 alone, O2 alone, O1 plus O2 supplied as separate DNA fragments and by O1L (containing O1 + IHF-binding site + nonspecific DNA) even at a 50-fold molar excess over the att sites (see also Ref.15Surette M.G. Chaconas G. Cell. 1992; 68: 1101-1108Abstract Full Text PDF PubMed Scopus (60) Google Scholar). An inactive derivative of pJMM lacking O1 (pJH28) could be partially rescued by O1-O2 (MM) in trans but not by O1 alone or by O1L. In a 30-min period, a plasmid deleted for O2 alone (pJH27) retained ∼60% of wild-type pJMM activity, which saturates more rapidly (see also Ref. 10Surette M.G. Lavoie B.D. Chaconas G. EMBO J. 1989; 8: 3483-3489Crossref PubMed Scopus (59) Google Scholar). By comparison, the corresponding relative activity at 5′ was only 20%. The pJHN reactions with the MM enhancer in trans also displayed similar slow kinetics (data not shown; see Ref. 15Surette M.G. Chaconas G. Cell. 1992; 68: 1101-1108Abstract Full Text PDF PubMed Scopus (60) Google Scholar). Addition of MM in trans in the presence or absence of IHF did not improve the" @default.
• W2007332550 created "2016-06-24" @default.
• W2007332550 creator A5043620679 @default.
• W2007332550 creator A5049473122 @default.
• W2007332550 date "2001-02-01" @default.
• W2007332550 modified "2023-10-15" @default.
• W2007332550 title "The Mu Enhancer Is Functionally Asymmetric Both incis and in trans" @default.
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