Structural insights into the retroviral DNA integration apparatus

Retroviral replication depends on successful integration of the viral genetic material into a host cell chromosome. Virally encoded integrase, an enzyme from the DDE(D) nucleotidyltransferase superfamily, is responsible for the key DNA cutting and joining steps associated with this process. Insights into the structural and mechanistic aspects of integration are directly relevant for the development of antiretroviral drugs. Recent breakthroughs have led to biochemical and structural characterization of the principal integration intermediates revealing the tetramer of integrase that catalyzes insertion of both 3' viral DNA ends into a sharply bent target DNA. This review discusses the mechanism of retroviral DNA integration and the mode of action of HIV-1 integrase strand transfer inhibitors in light of the recent visualization of the prototype foamy virus intasome, target DNA capture and strand transfer complexes.     

The DDE motif in RAG-1 is contributed in trans to a single active site that catalyzes the nicking and transesterification steps of V(D)J recombination

The process of assembling immunoglobulin and T-cell receptor genes from variable (V), diversity (D), and joining (J) gene segments, called V(D)J recombination, involves the introduction of DNA breaks at recombination signals. DNA cleavage is catalyzed by RAG-1 and RAG-2 in two chemical steps: first-strand nicking, followed by hairpin formation via direct transesterification. In vitro, these reactions minimally proceed in discrete protein-DNA complexes containing dimeric RAG-1 and one or two RAG-2 monomers bound to a single recombination signal sequence. Recently, a DDE triad of carboxylate residues essential for catalysis was identified in RAG-1. This catalytic triad resembles the DDE motif often associated with transposase and retroviral integrase active sites. To investigate which RAG-1 subunit contributes the residues of the DDE triad to the recombinase active site, cleavage of intact or prenicked DNA substrates was analyzed in situ in complexes containing RAG-2 and a RAG-1 heterodimer that carried an active-site mutation targeted to the same or opposite RAG-1 subunit mutated to be incompetent for DNA binding. The results show that the DDE triad is contributed to a single recombinase active site, which catalyzes the nicking and transesterification steps of V(D)J recombination by a single RAG-1 subunit opposite the one bound to the nonamer of the recombination signal undergoing cleavage (cleavage in trans). The implications of a trans cleavage mode observed in these complexes on the organization of the V(D)J synaptic complex are discussed.     

piggyBac can bypass DNA synthesis during cut and paste transposition

DNA synthesis is considered a defining feature in the movement of transposable elements. In determining the mechanism of piggyBac transposition, an insect transposon that is being increasingly used for genome manipulation in a variety of systems including mammalian cells, we have found that DNA synthesis can be avoided during piggyBac transposition, both at the donor site following transposon excision and at the insertion site following transposon integration. We demonstrate that piggyBac transposon excision occurs through the formation of transient hairpins on the transposon ends and that piggyBac target joining occurs by the direct attack of the 3'OH transposon ends on to the target DNA. This is the same strategy for target joining used by the members of DDE superfamily of transposases and retroviral integrases. Analysis of mutant piggyBac transposases in vitro and in vivo using a piggyBac transposition system we have established in Saccharomyces cerevisiae suggests that piggyBac transposase is a member of the DDE superfamily of recombinases, an unanticipated result because of the lack of sequence similarity between piggyBac and DDE family of recombinases.     

Tn5 transposase active site mutations suggest position of donor backbone DNA in synaptic complex

Tn5 transposase (Tnp), a 53.3-kDa protein, enables the movement of transposon Tn5 by a conservative mechanism. Within the context of a protein and DNA synaptic complex, a single Tnp molecule catalyzes four sequential DNA breaking and joining reactions at the end of a single transposon. The three amino acids of the DDE motif (Asp-97, Asp-188, and Glu-326), which are conserved among transposases and retroviral integrases, have been shown previously to be absolutely required for all catalytic steps. To probe the effect of active site geometry on the ability to form synaptic complexes and perform catalysis, single mutations at each position of the DDE motif were constructed. The aspartates were changed to glutamates, and the glutamate was changed to an aspartate. These mutants were studied by performing in vitro binding assays using short oligonucleotide substrates simulating the natural substrates for the synaptic complex formation and subsequent transposition steps. The results indicate that the aspartate to glutamate mutations restrict synaptic complex formation with substrates resembling the natural transposon prior to transferred strand nicking. This suggests a structural model in which the donor backbone DNA, prior to nicking, occupies the same space that is invaded by the longer side chains present in the aspartate to glutamate mutants. Additionally, catalytic assays support the previous proposal that the active site coordinates two divalent metal ions.     

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.     

The Structure of DNA within Cationic Lipid/DNA Complexes

The structure of DNA within CLDCs used for gene delivery is controversial. Previous studies using CD have been interpreted to indicate that the DNA is converted from normal B to C form in complexes. This investigation reexamines this interpretation using CD of model complexes, FTIR as well as Raman spectroscopy and molecular dynamics simulations to address this issue. CD spectra of supercoiled plasmid DNA undergo a significant loss of rotational strength in the signal near 275 nm upon interaction with either the cationic lipid dimethyldioctadecylammonium bromide or 1,2-dioleoyltrimethylammonium propane. This loss of rotational strength is shown, however, by both FTIR and Raman spectroscopy to occur within the parameters of the B-type conformation. Contributions of absorption flattening and differential scattering to the CD spectra of complexes are unable to account for the observed spectra. Model studies of the CD of complexes prepared from synthetic oligonucleotides of varying length suggest that significant reductions in rotational strength can occur within short stretches of DNA. Furthermore, some alteration in the hydrogen bonding of bases within CLDCs is indicated in the FTIR and Raman spectroscopy results. In addition, alterations in base stacking interactions as well as hydrogen bonding are suggested by molecular dynamics simulations. A global interpretation of all of the data suggests the DNA component of CLDCs remains in a variant B form in which base/base interactions are perturbed.     

Reorganization of the Mu transpososome active sites during a cooperative transition between DNA cleavage and joining

Transposition of mobile genetic elements proceeds through a series of DNA phosphoryl transfer reactions, with multiple reaction steps catalyzed by the same set of active site residues. Mu transposase repeatedly utilizes the same active site DDE residues to cleave and join a single DNA strand at each transposon end to a new, distant DNA location (the target DNA). To better understand how DNA is manipulated within the Mu transposase-DNA complex during recombination, the impact of the DNA immediately adjacent to the Mu DNA ends (the flanking DNA) on the progress of transposition was investigated. We show that, in the absence of the MuB activator, the 3 '-flanking strand can slow one or more steps between DNA cleavage and joining. The presence of this flanking DNA strand in just one active site slows the joining step in both active sites. Further evidence suggests that this slow step is not due to a change in the affinity of the transpososome for the target DNA. Finally, we demonstrate that MuB activates transposition by stimulating the reaction step between cleavage and joining that is otherwise slowed by this flanking DNA strand. Based on these results, we propose that the 3 '-flanking DNA strand must be removed from, or shifted within, both active sites after the cleavage step; this movement is coupled to a conformational change within the transpososome that properly positions the target DNA simultaneously within both active sites and thereby permits joining.     

DNA Sequence Determinants Controlling Affinity,Stability and Shape of DNA Complexes Bound by the Nucleoid Protein Fis

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequences in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. The affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.     

RAG transposase can capture and commit to target DNA before or after donor cleavage

The discovery that the V(D)J recombinase functions as a transposase in vitro suggests that transposition by this system might be a potent source of genomic instability. To gain insight into the mechanisms that regulate transposition, we investigated a phenomenon termed target commitment that reflects a functional association between the RAG transposase and the target DNA. We found that the V(D)J recombinase is quite promiscuous, forming productive complexes with target DNA both before and after donor cleavage, and our data indicate that the rate-limiting step for transposition occurs after target capture. Formation of stable target capture complexes depends upon the presence of active-site metal binding residues (the DDE motif), suggesting that active-site amino acids in RAG-1 are critical for target capture. The ability of the RAG transposase to commit to target prior to cleavage may result in a preference for transposition into nearby targets, such as immunoglobulin and T-cell receptor loci. This could bias transposition toward relatively "safe" regions of the genome. A preference for localized transposition may also have influenced the evolution of the antigen receptor loci.     

Amino acid residues in Rag1 crucial for DNA hairpin formation

The Rag proteins carry out V(D)J recombination through a process mechanistically similar to cut-and-paste transposition. Specifically, Rag complexes form DNA hairpins through direct transesterification, using a catalytic Asp-Asp-Glu (DDE) triad in Rag1. How is sufficient DNA distortion introduced to allow hairpin formation? We hypothesized that, like certain transposases, the Rag proteins might use aromatic amino acid residues to stabilize a flipped-out base. Through in vivo and in vitro experiments and structural predictions, we identified residues in Rag1 crucial for hairpin formation. One of these, a conserved tryptophan (Trp893), probably participates in base-stacking interactions near the cleavage site, as do Trp298, Trp265 and Trp319 in the Tn5, Tn10 and Hermes transposases, respectively. Other residues surrounding the catalytic glutamate (YKEFRK) may share functional similarities with the YREK motif in IS4 family transposases.     

Retroviral DNA integration: reaction pathway and critical intermediates

The key DNA cutting and joining steps of retroviral DNA integration are carried out by the viral integrase protein. Structures of the individual domains of integrase have been determined, but their organization in the active complex with viral DNA is unknown. We show that HIV-1 integrase forms stable synaptic complexes in which a tetramer of integrase is stably associated with a pair of viral DNA ends. The viral DNA is processed within these complexes, which go on to capture the target DNA and integrate the viral DNA ends. The joining of the two viral DNA ends to target DNA occurs sequentially, with a stable intermediate complex in which only one DNA end is joined. The integration product also remains stably associated with integrase and likely requires disassembly before completion of the integration process by cellular enzymes. The results define the series of stable nucleoprotein complexes that mediate retroviral DNA integration.     

Characterization of the DNA binding activity of stable RecA-DNA complexes. Interaction between the two DNA binding sites within RecA helical filaments

The DNA-binding, annealing and recombinational activities of purified RecA-DNA complexes stabilized by ATP gamma S (a slowly hydrolysable analog of ATP) are described. Electrophoretic analysis, DNase protection experiments and observations by electron microscopy suggest that saturated RecA complexes formed with single- or double-stranded DNA are able to accommodate an additional single strand of DNA with a stoichiometry of about one nucleotide of added single-stranded DNA per nucleotide or base-pair, respectively, of DNA resident in the complex. This strand uptake is independent of complementarity or homology between the added and resident DNA molecules. In the complex, the incoming and resident single-stranded DNA molecules are in close proximity as the two strands can anneal in case of their complementarity. Stable RecA complexes formed with single-stranded DNA bind double-stranded DNA efficiently when the added DNA is homologous to the complexed strand and then initiate a strand exchange reaction between the partner DNA molecules. Electron microscopy of the RecA-single-stranded DNA complexes associated with homologous double-stranded DNA suggests that a portion of duplex DNA is taken into the complex and placed in register with the resident single strand. Our experiments indicate that both DNA binding sites within RecA helical filaments can be occupied by either single- or double-stranded DNA. Presumably, the same first DNA binding site is used by RecA during its polymerization on single- or double-stranded DNA and the second DNA binding site becomes available for subsequent interaction of the protein-saturated complexes with naked DNA. The way by which additional DNA is taken into RecA-DNA complexes shows co-operative character and this helps to explain how topological problems are avoided during RecA-mediated homologous recombination.     

Binding of glucocorticoid receptors to DNA.

DNA has been implicated as the nuclear acceptor for receptor-glucocorticoid complexes. The present study concerns the interaction of these complexes, isolated from cultured rat hepatoma cells, with purified DNA. This association is rapid, reaching a maximum within a few minutes at 0 degrees, whereas dissociation requires several hours. DNA binds neither free glucocorticoids nor those complexed with transcortin or cytosol proteins different from the receptor. Receptors which are not complexed by steroid have little or no affinity for DNA. "Activation," necessary for the binding of receptor-steroid complexes to isolated nuclei, also enhances DNA binding. The capacity of DNA for binding receptor-steroid complexes is large; saturation was not observed at the complex concentrations studied, using either crude or partially purified receptor preparations. The association of complexes with DNA is inhibited by divalent cations, at increasing ionic strengths, and by mercurial reagents. Complexes bind equally well to bacterial, bacteriophage, or rat DNA; however, there was either no or substantially reduced binding by bacterial 23 S rRNA. The binding of complexes to native DNA is roughly 3-fold greater than to denatured DNA. These characteristics are consistent with the possibility that DNA is the nuclear acceptor for receptor-glucocorticoid complexes; however, the actual composition of the acceptor sites remains unknown.     

DNA strand arrangement within the SfiI-DNA complex: atomic force microscopy analysis

The SfiI restriction enzyme binds to DNA as a tetramer holding two usually distant DNA recognition sites together before cleavage of the four DNA strands. To elucidate structural properties of the SfiI-DNA complex, atomic force microscopy (AFM) imaging of the complexes under noncleaving conditions (Ca2+ instead of Mg2+ in the reaction buffer) was performed. Intramolecular complexes formed by protein interaction between two binding sites in one DNA molecule (cis interaction) as well as complexes formed by the interaction of two sites in different molecules (trans interaction) were analyzed. Complexes were identified unambiguously by the presence of a tall spherical blob at the DNA intersections. To characterize the path of DNA within the complex, the angles between the DNA helices in the proximity of the complex were systematically analyzed. All the data show clear-cut bimodal distributions centered around peak values corresponding to 60 degrees and 120 degrees. To unambiguously distinguish between the crossed and bent models for the DNA orientation within the complex, DNA molecules with different arm lengths flanking the SfiI binding site were designed. The analysis of the AFM images for complexes of this type led to the conclusion that the DNA recognition sites within the complex are crossed. The angles of 60 degrees or 120 degrees between the DNA helices correspond to a complex in which one of the helices is flipped with respect to the orientation of the other. Complexes formed by five different recognition sequences (5'-GGCCNNNNNGGCC-3'), with different central base pairs, were also analyzed. Our results showed that complexes containing the two possible orientations of the helices were formed almost equally. This suggests no preferential orientation of the DNA cognate site within the complex, suggesting that the central part of the DNA binding site does not form strong sequence specific contacts with the protein.     

Formation of DNA replication structures in herpes virus-infected cells requires a viral DNA binding protein

Eukaryotic DNA synthesis is thought to occur in multienzyme complexes present at numerous discrete sites throughout the nucleus. We demonstrate here that cellular DNA replication sites identified by bromodeoxyuridine labeling are relocated in cells infected with herpes simplex virus such that they correspond to viral prereplicative structures containing the HSV DNA replication protein, ICP8. Thus components of the cellular DNA replication apparatus are present at viral prereplicative sites. Mutant virus strains expressing defective ICP8 do not alter the pattern of host cell DNA replication sites, indicating that functional ICP8 is required for the redistribution of cellular DNA replication complexes. This demonstrates that a specific protein molecule can play a role in the organization of DNA replication proteins at discrete sites within the cell nucleus.     

Characterization of a new class of DNA delivery complexes formed by the local anesthetic bupivacaine

Bupivacaine, a local anesthetic and cationic amphiphile, forms stable liposomal-like structures upon direct mixing with plasmid DNA in aqueous solutions. These structures are on the order of 50-70 nm as determined by scanning electron microscopy, and are homogeneous populations as analyzed by density gradient centrifugation. The DNA within these structures is protected from nuclease degradation and UV-induced damage in vitro. Bupivacaine:DNA complexes have a negative zeta potential (surface charge), homogeneous nature, and an ability to rapidly assemble in aqueous solutions. Bupivacaine:DNA complexes, as well as similar complexes of DNA with other local anesthetics, have the potential to be a novel class of DNA delivery agents for gene therapy and DNA vaccines.     

Electron microscopy of SV40 DNA cross-linked by anti-Z DNA IgG.

Electron microscopy has revealed the specific binding of bivalent anti-Z DNA immunoglobulin G (IgG) to different sites on supercoiled Form I SV40 DNA. The anti-Z IgG links together left-handed regions located within individual or on multiple SV40 DNA molecules at the superhelix density obtained upon extraction. Velocity sedimentation, electrophoresis, and electron microscopy all show that two or more Z DNA sites in the SV40 genome can be intermolecularly cross-linked with bivalent IgG into high mol. wt. complexes. The formation and stability of the intermolecular antibody-DNA complexes are dependent on DNA superhelix density, as judged by three criteria: (1) relaxed circular (Form II) DNA does not react; (2) release of torsional stress by intercalation of 0.25 microM ethidium bromide removes the antibody; and (3) linearization with specific restriction endonucleases reverses antibody binding and DNA cross-linking. Non-immune IgG does not bind to negatively supercoiled SV40 Form I DNA, nor are complexes observed in the presence of competitive synthetic polynucleotides constitutively in the left-handed Z conformation; B DNA has no effect. Using various restriction endonucleases, three major sites of anti-Z IgG binding have been mapped by electron microscopy to the 300-bp region containing nucleotide sequences controlling SV40 gene expression. A limited number of minor sites may also exist (at the extracted superhelix density).     

Archaeal histone tetramerization determines DNA affinity and the direction of DNA supercoiling

DNA binding and the topology of DNA have been determined in complexes formed by >20 archaeal histone variants and archaeal histone dimer fusions with residue replacements at sites responsible for histone fold dimer:dimer interactions. Almost all of these variants have decreased affinity for DNA. They have also lost the flexibility of the wild type archaeal histones to wrap DNA into a negative or positive supercoil depending on the salt environment; they wrap DNA into positive supercoils under all salt conditions. The histone folds of the archaeal histones, HMfA and HMfB, from Methanothermus fervidus are almost identical, but (HMfA)(2) and (HMfB)(2) homodimers assemble into tetramers with sequence-dependent differences in DNA affinity. By construction and mutagenesis of HMfA+HMfB and HMfB+HMfA histone dimer fusions, the structure formed at the histone dimer:dimer interface within an archaeal histone tetramer has been shown to determine this difference in DNA affinity. Therefore, by regulating the assembly of different archaeal histone dimers into tetramers that have different sequence affinities, the assembly of archaeal histone-DNA complexes could be localized and used to regulate gene expression.     

Unexpected structural diversity in DNA recombination: the restriction endonuclease connection

Transposition requires a coordinated series of DNA breakage and joining reactions. The Tn7 transposase contains two proteins: TnsA, which carries out DNA breakage at the 5' ends of the transposon, and TnsB, which carries out breakage and joining at the 3' ends of the transposon. TnsB is a member of the retroviral integrase superfamily whose hallmark is a conserved DDE motif. We report here the structure of TnsA at 2.4 A resolution. Surprisingly, the TnsA fold is that of a type II restriction endonuclease. Thus, Tn7 transposition involves a collaboration between polypeptides, one containing a DDE motif and one that does not. This result indicates that the range of biological processes that utilize restriction enzyme-like folds also includes DNA transposition.     

Radiolabelling of DNA/polypeptide complexes in isolated bulk DNA and in residual nuclear matrix DNA by nick-translation

Conditions are described that allow 32P-radiolabelling and detection of tight complexes between DNA and polypeptides by nick-translation. Prolonged nick-translation of purified bulk DNA results in radiolabelled complexes migrating on SDS-polyacrylamide gels with apparent molecular weights of 68 kd and 54 kd respectively. Residual nuclear matrix DNA which is not accessible to DNase I on the nuclear level becomes accessible to radiolabelling by nick-translation on the nuclear matrix level. In this case the in situ radiolabelled complexes migrate on SDS-polyacrylamide gels with apparent molecular weights of 68 kd and 100 kd. The DNA/polypeptide complexes are stable during treatments with SDS, beta-mercapto ethanol and alkali which points to covalent bonds between the polypeptides and DNA strands.