The basis of asymmetry in IS2 transposition.

In the first step of IS2 transposition, the formation of an IS2 minicircle, the roles of the two IS ends differ. Terminal cleavage initiates exclusively at the right inverted repeat (IRR) - the donor end - whereas IRL is always the target. At the resulting minicircle junction, the two abutted ends are separated by a spacer of 1 or 2 basepairs. In this study, we have identified the determinants of donor and target function. The inability of IRL to act as a donor results largely from two sequence differences between IRL and IRR - an extra basepair between the conserved transposase binding sequences and the end of the element, and a change of the terminal dinucleotide from CA-3' to TA-3'. These two changes also impose a characteristic size on the minicircle junction spacer. The only sequences required for the efficient target function of IRL appear to be contained within the segment from position 11-42. Although IRR can function as a target, its shorter length and additional contacts with transposase (positions 1-7) result in minicircles with longer, and inappropriate, spacers. We propose a model for the synaptic complex in which the terminus of IRL makes different contacts with the transposase for the initial and final strand transfer steps. The sequence differences between IRR and IRL, and the behavioural characteristics of IRL that result from them, have probably been selected because they optimize expression of transposase from the minicircle junction promoter, Pjunc.

A model for the gamma delta resolvase synaptic complex.

The serine recombinase gamma delta resolvase performs site-specific recombination in an elaborate synaptic complex containing 12 resolvase subunits and two 114-base pair res sites. Here we present an alternative structural model for the synaptic complex. Resolvase subunits in the complex contact their neighbors in equivalent ways, using three principal interactions, one of which is a newly proposed synaptic interaction. Evidence in support of this interaction is provided by mutations at the interface that either enable resolvase to synapse two copies of site I or inhibit synapsis of complete res sites. In our model, the two crossover sites are far apart, separated by the resolvase catalytic domains bound to them. Thus, recombination would require a substantial rearrangement of resolvase subunits or domains.

Contacts between the 5' nuclease of DNA polymerase I and its DNA substrate.

The 5' nuclease of DNA polymerase I (Pol I) of Escherichia coli is a member of an important class of prokaryotic and eukaryotic nucleases, involved in DNA replication and repair, with specificity for the junction between single-stranded and duplex DNA. We have investigated the interaction of the 5' nuclease domain with DNA substrates from the standpoint of both the protein and the DNA. Phosphate ethylation interference showed that the nuclease binds to the nucleotides immediately surrounding the cleavage site and also contacts the complementary strand one-half turn away, indicating that contacts are made to one face only of the duplex portion of the DNA substrate. Phosphodiester contacts were investigated further using DNA substrates carrying unique methylphosphonate substitutions, together with mutations in the 5' nuclease. These experiments suggested that two highly conserved basic residues, Lys(78) and Arg(81), are close to the phosphodiester immediately 5' to the cleavage site, while a third highly conserved residue, Arg(20), may interact with the phosphodiester 3' to the cleavage site. Our results provide strong support for a DNA binding model proposed for the related exonuclease from bacteriophage T5, in which the conserved basic residues mentioned above define the two ends of a helical arch that forms part of the single-stranded DNA-binding region. The nine highly conserved carboxylates in the active site region appear to play a relatively minor role in substrate binding, although they are crucial for catalysis. In addition to binding the DNA backbone around the cleavage point, the 5' nuclease also has a binding site for one or two frayed bases at the 3' end of an upstream primer strand. In agreement with work in related systems, 5' nuclease cleavage is blocked by duplex DNA in the 5' tail, but the enzyme is quite tolerant of abasic DNA or polarity reversal within the 5' tail.

In vitro transposition system for efficient generation of random mutants of Campylobacter jejuni.

Campylobacter jejuni is the most common cause of food-borne illnesses in the United States. Despite the fact that the entire nucleotide sequence of its genome has recently become available, its mechanisms of pathogenicity are poorly understood. This is in part due to the lack of an efficient mutagenesis system. Here we describe an in vitro transposon mutagenesis system based on the Staphylococcus aureus transposable element Tn552 that allows the efficient generation of insertion mutants of C. jejuni. Insertions occur randomly and throughout the entire bacterial genome. We have tested this system in the isolation of nonmotile mutants of C. jejuni. Demonstrating the utility of the system, six nonmotile mutants from a total of nine exhibited insertions in genes known to be associated with motility. An additional mutant had an inactivating insertion in sigma 54, implicating this transcription factor in flagellum regulation. The availability of this efficient system will greatly facilitate the study of the mechanisms of pathogenesis of this important pathogen.

Coordination between the polymerase and 5'-nuclease components of DNA polymerase I of Escherichia coli.

The polymerase and 5'-nuclease components of DNA polymerase I must collaborate in vivo so as to generate ligatable structures. Footprinting shows that the polymerase and 5'-nuclease cannot bind simultaneously to a DNA substrate and appear to compete with one another, suggesting that the two active sites are physically separate and operate independently. The desired biological end point, a ligatable nick, results from the substrate specificities of the polymerase and 5'-nuclease. The preferred substrate of the 5'-nuclease is a "double-flap" structure having a frayed base at the primer terminus overlapping the displaced strand that is to be cleaved by the 5'-nuclease. Cleavage of this structure occurs almost exclusively between the first two paired bases of the downstream strand, yielding a ligatable nick. In whole DNA polymerase I, the polymerase and 5'-nuclease activities are coupled such that the majority of molecules cleaved by the 5'-nuclease have also undergone polymerase-catalyzed addition to the primer terminus. This implies that the 5'-nuclease can capture a DNA molecule from the polymerase site more efficiently than from the bulk solution.

Identification of genes encoding exported Mycobacterium tuberculosis proteins using a Tn552'phoA in vitro transposition system.

Secreted and cell envelope-associated proteins are important to both Mycobacterium tuberculosis pathogenesis and the generation of protective immunity to M. tuberculosis. We used an in vitro Tn552'phoA transposition system to identify exported proteins of M. tuberculosis. The system is simple and efficient, and the transposon inserts randomly into target DNA. M. tuberculosis genomic libraries were targeted with Tn552'phoA transposons, and these libraries were screened in M. smegmatis for active PhoA translational fusions. Thirty-two different M. tuberculosis open reading frames were identified; eight contain standard signal peptides, six contain lipoprotein signal peptides, and seventeen contain one or more transmembrane domains. Four of these proteins had not yet been assigned as exported proteins in the M. tuberculosis databases. This collection of exported proteins includes factors that are known to participate in the immune response of M. tuberculosis and proteins with homologies, suggesting a role in pathogenesis. Nine of the proteins appear to be unique to mycobacteria and represent promising candidates for factors that participate in protective immunity and virulence. This technology of creating comprehensive fusion libraries should be applicable to other organisms.

In vitro transposition of Tn552: a tool for DNA sequencing and mutagenesis.

We have explored the potential of the Tn 552 in vitro transposition reaction as a genetic tool. The reaction is simple (requiring a single protein component), robust and efficient, readily producing insertions into several percent of target DNA. Most importantly, Tn 552 insertions in vitro appear to be essentially random. Extensive analyses indicate that the transposon exhibits no significant regional or sequence specificity for target DNA and leaves no discernible 'cold' spots devoid of insertions. The utility of the in vitro reaction for DNA sequencing was demonstrated with a cosmid containing the Mycobacterium smegmatis recBCD gene cluster. The nucleotide sequence of the entire operon was determined using 71 independent Tn 552 insertions, which generated over 13.5 kb of unique sequence and simultaneously provided a comprehensive collection of insertion mutants. The relatively short ends of Tn 552 make construction of novel transposons a simple process and we describe several useful derivatives. The data presented suggest that Tn 552 transposition is a valuable addition to the arsenal of tools available for molecular biology and genomics.

Mutants of Tn3 resolvase which do not require accessory binding sites for recombination activity.

Tn3 resolvase promotes site-specific recombination between two res sites, each of which has three resolvase dimer-binding sites. Catalysis of DNA-strand cleavage and rejoining occurs at binding site I, but binding sites II and III are required for recombination. We used an in vivo screen to detect resolvase mutants that were active on res sites with binding sites II and III deleted (that is, only site I remaining). Mutations of amino acids Asp102 (D102) or Met103 (M103) were sufficient to permit catalysis of recombination between site I and a full res, but not between two copies of site I. A double mutant resolvase, with a D102Y mutation and an additional activating mutation at Glu124 (E124Q), recombined substrates containing only two copies of site I, in vivo and in vitro. In these novel site Ixsite I reactions, product topology is no longer restricted to the normal simple catenane, indicating synapsis by random collision. Furthermore, the mutants have lost the normal specificity for directly repeated sites and supercoiled substrates; that is, they promote recombination between pairs of res sites in linear molecules, or in inverted repeat in a supercoiled molecule, or in separate molecules.

Architecture of the gamma delta resolvase synaptosome: oriented heterodimers identity interactions essential for synapsis and recombination.

Gammadelta resolvase catalyzes recombination within a complex nucleoprotein structure containing at least 12 resolvase subunits bound to two 114 bp res sites. The 2,3' interaction between resolvase dimers is essential for synapsis and recombination. Using oriented resolvase heterodimers, we have identified half sites of res that must be occupied by a 2,3'-proficient protomer. For synapsis, only four of the eight subunits bound to sites II and III, those at II-L and III-L, require a 2,3'-proficient interface. This 2,3' interaction, apparently between protomers bound to adjacent sites, may nucleate assembly of the core synaptic complex. For recombination, 2,3'-proficient subunits are also required at sites I-R and III-R, suggesting an important communication between the crossover site and the core of the synapse.

Tn552 transposase catalyzes concerted strand transfer in vitro.

The Tn552 transposase, a member of the DDE superfamily of transposase and retroviral integrase proteins, has been expressed in soluble form. The purified protein performs concerted strand transfer in vitro, efficiently pairing two preprocessed transposon ends and inserting them into target DNA. For maximum efficiency, both participating DNA ends must contain the two adjacent transposase-binding sites that are the normal constituents of the Tn552 termini. As is the case with transposition in vivo, the insertions recovered from the reaction in vitro are flanked by repeats of a short target sequence, most frequently 6 bp. The reaction has stringent requirements for a divalent metal ion. Concerted strand transfer is most efficient with Mg2+. Although it stimulates strand transfer overall, Mn2+ promotes uncoupled, single-ended events at the expense of concerted insertions. The simplicity and efficiency of the Tn552 transposition system make it an attractive subject for structural and biochemical studies and a potentially useful genetic tool.

A single side chain prevents Escherichia coli DNA polymerase I (Klenow fragment) from incorporating ribonucleotides.

Although nucleic acid polymerases from different families show striking similarities in structure, they maintain stringent specificity for the sugar structure of the incoming nucleoside triphosphate. The Klenow fragment of E. coli DNA polymerase I selects its natural substrates, deoxynucleotides, over ribonucleotides by several thousand fold. Analysis of mutant Klenow fragment derivatives indicates that discrimination is provided by the Glu-710 side chain which sterically blocks the 2'-OH of an incoming rNTP. A nearby aromatic side chain, at position 762, plays an important role in constraining the nucleotide so that the Glu-710 "steric gate" can be fully effective. Even with the E710A mutation, which is extremely permissive for addition of a single ribonucleotide to a DNA primer, Klenow fragment does not efficiently synthesize pure RNA, indicating that additional barriers prevent the incorporation of successive ribonucleotides.

How E. coli DNA polymerase I (Klenow fragment) distinguishes between deoxy- and dideoxynucleotides.

Deoxy- and dideoxynucleotides differ only in whether they have a hydroxyl substituent at C-3' of the ribose moiety, and yet the Klenow fragment DNA polymerase prefers the natural (dNTP) substrate by several thousandfold. We have used this preference in order to investigate how Klenow fragment interacts with the sugar portion of an incoming dNTP. We screened mutant derivatives of Klenow fragment so as to identify those amino acid residues that play important roles in distinguishing between dNTPs and ddNTPs. Substitution of Phe762 with Ala or Tyr caused a dramatic decrease in the discrimination against ddNTPs, while mutations in Tyr766 and Glu710 had a smaller effect, suggesting that these two side-chains play secondary roles in the selection of dNTPs over ddNTPs. In order to understand the interactions in the enzyme-DNA-dNTP ternary complex, pre-steady-state kinetic parameters for the incorporation of dNTPs and ddNTPs were determined for wild-type Klenow fragment and for mutant derivatives that showed changes in dNTP/ddNTP discrimination. From elemental effect measurements we infer that selection against dideoxynucleotides takes place in the transition state for the conformational change that precedes phosphoryl transfer. The crucial role of the Phe762 side-chain appears to be to constrain the dNTP molecule so that the 3'-OH can make an interaction with another group within the ternary complex. When Tyr is substituted at position 762, the same interactions can take place to position the dNTP, but specificity against the ddNTP is lost because the phenolic OH can compensate for the missing 3'-OH of the nucleotide. Substitution of the smaller Ala side-chain results in a loss in specificity because the dNTP is no longer appropriately constrained. Measurement of reaction rates as a function of magnesium ion concentration suggests that the interaction made with the dNTP 3'-OH may involve a metal ion and the Glu710 side-chain, the simplest scenario being that both the 3'-OH and the carboxylate of Glu710 are ligands to the same metal ion.

Site-specific recombination: synapsis and strand exchange revealed.

Recently determined crystal structures of several members of the lambda integrase family of site-specific recominases have provided insights into the cis versus trans action of active site constituents, and hte processes of synapsis and strand exchange.

Two abundant intramolecular transposition products, resulting from reactions initiated at a single end, suggest that IS2 transposes by an unconventional pathway.

The Escherichia coli insertion sequence, IS2, is a member of the IS3 family of bacterial transposable elements. Its transposase is a fusion protein, OrfAB, made by a programmed -1 translational frameshift near to the end of orfA and just after the start of orfB. We have characterized two major products of IS2 intramolecular transposition, which accumulate in cells that express the IS2 OrfAB fusion protein at elevated levels. The more abundant product is a minicircle composed of the complete IS2 with just a single basepair (occasionally 2bp) separating the two IS ends. In all cases, this basepair is derived from the vector sequence immediately adjacent to the left IS2 end (IRL). The second product is a figure-eight molecule that contains all the IS2 and vector sequences present in the parental plasmid. One DNA strand contains the parental sequences unrearranged. The other contains a single-stranded version of the minicircle junction--the precise 3' end of IRR has been cleaved and joined to a target just outside the 5' end of IRL; the remaining vector sequences have a free 5' end, derived from cleavage at the 3' end of IRR, and a free 3' end, released upon cleavage of the target site adjacent to IRL. We propose that figure-eight molecules are the precursor to IS2 minicircles and that the formation of these two products is the initial step in IS2 intermolecular transposition. This proposed transposition pathway provides a means for a transposase that can cleave only one strand at each IS end to produce simple insertions and avoid forming co-integrates.

Biochemical and mutational studies of the 5'-3' exonuclease of DNA polymerase I of Escherichia coli.

In order to improve our understanding of the 5'-3' exonuclease reaction catalyzed by Escherichia coli DNA polymerase I, we have constructed expression plasmids and developed purification methods for whole DNA polymerase I and its 5'-3' exonuclease domain that allow the production of large quantities of highly purified material suitable for biophysical and other studies. We have studied the enzymatic properties of the 5'-3' exonuclease, both as an isolated domain and in the context of the whole polymerase, using a variety of model oligonucleotides to explore the enzyme-substrate interaction. The 5'-3' exonuclease is known to be a structure-specific nuclease that cleaves a 5' displaced strand at the junction between single-stranded and duplex regions. Since the isolated domain shows the same structure specificity as the whole polymerase, the correct geometry of substrate binding is achieved without the assistance of the polymerase domain. The 5'-3' exonuclease reaction has a strict requirement for a free 5' end on the displaced strand; however, the upstream template and primer strands are dispensable. Site-directed mutagenesis of the ten carboxylate residues that are highly conserved among bacterial and bacteriophage 5'-3' exonucleases indicates that nine of them are important in the reaction. This finding is discussed in relation to structural and mutational data for related 5' nucleases.

Cis preference of the IS903 transposase is mediated by a combination of transposase instability and inefficient translation.

The transposase protein encoded by the insertion element IS903 belongs to an unusual class of DNA-binding proteins, termed cis-acting proteins, that act preferentially at their site of synthesis. Previous work had led us to propose that instability of the IS903 transposase was a major determinant of its cis preference. Here we describe the isolation of two classes of mutations within the transposase gene that increased action in trans. One class specifically increased trans action without increasing the level of transposition when the mutant gene was located in cis to the transposon. In particular, a threonine-to-proline substitution at amino acid 25 (T25P) reduced cis preference about 60-fold. The half-life of this mutant transposase was significantly longer than that of the wild-type transposase, confirming the critical role of protein instability. The second, larger, class of mutations increased the level of transposition both in trans and in cis. The behaviour and location of these mutations were consistent with an increase in gene expression by improving translational initiation. Several of these mutations exerted a disproportionate effect on the action of transposase in trans, implying that translation efficiency may affect more than just the amount of transposase made. Our results indicate that cis preference of the IS903 transposase is mediated by a combination of transposase instability and inefficient translation initiation.

Catalytic residues of gamma delta resolvase act in cis.

The resolvase protein of the gamma delta transposon is a site-specific recombinase that acts by a concerted break-and-join mechanism. To analyse the role of individual resolvase subunits in DNA strand cleavage, we have directed the binding of catalytic mutants to specific recombination crossover sites or half-sites. Our results demonstrate that the resolvase subunit bound at the half-site proximal to each scissile phosphodiester bond provides the Ser10 nucleophile and Arg8, Arg68 and Arg71 residues essential for cleavage and covalent attachment to the DNA. Several other residues near the presumptive active site are also shown to act in cis. Double-strand cleavage at one crossover site can proceed independently of cleavage at the other site, although interactions between the resolvase dimers bound at the two crossover sites remain essential. An appropriately oriented heterodimer of active and inactive protomers can in most cases mediate either a 'top' or 'bottom' single-strand cleavage, suggesting that there is no obligatory order of strand cleavages. Top-strand cleavage is associated with the topoisomerase I activity of resolvase, suggesting that a functional asymmetry may be imposed on the crossover site by the structure of the active synapse.

A functional analysis of the inverted repeat of the gamma delta transposable element.

We have constructed a library of point mutants of the 35 base-pair terminal inverted repeat (IR) of the bacterial transposon gamma delta, a member of the Tn3 family of transposable elements. The effect of the mutant ends, both on the immunity conferred on an IR-containing target plasmid and on the transposition of model transposons, was determined. The region important for immunity was shown to be a 30 base-pair stretch of DNA, running from G8 and A9 to G38; mutations in the outermost seven or eight base-pairs did not significantly affect immunity. Positions at which mutations disrupted immunity chiefly coincided with positions previously determined to constitute three segments of the IR with which gamma delta tranposase protein interacts via major groove contacts. We conclude that sequence-specific binding contacts between gamma delta transposase and its cognate IR are limited to a specific subset of positions (those sensitive to mutation in the immunity assay) within this 30 base-pair region. We found that the innermost of the three major groove contact regions was the most susceptible to mutation, while the outermost was the least. Indications of minor groove contacts were also found. Very few point mutations within the 30 base-pair sequence-specific binding region had much effect on transposition when the mutant ends were in the "wild-type" context with the adjacent integration host factor (IHF) binding site. However, deletion of the IHF site, in some cases, revealed a transposition defect, suggesting that for transposition (but not immunity), IHF-transposase cooperation can largely overcome the effects of reduced transposase binding. Although the outer seven base-pairs were not important for immunity, mutations in the outer three or four eliminated or reduced transposition activity, suggesting that these positions are involved in a step in transposition that follows transposase binding.

The tyrosine-6 hydroxyl of gamma delta resolvase is not required for the DNA cleavage and rejoining reactions.

Site-specific recombinases of the resolvase and DNA invertase family all contain a tyrosine residue close to the N-terminus, and four residues away from a serine that has been implicated in catalysis of DNA strand breakage and reunion. To examine the role of this tyrosine in recombination, we have constructed a mutant of gamma delta resolvase in which the tyrosine (residue 6) is replaced by phenylalanine. Characterization of the Y6F mutant protein in vitro indicated that although it was highly defective in recombination, it could cleave DNA at the cross-over site, form a covalent resolvase-DNA complex and rejoin the cleaved cross-over site (usually restoring the parental site). These data rule out a direct role of the Tyr-6 hydroxyl as the nucleophile in the DNA cleavage reaction and strengthen the conclusion that this nucleophile is the nearby invariant serine residue, Ser-10. We conclude that Tyr-6 is essential for fully co-ordinated strand cleavage and exchange, but is dispensable for individual strand cleavage and religation reactions.