Solution structure of the Mu end DNA-binding ibeta subdomain of phage Mu transposase: modular DNA recognition by two tethered domains.

The phage Mu transposase (MuA) binds to the ends of the Mu genome during the assembly of higher order nucleoprotein complexes. We investigate the structure and function of the MuA end-binding domain (Ibetagamma). The three-dimensional solution structure of the Ibeta subdomain (residues 77-174) has been determined using multidimensional NMR spectroscopy. It comprises five alpha-helices, including a helix-turn-helix (HTH) DNA-binding motif formed by helices 3 and 4, and can be subdivided into two interacting structural elements. The structure has an elongated disc-like appearance from which protrudes the recognition helix of the HTH motif. The topology of helices 2-4 is very similar to that of helices 1-3 of the previously determined solution structure of the MuA Igamma subdomain and to that of the homeodomain family of HTH DNA-binding proteins. We show that each of the two subdomains binds to one half of the 22 bp recognition sequence, Ibeta to the more conserved Mu end distal half (beta subsite) and Igamma to the Mu end proximal half (gamma subsite) of the consensus Mu end-binding site. The complete Ibetagamma domain binds the recognition sequence with a 100- to 1000-fold higher affinity than the two subdomains independently, indicating a cooperative effect. Our results show that the Mu end DNA-binding domain of MuA has a modular organization, with each module acting on a specific part of the 22 bp binding site. Based on the present binding data and the structures of the Ibeta and Igamma subdomains, a model for the interaction of the complete Ibetagamma domain with DNA is proposed.     

The <Emphasis Type="Italic">Drosophila</Emphasis> Pipsqueak protein defines a new family of helix-turn-helix DNA-binding proteins

Many prokaryotic and eukaryotic DNA-binding proteins use a helix-turn-helix (HTH) structure for DNA recognition. Here we describe a new family of eukaryotic HTH proteins, the Pipsqueak (Psq) family, which includes proteins from fungi, sea urchins, nematodes, insects, and vertebrates. Three subgroups of the Psq family can be distinguished. Like the HTH proteins of the prokaryotic resolvase family, members of the CENP-B/transposase subgroup catalyze site-specific recombination reactions. This functional conservation, together with a primary sequence similarity between the resolvase and Psq DNA-binding domains, suggests that the resolvase and Psq families are evolutionarily linked. More than half of the newly identified Drosophila Psq proteins contain a BTB protein-protein interaction domain. All proteins of this BTB subgroup belong to the conserved Tramtrack group of BTB-domain proteins. About half of the members of the Tramtrack group contain a Psq domain, while the other half is made up of proteins that contain a zinc finger domain. Thus, nearly all members of this group appear to be DNA-binding proteins. Among other developmental regulators, the Drosophila cell death protein E93 was found to contain a Psq motif and to define a third subgroup of Psq domain proteins. The high sequence conservation of the E93 Psq motif allowed the identification of E93 orthologs in humans and lower metazoans.     

Solution conformations of early intermediates in Mos1 transposition

DNA transposases facilitate genome rearrangements by moving DNA transposons around and between genomes by a cut-and-paste mechanism. DNA transposition proceeds in an ordered series of nucleoprotein complexes that coordinate pairing and cleavage of the transposon ends and integration of the cleaved ends at a new genomic site. Transposition is initiated by transposase recognition of the inverted repeat sequences marking each transposon end. Using a combination of solution scattering and biochemical techniques, we have determined the solution conformations and stoichiometries of DNA-free Mos1 transposase and of the transposase bound to a single transposon end. We show that Mos1 transposase is an elongated homodimer in the absence of DNA and that the N-terminal 55 residues, containing the first helix-turn-helix motif, are required for dimerization. This arrangement is remarkably different from the compact, crossed architecture of the dimer in the Mos1 paired-end complex (PEC). The transposase remains elongated when bound to a single-transposon end in a pre-cleavage complex, and the DNA is bound predominantly to one transposase monomer. We propose that a conformational change in the single-end complex, involving rotation of one half of the transposase along with binding of a second transposon end, could facilitate PEC assembly.     

DNA recognition for virus assembly through multiple sequence-independent interactions with a helix-turn-helix motif

The helix-turn-helix (HTH) motif features frequently in protein DNA-binding assemblies. Viral pac site-targeting small terminase proteins possess an unusual architecture in which the HTH motifs are displayed in a ring, distinct from the classical HTH dimer. Here we investigate how such a circular array of HTH motifs enables specific recognition of the viral genome for initiation of DNA packaging during virus assembly. We found, by surface plasmon resonance and analytical ultracentrifugation, that individual HTH motifs of the Bacillus phage SF6 small terminase bind the packaging regions of SF6 and related SPP1 genome weakly, with little local sequence specificity. Nuclear magnetic resonance chemical shift perturbation studies with an arbitrary single-site substrate suggest that the HTH motif contacts DNA similarly to how certain HTH proteins contact DNA non-specifically. Our observations support a model where specificity is generated through conformational selection of an intrinsically bent DNA segment by a ring of HTHs which bind weakly but cooperatively. Such a system would enable viral gene regulation and control of the viral life cycle, with a minimal genome, conferring a major evolutionary advantage for SPP1-like viruses.     

Tc1 transposase of Caenorhabditis elegans is an endonuclease with a bipartite DNA binding domain.

The Tc1 transposon of Caenorhabditis elegans is a member of the Tc1/mariner family of mobile elements. These elements have inverted terminal repeats that flank a single transposase gene. Here we show that Tc1 transposase, Tc1A, has a bipartite DNA binding domain related to the paired domain of mammalian and Drosophila genes. Both the DNA binding domain of Tc1A and the DNA binding site in the inverted repeat of Tc1 can be divided into two subdomains. Methylation interference studies demonstrate adjacent minor and major groove contacts at the inner part of the binding site by the N-terminal 68 amino acids of the DNA binding domain. In addition, Tc1A amino acids 69-142 are essential for major groove contacts at the outer part of the binding site. Recombinant Tc1A is found to be able to introduce a single strand nick at the 5' end of the transposon in vitro. Furthermore, Tc1A can mediate a phosphoryl transfer reaction. A mutation in a DDE motif abolishes both endonucleolytic and phosphoryl transfer activities, suggesting that Tc1A carries a catalytic core common to retroviral integrases and IS transposases.     

Major groove recognition by three-stranded beta-sheets: affinity determinants and conserved structural features

We present the results of a rational mutagenesis and binding-affinity study of the three-stranded beta-sheet-DNA interface in the complex formed by the amino-terminal DNA-binding domain of the Tn916 integrase protein and its cognate binding site. The relative importance of interfacial contacts present in its NMR-derived solution structure have been tested through mutagenesis, fluorescence anisotropy, and intrinsic quenching DNA-binding assays. We find that seven protein-DNA hydrogen bonds (two base-specific and five to phosphate groups) significantly contribute to the level of affinity. These interactions span the entire DNA-binding surface on the protein, but primarily originate from residues in only two strands of the sheet and loop L2. Interestingly, we show that highly populated, precisely defined intermolecular hydrogen bonds in the ensemble of conformers are invariably important for DNA-binding, implying that NMR-derived solution structures provide direct insight into the energetics of recognition. Unusual three-stranded beta-sheet-DNA interfaces have recently been discovered in three unrelated protein-DNA complexes. A comparative analysis of these structures reveals similar sheet positioning, the presence of two invariant interfacial contacts to the phosphodiester backbone, and two semi-conserved base-specific hydrogen bonds. Two of these conserved contacts significantly contribute to the affinity of the integrase-DNA complex, suggesting that the three-stranded beta-sheet DNA-binding motif exhibits conserved principles of recognition.     

Structural and functional studies of an intermediate on the pathway to operator binding by Escherichia coli MetJ

We present the results of in vitro DNA-binding assays for a mutant protein (Q44K) of the E. coli methionine repressor, MetJ, as well as the crystal structure at 2.2 A resolution of the apo-mutant bound to a 10-mer oligonucleotide encompassing an 8 bp met-box sequence. The wild-type protein binds natural operators co-operatively with respect to protein concentration forming at least a dimer of repressor dimers along operator DNAs. The minimum operator length is thus 16 bp, each MetJ dimer interacting with a single met-box site. In contrast, the Q44K mutant protein can also bind stably as a single dimer to 8 bp target sites, in part due to additional contacts made to the phosphodiester backbone outside the 8 bp target via the K44 side-chains. Protein-protein co-operativity in the mutant is reduced relative to the wild-type allowing the properties of an intermediate on the pathway to operator site saturation to be examined for the first time. The crystal structure of the decamer complex shows a unique conformation for the protein bound to the single met-box site, possibly explaining the reduced protein-protein co-operativity. In both the extended and minimal DNA complexes formed, the mutant protein makes slightly different contacts to the edges of DNA base-pairs than the wild-type, even though the site of amino acid substitution is distal from the DNA-binding motif. Quantitative binding assays suggest that this is not due to artefacts caused by the crystallisation conditions but is most likely due to the relatively small contribution of such direct contacts to the overall binding energy of DNA-protein complex formation, which is dominated by sequence-dependent distortions of the DNA duplex and by the protein-protein contact between dimers.     

The human SETMAR protein preserves most of the activities of the ancestral Hsmar1 transposase

Transposons have contributed protein coding sequences to a unexpectedly large number of human genes. Except for the V(D)J recombinase and telomerase, all remain of unknown function. Here we investigate the activity of the human SETMAR protein, a highly expressed fusion between a histone H3 methylase and a mariner family transposase. Although SETMAR has demonstrated methylase activity and a DNA repair phenotype, its mode of action and the role of the transposase domain remain obscure. As a starting point to address this problem, we have dissected the activity of the transposase domain in the context of the full-length protein and the isolated transposase domain. Complete transposition of an engineered Hsmar1 transposon by the transposase domain was detected, although the extent of the reaction was limited by a severe defect for cleavage at the 3' ends of the element. Despite this problem, SETMAR retains robust activity for the other stages of the Hsmar1 transposition reaction, namely, site-specific DNA binding to the transposon ends, assembly of a paired-ends complex, cleavage of the 5' end of the element in Mn(2+), and integration at a TA dinucleotide target site. SETMAR is unlikely to catalyze transposition in the human genome, although the nicking activity may have a role in the DNA repair phenotype. The key activity for the mariner domain is therefore the robust DNA-binding and looping activity which has a high potential for targeting the histone methylase domain to the many thousands of specific binding sites in the human genome provided by copies of the Hsmar1 transposon.     

Tdd-4, a DNA transposon of Dictyostelium that encodes proteins similar to LTR retroelement integrases.

Tdd-4 is the first DNA transposon to be isolated from Dictyostelium discoideum. This element was isolated by insertion into a target plasmid. Two classes of elements were identified which include a 3.8 kb version and a 3.4 kb deleted version. Sequence analysis reveals that the 145 bp inverted terminal repeats contain the 5'-TGellipsisCA-3' conserved terminal dinucleotides found in prokaryotic transposons and integrated LTR retroelement DNA sequences. Tdd-4 open reading frames are assembled by removal of six introns. Introns 1-5 conform to the GT-AG rule, whereas intron 6 appears to be an AT-AA intron. Also, intron 6 undergoes an alternative 5' splicing reaction. The alternatively spliced region encodes 15 tandem SPXX repeats that are proposed to function as a DNA binding motif. By analogy to other transposons that encode two proteins from the same gene, the full-length Tdd-4 protein is the putative transposase and the truncated Tdd-4 protein is the putative transposition inhibitor. Protein database searches demonstrate Tdd-4 encoded proteins are unique for a DNA element by containing similarities to retroviral/retrotransposon integrases. The putative Tdd-4 transposase contains the same structural relationship as integrases by possessing an N-terminal HHCC motif, a central DDE motif and a C-terminal DNA-binding domain composed of the SPXX motif.     

Mechanism of IS200/IS605 family DNA transposases: activation and transposon-directed target site selection

The smallest known DNA transposases are those from the IS200/IS605 family. Here we show how the interplay of protein and DNA activates TnpA, the Helicobacter pylori IS608 transposase, for catalysis. First, transposon end binding causes a conformational change that aligns catalytically important protein residues within the active site. Subsequent precise cleavage at the left and right ends, the steps that liberate the transposon from its donor site, does not involve a site-specific DNA-binding domain. Rather, cleavage site recognition occurs by complementary base pairing with a TnpA-bound subterminal transposon DNA segment. Thus, the enzyme active site is constructed from elements of both protein and DNA, reminiscent of the interdependence of protein and RNA in the ribosome. Our structural results explain why the transposon ends are asymmetric and how the transposon selects a target site for integration, and they allow us to propose a molecular model for the entire transposition reaction.     

Intertwined structure of the DNA-binding domain of intron endonuclease I-TevI with its substrate.

I-TevI is a site-specific, sequence-tolerant intron endonuclease. The crystal structure of the DNA-binding domain of I-TevI complexed with the 20 bp primary binding region of its DNA target reveals an unusually extended structure composed of three subdomains: a Zn finger, an elongated segment containing a minor groove-binding alpha-helix, and a helix-turn-helix. The protein wraps around the DNA, mostly following the minor groove, contacting the phosphate backbone along the full length of the duplex. Surprisingly, while the minor groove-binding helix and the helix-turn- helix subdomain make hydrophobic contacts, the few base-specific hydrogen bonds occur in segments that lack secondary structure and flank the intron insertion site. The multiple base-specific interactions over a long segment of the substrate are consistent with the observed high site specificity in spite of sequence tolerance, while the modular composition of the domain is pertinent to the evolution of homing endonucleases.     

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.     

Structural basis of replication origin recognition by the DnaA protein

Escherichia coli DnaA binds to 9 bp sequences (DnaA boxes) in the replication origin, oriC, to form a complex initiating chromosomal DNA replication. In the present study, we determined the crystal structure of its DNA-binding domain (domain IV) complexed with a DnaA box at 2.1 Å resolution. DnaA domain IV contains a helix–turn–helix motif for DNA binding. One helix and a loop of the helix– turn–helix motif are inserted into the major groove and 5 bp (3′ two-thirds of the DnaA box sequence) are recognized through base-specific hydrogen bonds and van der Waals contacts with the C5-methyl groups of thymines. In the minor groove, Arg399, located in the loop adjacent to the motif, recognizes three more base pairs (5′ one-third of the DnaA box sequence) by base-specific hydrogen bonds. DNA bending by ~28° was also observed in the complex. These base-specific interactions explain how DnaA exhibits higher affinity for the strong DnaA boxes (R1, R2 and R4) than the weak DnaA boxes (R3 and M) in the replication origin.     

Transposase makes critical contacts with, and is stimulated by, single-stranded DNA at the P element termini in vitro.

P elements transpose by a cut-and-paste mechanism. Donor DNA cleavage mediated by transposase generates 17 nucleotide (nt) 3' single-strand extensions at the P element termini which, when present on oligonucleotide substrates, stimulate both the strand-transfer and disintegration reactions in vitro. A significant amount of the strand-transfer products are the result of double-ended integration. Chemical DNA modification-interference experiments indicate that during the strand-transfer reaction, P element transposase contacts regions of the substrate DNA that include the transposase binding site and the duplex portion of the 31 bp inverted repeat, as well as regions of the terminal 17 nt single-stranded DNA. Together these data suggest that the P element transposase protein contains two DNA-binding sites and that the active oligomeric form of the transposase protein is at least a dimer.