Defining functional regions of the IS903 transposase

The insertion sequence IS903 encodes a 307 amino acid residue protein, transposase, that is essential for transposition. It is a multi-functional DNA-binding protein that specifically recognizes the 18 bp inverted repeats at the ends of the element and also recognizes DNa non-specifically when it captures a target site. In addition, transposase performs catalytic functions when it mediates the cleavage and religation steps of transposition. We have carried out deletion and mutational analyses to define functional domains of the transposase protein. The deletion studies delineate a 99 residue region of the protein (residues 31 to 129) that specifies binding to the inverted repeat. A slightly larger maltose-binding protein-transposase fusion that includes residues 22 to 139 (Tnp 22-139) binds as efficiently and with the same specificity as the full-length transposase protein. Tnp 22-139 also induces a DNA bend similar to that of the wild-type protein, and so we conclude that all binding and bending specificity is contained within the N-terminal domain of the protein. Unlike full-length transposase, Tnp 22-139 forms additional higher-order complexes in band-shift gels suggesting that the deletion has exposed a surface(s) capable of participating in protein-protein interactions. Six highly conserved residues in the C-terminal portion of the protein were mutated to alanine. Each mutant protein was binding-proficient but defective in transposition. The phenotype of these substitutions, and their alignment with residues shown to abolish catalysis of other transposases and integrases, suggest that these are residues responsible for catalytic steps in transposition of IS903; we believe three of these residues comprise the DDE motif, conserved in transposases and integrases. Our data are consistent with IS903 transposase being composed of two domains: an N-terminal domain primarily involved in DNA binding and a C-terminal domain that is involved in catalysis.     

DNA binding by the KP repressor protein inhibits P-element transposase activity in vitro

P elements are a family of mobile DNA elements found in Drosophila. P-element transposition is tightly regulated, and P-element-encoded repressor proteins are responsible for inhibiting transposition in vivo. To investigate the molecular mechanisms by which one of these repressors, the KP protein, inhibits transposition, a variety of mutant KP proteins were prepared and tested for their biochemical activities. The repressor activities of the wild-type and mutant KP proteins were tested in vitro using several different assays for P-element transposase activity. These studies indicate that the site-specific DNA-binding activity of the KP protein is essential for repressing transposase activity. The DNA-binding domain of the KP repressor protein is also shared with the transposase protein and resides in the N-terminal 88 amino acids. Within this region, there is a C2HC putative metal-binding motif that is required for site-specific DNA binding. In vitro the KP protein inhibits transposition by competing with the transposase enzyme for DNA-binding sites near the P-element termini.     

Effects of the human immunodeficiency virus type 1 Rev protein on reporter gene and host T-cell gene expression

The mechanism of DNA mismatch repair has been modeled upon biochemical studies of the E. coli DNA adenine methylation-instructed pathway where the initial recognition of mismatched nucleotides is performed by the MutS protein. MutS homologs (MSH) have been identified based on a highly conserved region containing a Walker-A adenine nucleotide binding motif. Here we show that adenine nucleotide binding and hydrolysis by the human mismatch recognition complex hMSH2-hMSH6 functions as a novel molecular switch. The hMSH2-hMSH6 complex is ON (binds mismatched nucleotides) in the ADP-bound form and OFF in the ATP-bound form. These results suggest a new model for the function of MutS proteins during mismatch repair in which the switch determines the timing of downstream events.     

Factors responsible for target site selection in Tn10 transposition: a role for the DDE motif in target DNA capture

Tn10, like several other transposons, exhibits a marked preference for integration into particular target sequences. Such sequences are referred to as integration hotspots and have been used to define a consensus target site in Tn10 transposition. We demonstrate that a Tn10 hotspot called HisG1, which was identified originally in vivo, also functions as an integration hotspot in vitro in a reaction where the HisG1 sequence is present on a short DNA oligomer. We use this in vitro system to define factors which are important for the capture of the HisG1 target site. We demonstrate that although divalent metal ions are not essential for HisG1 target capture, they greatly facilitate capture of a mutated HisG1 site. Analysis of catalytic transposase mutants further demonstrates that the DDE motif plays a critical role in 'divalent metal ion-dependent' target capture. Analysis of two other classes of transposase mutants, Exc+ Int- (which carry out transposon excision but not integration) and ATS (altered target specificity), demonstrates that while a particular ATS transposase binds HisG1 mutants better than wild-type transposase, Exc+ Int- mutants are defective in HisG1 capture, further defining the properties of these classes of mutants. Possible mechanisms for the above observations are considered.     

A functional analysis of the Tn5 transposase. Identification of domains required for DNA binding and multimerization

A series of deletions were constructed in the 476 amino acid Tn5 transposase in order to assemble an initial domain structure for this protein. The first four amino acids were found to be important for transposition activity but not for DNA binding to the Tn5 outside end (OE). Larger amino-terminal deletions result in the complete loss of transposition in vivo and the concomitant loss of specific DNA binding. Four point mutants and a six base-pair deletion in the amino terminus between residues 20 and 36 were also found to impair DNA binding to the OE. Analysis of a series of carboxy-terminal deletions has revealed that the carboxy terminus may actually mask the DNA binding domain, since deletions to residues 388 and 370 result in a large increase in DNA binding activity. In addition, the carboxy-terminal deletion to residue 370 results in a significant increase in the mobility of the Tnp-OE complex indicative of a change in the oligomeric state of this complex. Further carboxy-terminal deletions beyond residue 370 also abolished DNA binding activity. These results indicate that the first four amino acids of Tnp are important for transposition but not DNA binding, a region between residues 5 and 36 is critical for DNA binding, the wild-type carboxy terminus acts to inhibit DNA binding, and that a region towards the carboxy terminus, defined by residues 370 to 387, is critical for Tnp multimeric interactions.     

Mutational analysis of the Mu transposase. Contributions of two distinct regions of domain II to recombination

Mu transposase is a member of a protein family that includes many transposases and the retroviral integrases. These recombinases catalyze the DNA cleavage and joining reactions essential for transpositional recombination. Here we demonstrate that, consistent with structural predictions, aspartate 336 of Mu transposase is required for catalysis of both DNA cleavage and DNA joining. This residue, although located 55 rather than 35 residues NH2-terminal of the essential glutamate, is undoubtedly the analog of the second aspartate of the Asp-Asp-35-Glu motif found in other family members. The core domain of Mu transposase consists of two subdomains: the NH2-terminal subdomain (IIA) contains the conserved Asp-Asp-Glu motif residues, whereas the smaller COOH-terminal subdomain (IIB) contains a large positively charged region exposed on its surface. To probe the function of domain IIB, we constructed mutant proteins carrying deletion or substitution mutations within this region. The activity of the deletion proteins revealed that domains IIA and IIB can be provided by different subunits in the transposase tetramer. Substitution mutations at two pairs of exposed lysine residues within the positively charged surface of domain IIB render transposase defective in transposition at a reaction step after DNA cleavage but prior to DNA joining. The severity of this defect depends on the structure of the DNA flanking the cleavage site. Thus, these data suggest that domain IIB is involved in manipulating the DNA near the cleavage site and that this function is important during the transition between the DNA cleavage and the DNA joining steps of recombination.     

Transposase is the only nematode protein required for in vitro transposition of Tc1

The Tc1 element of Caenorhabditis elegans is a member of the most widespread class of DNA transposons known in nature. Here, we describe efficient and precise transposition of Tc1 in a cell-free system. Tc1 appears to jump by a cut-and-paste mechanism of transposition. The terminal 26 bp of the Tc1 terminal repeats together with the flanking TA sequence are sufficient for transposition. The target site choice in vitro is similar to that in vivo. Transposition is achieved with an extract prepared from nuclei of transgenic nematodes that overexpress Tc1 transposase but also by recombinant transposase purified from Escherichia coli. The simple reaction requirements explain why horizontal spread of Tc1/mariner transposons can occur. They also suggest that Tcl may be a good vector for transgenesis of diverse animal species.     

PCNA binding proteins in Drosophila melanogaster : the analysis of a conserved PCNA binding domain

The eukaryotic polymerase processivity factor, PCNA, interacts with cell cycle regulatory proteins such as p21(WAF1/Cip1) and Gadd45, as well as with proteins involved in the mechanics of DNA repair and replication. A conserved PCNA-binding motif is found in a subset of PCNA-interacting proteins, including p21, suggesting that the regulation of these interactions is important for the co-ordination of DNA replication and repair. We have identified several classes of protein which bind to Drosophila PCNA. Two of these proteins contain the consensus PCNA-binding domain: one is the Dacapo protein, a Drosophila homologue of p21(WAF1/Cip1), and the second is the transposase encoded by the Pogo DNA transposon . A conserved PCNA-binding domain is also present in a human relative of Pogo , named Tigger , suggesting that this domain has a functional role in this class of transposable element. This raises interesting possibilities for a novel method of transposition in which the transposase might be targeted to replicating DNA. Finally, we have investigated the use of this conserved PCNA-binding domain as a predictor of PCNA-binding capacity.     

The Tn7 transposase is a heteromeric complex in which DNA breakage and joining activities are distributed between different gene products

The bacterial transposon Tn7 translocates by a cut and paste mechanism: excision from the donor site results from double-strand breaks at each end of Tn7 and target insertion results from joining of the exposed 3' Tn7 tips to the target DNA. Through site-directed mutagenesis of the Tn7-encoded transposition proteins TnsA and TnsB, we demonstrate that the Tn7 transposase is a heteromeric complex of these proteins, each protein executing different DNA processing reactions. TnsA mediates DNA cleavage reactions at the 5' ends of Tn7, and TnsB mediates DNA breakage and joining reactions at the 3' ends of Tn7. Thus the double-strand breaks that underlie Tn7 excision result from a collaboration between two active sites, one in TnsA and one in TnsB; the same (or a closely related) active site in TnsB also mediates the subsequent joining of the 3' ends to the target. Both TnsA and TnsB appear to be members of the retroviral integrase superfamily: mutation of their putative DD(35)E motifs blocks catalytic activity. Recombinases of this class require a divalent metal cofactor that is thought to interact with these acidic residues. Through analysis of the metal ion specificity of a TnsA mutant containing a sulfur (cysteine) substitution, we provide evidence that a divalent metal actually interacts with these acidic amino acids.     

Screening for germ-line rearrangements and regulatory mutations in BRCA1 led to the identification of four new deletions

Pogo is a transposable element with short terminal inverted repeats. It contains two open reading frames that are joined by splicing and code for the putative pogo transposase, the sequence of which indicates that it is related to the transposases of members of the Tc1/mariner family as well as proteins that have no known transposase activity including the centromere binding protein CENP-B. We have shown that the N-terminal region of pogo transposase binds in a sequence-specific manner to the ends of pogo and have identified residues essential for this. The results are consistent with a prediction that DNA binding is due to a helix-turn-helix motif within this region. The transposase recognises a 12 bp sequence, two copies of which are present at each end of pogo DNA. The outer two copies occur as inverted repeats 14 nucleotides from each end of the element, and contain a single base mismatch and indicate the inverted repeats of pogo are 26 nucleotides long. The inner copies occur as direct repeats, also with a single mismatch.     

Negative and positive regulation of Tn10/IS10-promoted recombination by IHF: two distinguishable processes inhibit transposition off of multicopy plasmid replicons and activate chromosomal events that favor evolution of new transposons

Tn10 is a composite transposon; inverted repeats of insertion sequence IS10 flank a tetracycline-resistance determinant. Previous work has identified several regulatory processes that modulate the interaction between Tn10 and its host. Among these, host-specified DNA adenine methylation, an IS10-encoded antisense RNA and preferential cis action of transposase are particularly important. We now find that the accessory host protein IHF and the sequences that encode the IHF-binding site in IS10 are also important regulators of the Tn10 transposition reaction in vivo and that these determinants are involved in two distinguishable regulatory processes. First, IHF and the IHF-binding site of IS10, together with other host components (e.g., HU), negatively regulate the normal intermolecular transposition process. Such negative regulation is prominent only for elements present on multicopy plasmid replicons. This multicopy plasmid-specific regulation involves effects both on the transposition reaction per se and on transposase gene expression. Second, specific interaction of IHF with its binding site stimulates transposon-promoted chromosome rearrangements but not transposition of a short Tn10-length chromosomal element. However, additional considerations predict that IHF action should favor chromosomal transposition for very long composite elements. On the basis of these and other observations we propose that, for chromosomal events, the major role of IHF is to promote the evolution of new IS10-based composite transposons.     

Assembly of a strong promoter following IS911 circularization and the role of circles in transposition

When supplied with high levels of the IS911-encoded transposase, IS911-based transposons can excise as circles in which the right and left terminal inverted repeats are abutted. Formation of the circle junction is shown here to create a promoter, p(junc), which is significantly stronger than the indigenous promoter, pIRL, and is also capable of driving expression of the IS911 transposition proteins. High transposase expression from the circular transposon may promote use of the circle as an integration substrate. The results demonstrate that IS911 circles are highly efficient substrates for insertion into a target molecule in vivo. Insertion leads to the disassembly of p(junc) and thus to a lower level of synthesis of the transposition proteins. The observation that normal levels of IS911 transposition proteins supplied by wild-type copies of IS911 are also capable of generating transposon circles, albeit at a low level, reinforces the idea that the transposon circles might form part of the natural transposition cycle of IS911. These observations form the elements of a feedback control mechanism and have been incorporated into a model describing one possible pathway of IS911 transposition.     

Functional characterization of the Tn5 transposase by limited proteolysis

The 476 amino acid Tn5 transposase catalyzes DNA cutting and joining reactions that cleave the Tn5 transposon from donor DNA and integrate it into a target site. Protein-DNA and protein-protein interactions are important for this tranposition process. A truncated transposase variant, the inhibitor, decreases transposition rates via the formation of nonproductive complexes with transposase. Here, the inhibitor and the transposase are shown to have similar secondary and tertiary folding. Using limited proteolysis, the transposase has been examined structurally and functionally. A DNA binding region was localized to the N-terminal 113 amino acids. Generally, the N terminus of transposase is sensitive to proteolysis but can be protected by DNA. Two regions are predicted to contain determinants for protein-protein interactions, encompassing residues 114-314 and 441-476. The dimerization regions appear to be distinct and may have separate functions, one involved in synaptic complex formation and one involved in nonproductive multimerization. Furthermore, predicted catalytic regions are shown to lie between major areas of proteolysis.     

A novel DNA binding and nuclease activity in domain III of Mu transposase: evidence for a catalytic region involved in donor cleavage

The Mu A protein is a 75 kDa transposase organized into three structural domains. By severing the C-terminal region (domain III) from the remainder of the protein, we unmasked a novel non-specific DNA binding and nuclease activity in this region. Deletion analysis localized both activities to a 26 amino acid stretch (aa 575-600) which remarkably remained active in DNA binding and cleavage. The two activities were shown to be tightly linked by site-directed mutagenesis. To study the importance of these activities in the transposition process, an intact mutant transposase lacking the DNA binding and nuclease activity of domain III was constructed and purified. The mutant transposase was indistinguishable from wild-type Mu A in binding affinity for both the Mu ends and the enhancer, and in strand transfer activity when the cleavage step was bypassed. In contrast, the mutant transposase displayed defects in both synapsis and donor cleavage. Our results strongly suggest that the 26 amino acid region in domain III carries catalytic residues required for donor DNA cleavage by Mu A protein. Furthermore, our data suggest that an active site for donor cleavage activity in the Mu tetramer is assembled from domain II (metal ion binding) in one A monomer and domain III (DNA cleavage) in a separate A monomer. This proposal for active site assembly is in agreement with the recently proposed domain sharing model by Yang et al. (Yang, J.Y., Kim, K., Jayaram, M. and Harshey, R.M. [1995] EMBO J., 14, 2374-2384).     

The same two monomers within a MuA tetramer provide the DDE domains for the strand cleavage and strand transfer steps of transposition

The chemistry of Mu transposition is executed within a tetrameric form of the Mu transposase (MuA protein). A triad of DDE (Asp, Asp35Glu motif) residues in the central domain of MuA (DDE domain) is essential for both the strand cleavage and strand transfer steps of transposition. Previous studies had suggested that complete Mu transposition requires all four subunits in the MuA tetramer to carry an active DDE domain. Using a mixture of MuA proteins with either wild-type or altered att-DNA binding specificities, we have now designed specific arrangements of MuA subunits carrying the DDE domain. From analysis of the abilities of oriented tetramers to carry out DNA cleavage and strand transfer from supercoiled DNA, a new picture of the disposition of DNA and protein partners during transposition has emerged. For DNA cleavage, two subunits of MuA located at attL1 and attR1 (sites that undergo cleavage) provide DDE residues in trans. The same two subunits contribute DDE residues for strand transfer, also in trans. Thus, only two active DDE+ monomers within the tetramer carry out complete Mu transposition. We also show that when the attR1-R2 arrangement used on supercoiled substrates is tested for cleavage on linear substrates, alternative chemically competent DNA-protein associations are produced, wherein the functional DDE subunits are positioned at R2 rather than at R1.     

Identification of cGMP-dependent protein kinase anchoring proteins (GKAPs)

To promote both efficiency and selectivity, many protein kinases and phosphatases are maintained in specific subcellular microenvironments through their association with anchoring proteins. In this study, we describe a new class of proteins, called GKAPS, that specifically bind the Type II cGMP-dependent protein kinase (PKG). GKAPs were detected in rat aorta, brain, and intestine using a protein overlay technique. The PKG binding proteins were distinct from AKAPs, proteins known to bind the cAMP-dependent protein kinase (PKA). Furthermore, a synthetic peptide that blocks association of PKA with AKAPs did not affect the PKG-GKAP interaction. Deletion mutagenesis was used to map the GKAP binding determinants within PKG to the N-terminal regulatory region. While most GKAPs were tissue-specific, a ubiquitous PKG-binding protein was detected and identified as myosin. Analysis of myosin fragments revealed that PKG binds within Subfragment 2. The results define a novel class of anchoring proteins that may target PKG for specific functional roles.     

(+)-CC-1065 as a structural probe of Mu transposase-induced bending of DNA: overcoming limitations of hydroxyl-radical footprinting

Phage Mu transposase (A-protein) is primarily responsible for transposition of the Mu genome. The protein binds to six att sites, three at each end of Mu DNA. At most att sites interaction of a protein monomer with DNA is seen to occur over three minor and two consecutive major grooves and to result in bending up to about 90 degrees. To probe the directionality and locus of these A-protein-induced bends, we have used the antitumor antibiotic (+)-CC-1065 as a structural probe. As a consequence of binding within the minor groove, (+)-CC-1065 is able to alkylate N3 of adenine in a sequence selective manner. This selectivity is partially determined by conformational flexibility of the DNA sequence, and the covalent adduct has a bent DNA structure in which narrowing of the minor groove has occurred. Using this drug in experiments in which either gel retardation or DNA strand breakage are used to monitor the stability of the A-protein--DNA complex or the (+)-CC-1065 alkylation sites on DNA (att site L3), we have demonstrated that of the three minor grooves implicated in the interaction with A-protein, the peripheral two are 'open' or accessible to drug bonding following protein binding. These drug-bonding sites very likely represent binding at at least two A-protein-induced bending sites. Significantly, the locus of bending at these sites is spaced approximately two helical turns apart, and the bending is proposed to occur by narrowing of the minor groove of DNA. The intervening minor groove between these two peripheral sites is protected from (+)-CC-1065 alkylation. The results are discussed in reference to a proposed model for overall DNA bending in the A-protein att L3 site complex. This study illustrates the utility of (+)-CC-1065 as a probe for protein-induced bending of DNA, as well as for interactions of minor groove DNA bending proteins with DNA which may be masked in hydroxyl radical footprinting experiments.     

Tn5 in vitro transposition

This communication reports the development of an efficient in vitro transposition system for Tn5. A key component of this system was the use of hyperactive mutant transposase. The inactivity of wild type transposase is likely to be related to the low frequency of in vivo transposition. The in vitro experiments demonstrate the following: the only required macromolecules for most of the steps in Tn5 transposition are the transposase, the specific 19-bp Tn5 end sequences, and target DNA; transposase may not be able to self-dissociate from product DNAs; Tn5 transposes by a conservative "cut and paste" mechanism; and Tn5 release from the donor backbone involves precise cleavage of both 3' and 5' strands at the ends of the specific end sequences.