Triple interpolyelectrolyte complexes DNA-polycation-polyanion: a new approach to optimization of mixtures for transfection in eukaryotic cells

A new approach to optimization of mixtures for the condensation and introduction of plasmid DNA into eukaryotic cells is proposed, which is based on the formation of ternary interpolyelectrolyte complexes (IPEC) DNA/polycation/polyanion. Polyethyleneimine (PEI) with M 30-40 kDa as polycation and polyacrylic acid (PA) with M 20 kDa or its grafted copolymer with polyethyleneglycol (PEG) as polyanion were used, and ternary complexes with various ratios of the components were prepared. The PA-PEG incorporation into a ternary complex (by itself or as a 1:1 mixture with PA) was shown to confer the solubility onto complexes in a wide range of DNA/PEI ratios. Incorporation of even minute amounts of PA-PEG (as a 1:9 mixture with PA), while not completely preventing the aggregation of ternary IPEC, drastically changed their sorption characteristics. Using a beta-galactosidase-encoding plasmid, efficiencies of transfection of the CHO-AA8 and 293 cells for different IPEC and DNA/lipofectin complex were compared. The maximum efficiency was exhibited by ternary complex DNA/PEI/polyanion where a 1:1 mixture of PA and PA-PEG was used as polyanion. Possible reasons for this effect and further ways of optimization of mixtures for expression of plasmid DNA in the context of the new approach are discussed.     

Analysis of the interaction between the Saccharomyces cerevisiae MSH2-MSH6 and MLH1-PMS1 complexes with DNA using a reversible DNA end-blocking system

The Lac repressor-operator interaction was used as a reversible DNA end-blocking system in conjunction with an IAsys biosensor instrument (Thermo Affinity Sensors), which detects total internal reflectance and allows monitoring of binding and dissociation in real time, in order to develop a system for studying the ability of mismatch repair proteins to move along the DNA. The MSH2-MSH6 complex bound to a mispaired base was found to be converted by ATP binding to a form that showed rapid sliding along the DNA and dissociation via the DNA ends and also showed slow, direct dissociation from the DNA. In contrast, the MSH2-MSH6 complex bound to a base pair containing DNA only showed direct dissociation from the DNA. The MLH1-PMS1 complex formed both mispair-dependent and mispair-independent ternary complexes with the MSH2-MSH6 complex on DNA. The mispair-independent ternary complexes were formed most efficiently on DNA molecules with free ends under conditions where ATP hydrolysis did not occur, and only exhibited direct dissociation from the DNA. The mispair-dependent ternary complexes were formed in the highest yield on DNA molecules with blocked ends, required ATP and magnesium for formation, and showed both dissociation via the DNA ends and direct dissociation from the DNA.     

Interactions between DNA helicases and frozen topoisomerase IV-quinolone-DNA ternary complexes.

Collisions between replication forks and topoisomerase-drug-DNA ternary complexes result in the inhibition of DNA replication and the conversion of the normally reversible ternary complex to a nonreversible form. Ultimately, this can lead to the double strand break formation and subsequent cell death. To understand the molecular mechanisms of replication fork arrest by the ternary complexes, we have investigated molecular events during collisions between DNA helicases and topoisomerase-DNA complexes. A strand displacement assay was employed to assess the effect of topoisomerase IV (Topo IV)-norfloxacin-DNA ternary complexes on the DnaB, T7 gene 4 protein, SV40 T-antigen, and UvrD DNA helicases. The ternary complexes inhibited the strand displacement activities of these DNA helicases. Unlike replication fork arrest, however, this general inhibition of DNA helicases by Topo IV-norfloxacin-DNA ternary complexes did not require the cleavage and reunion activity of Topo IV. We also examined the reversibility of the ternary complexes after collisions with these DNA helicases. UvrD converted the ternary complex to a nonreversible form, whereas DnaB, T7 gene 4 protein, and SV40 T-antigen did not. These results suggest that the inhibition of DnaB translocation may be sufficient to arrest the replication fork progression but it is not sufficient to generate cytotoxic DNA lesion.     

Ternary complex consisting of DNA, polycation, and a natural polysaccharide of schizophyllan to induce cellular uptake by antigen presenting cells

A natural polysaccharide called schizophyllan (SPG) can form a complex with polynucleotides, and the complex has been shown to deliver biofunctional short DNAs such as antisense DNAs and CpG-DNAs. Although it is a novel and efficient method, there is a drawback: attachment of homo-polynucleotide tails [for example, poly(dA) or poly(C)] to the end of DNA is necessary to stabilize the complex, because DNA heterosequences cannot bind to SPG. The aim of this paper is to present an alternative method in which SPG/DNA complexes can be made without using the tails. The basic strategy is as follows: since SPG can form hydrophobic domains in aqueous solutions, hydrophobic objects should be encapsulated by this domain. DNA alone is highly hydrophilic; however, once DNA/polycation complexes are made, they should be included by the SPG hydrophobic domain. The aim of this paper is to prove the formation of the polycation/DNA/SPG ternary complex. Gel electrophoresis showed that presence of SPG influenced the migration pattern of polycation+DNA mixtures. With increasing the SPG ratio, the zeta potential (zeta) of the polycation+DNA+SPG mixture decreased drastically to reach almost zeta = 0 and the particle size distributions were altered due to the ternary complex formation. Confocal laser scanning microscopy revealed that the polycation/DNA/SPG ternary complexes showed high uptake efficiency when the complexes were exposed to macrophage-like cells (J774.A1). IL-12 secretion was enhanced when CpG-DNA was added as the ternary complex. These features can be ascribed to the fact that J774.A1 has a SPG recognition site called Dectin-1 on the cellular surface and the ternary complex can be ingested by this pathway.     

Investigation of Ternary Complexes: DNA–Phosphatidylcholine Liposomes–Mg<Superscript>2+</Superscript> by Freeze-Fracture Method and Their Role in the Formation of Some Cell Structures

Long-term investigations of ternary complexes: DNA–zwitterionic liposomes–divalent metal cations have revealed many details of their structure; but some questions need additional study. The conditions under which fusion or aggregation of liposomes occurs during such complex formation remain obscure. The DNA structure in the ternary complex is still unclear. In this work, using a freeze-fracture method, author demonstrate the thin structure of a complex (early attempts to observe this structure employing other electron microscopic methods, in particular cryo-TEM, have not met with success). After treatment of ternary complexes with nuclease S1, which is able to digest single-stranded DNA, local DNA unwinding in such complexes was confirmed. Author describe how the curvature of liposomes as the main factor may determine the interaction between liposomes and DNA, especially aggregation or fusion of liposomes during ternary complex formation. Therefore, interaction between lipids of membrane vesicles in cell and chromatin DNA can be the first stage of a nuclear envelope and pore complex assembly.     

Ternary Interpolyelectrolyte Complexes DNA–Polycation–Polyanion: A New Approach to Optimization of Mixtures for Transfection of Eukaryotic Cells

A new approach to optimization of mixtures for the condensation and introduction of plasmid DNA into eukaryotic cells is proposed, which is based on the formation of ternary interpolyelectrolyte complexes (IPEC) DNA/polycation/polyanion. Polyethyleneimine (PEI) with M30–40 kDa as polycation and polyacrylic acid (PA) with M20 kDa or its grafted copolymer with polyethyleneglycol (PEG) as polyanion were used, and ternary complexes with various ratios of the components were prepared. The PA–PEG incorporation into a ternary complex (by itself or as a 1 : 1 mixture with PA) was shown to confer the solubility onto complexes in a wide range of DNA/PEI ratios. Incorporation of even minute amounts of PA–PEG (as a 1 : 9 mixture with PA), while not completely preventing the aggregation of ternary IPEC, drastically changed their sorption characteristics. Using a -galactosidase-encoding plasmid, efficiencies of transfection of the CHO-AA8 and 293 cells for different IPEC and DNA/lipofectin complex were compared. The maximum efficiency was exhibited by ternary complex DNA/PEI/polyanion where a 1 : 1 mixture of PA and PA–PEG was used as polyanion. Possible reasons for this effect and further ways of optimization of mixtures for expression of plasmid DNA in the context of the new approach are discussed.     

Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation.

The crystal structures of two ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I (Klentaq1) with a primer/template DNA and dideoxycytidine triphosphate, and that of a binary complex of the same enzyme with a primer/template DNA, were determined to a resolution of 2.3, 2.3 and 2.5 A, respectively. One ternary complex structure differs markedly from the two other structures by a large reorientation of the tip of the fingers domain. This structure, designated 'closed', represents the ternary polymerase complex caught in the act of incorporating a nucleotide. In the two other structures, the tip of the fingers domain is rotated outward by 46 degrees ('open') in an orientation similar to that of the apo form of Klentaq1. These structures provide the first direct evidence in DNA polymerase I enzymes of a large conformational change responsible for assembling an active ternary complex.     

The RuvAB branch migration complex can displace topoisomerase IV.quinolone.DNA ternary complexes

Quinolone antimicrobial drugs target both DNA gyrase and topoisomerase IV (Topo IV) and convert these essential enzymes into cellular poisons. Topoisomerase poisoning results in the inhibition of DNA replication and the generation of double-strand breaks. Double-strand breaks are repaired by homologous recombination. Here, we have investigated the interaction between the RuvAB branch migration complex and the Topo IV.quinolone.DNA ternary complex. A strand-displacement assay is employed to assess the helicase activity of the RuvAB complex in vitro. RuvAB-catalyzed strand displacement requires both RuvA and RuvB proteins, and it is stimulated by a 3'-non-hybridized tail. Interestingly, Topo IV.quinolone.DNA ternary complexes do not inhibit the translocation of the RuvAB complex. In fact, Topo IV.quinolone.DNA ternary complexes are reversed and displaced from the DNA upon their collisions with the RuvAB complex. These results suggest that the RuvAB branch migration complex can actively remove quinolone-induced covalent topoisomerase.DNA complexes from DNA and complete the homologous recombination process in vivo.     

DNA gyrase-mediated wrapping of the DNA strand is required for the replication fork arrest by the DNA gyrase-quinolone-DNA ternary complex

The ability of DNA gyrase (Gyr) to wrap the DNA strand around itself allows Gyr to introduce negative supercoils into DNA molecules. It has been demonstrated that the deletion of the C-terminal DNA-binding domain of the GyrA subunit abolishes the ability of Gyr to wrap the DNA strand and catalyze the supercoiling reaction (Kampranis, S. C., and Maxwell, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14416-14421). By using this mutant Gyr, Gyr (A59), we have studied effects of Gyr-mediated wrapping of the DNA strand on its replicative function and its interaction with the quinolone antibacterial drugs. We find that Gyr (A59) can support oriC DNA replication in vitro. However, Gyr (A59)-catalyzed decatenation activity is not efficient enough to complete the decatenation of replicating daughter DNA molecules. As is the case with topoisomerase IV, the active cleavage and reunion activity of Gyr is required for the formation of the ternary complex that can arrest replication fork progression in vitro. Although the quinolone drugs stimulate the covalent Gyr (A59)-DNA complex formation, the Gyr (A59)-quinolone-DNA ternary complexes do not arrest the progression of replication forks. Thus, the quinolone-induced covalent topoisomerase-DNA complex formation is necessary but not sufficient to cause the inhibition of DNA replication. We also assess the stability of ternary complexes formed with Gyr (A59), the wild type Gyr, or topoisomerase IV. The ternary complexes formed with Gyr (A59) are more sensitive to salt than those formed with either the wild type Gyr or topoisomerase IV. Furthermore, a competition experiment demonstrates that the ternary complexes formed with Gyr (A59) readily disassociate from the DNA, whereas the ternary complexes formed with either the wild type Gyr or topoisomerase IV remain stably bound. Thus, Gyr-mediated wrapping of the DNA strand is required for the formation of the stable Gyr-quinolone-DNA ternary complex that can arrest replication fork progression.     

Elucidation of the mode of interaction in the UP1-telomerase RNA-telomeric DNA ternary complex which serves to recruit telomerase to telomeric DNA and to enhance the telomerase activity

We found that UP1, a proteolytic product of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), both enhances and represses the telomerase activity. The formation of the UP1–telomerase RNA–telomeric DNA ternary complex was revealed by a gel retardation experiment. The interactions in the ternary and binary complexes were elucidated by NMR. UP1 has two nucleic acid-binding domains, BD1 and BD2. In the UP1–telomerase RNA binary complex, both BD1 and BD2 interact with telomerase RNA. Interestingly, when telomeric DNA was added to the binary complex, telomeric DNA bound to BD1 in place of telomerase RNA. Thus, BD1 basically binds to telomeric DNA, while BD2 mainly binds to telomerase RNA, which resulted in the formation of the ternary complex. Here, UP1 bridges telomerase and telomeric DNA. It is supposed that UP1/hnRNP A1 serves to recruit telomerase to telomeric DNA through the formation of the ternary complex. A model has been proposed for how hnRNP A1/UP1 contributes to enhancement of the telomerase activity through recruitment and unfolding of the quadruplex of telomeric DNA.     

Crystallization of the yeast MATalpha2/MCM1/DNA ternary complex: general methods and principles for protein/DNA cocrystallization

We describe our efforts to crystallize binary MCM1/DNA and ternary MATalpha2/MCM1/DNA complexes, including the unsuccessful attempts to crystallize MCM1/DNA complexes and the successful design of DNA crystal packing that resulted in high-resolution crystals of the MATalpha2/MCM1/DNA complex. We detail general procedures useful for preparing protein/DNA cocrystals, including improved methods for producing and purifying DNA-binding proteins and DNA fragments, for purifying protein/DNA complexes, and for controlling pH conditions during crystallization. We also describe the rational design of DNA for protein/DNA cocrystallization attempts, based on our analysis of how straight and bent DNA with single base-pair overhangs can pack end-to-end in a crystal.     

DNA chain length dependence of formation and dynamics of hMutSalpha.hMutLalpha.heteroduplex complexes.

Formation of a ternary complex between human MutSalpha, MutLalpha, and heteroduplex DNA has been demonstrated by surface plasmon resonance spectroscopy and electrophoretic gel shift methods. Formation of the hMutLalpha.hMutSalpha.heteroduplex complex requires a mismatch and ATP hydrolysis, and depends on DNA chain length. Ternary complex formation was supported by a 200-base pair G-T heteroduplex, a 100-base pair substrate was somewhat less effective, and a 41-base pair heteroduplex was inactive. As judged by surface plasmon resonance spectroscopy, ternary complexes produced with the 200-base pair G-T DNA contained approximately 0.8 mol of hMutLalpha/mol of heteroduplex-bound hMutSalpha. Although the steady-state levels of the hMutLalpha.hMutSalpha. heteroduplex were substantial, this complex was found to turn over, as judged by surface plasmon resonance spectroscopy and electrophoretic gel shift analysis. With the former method, the majority of the complexes dissociated rapidly upon termination of protein flow, and dissociation occurred in the latter case upon challenge with competitor DNA. However, ternary complex dissociation as monitored by gel shift assay was prevented if both ends of the heteroduplex were physically blocked with streptavidin.biotin complexes. This observation suggests that, like hMutSalpha, the hMutLalpha.hMutSalpha complex can migrate along the helix contour to dissociate at DNA ends.     

Single-cycle kinetic analysis of ternary DNA complexes by surface plasmon resonance on a decaying surface

Complexes involving three DNA strands were used to demonstrate that the single-cycle kinetics (SCK) method, which consists in injecting sequentially samples at increasing concentrations and until now used exclusively to investigate bimolecular complexes by surface plasmon resonance, can be extended to the kinetic analysis of ternary complexes. DNA targets, B, were designed with sequences of variable lengths on their 3' sides that recognise a surface-immobilized biotinylated DNA anchor, A. These targets displayed on their 5' sides sequences that recognise DNA oligonucleotides of variable lengths, C, namely the analytes. Combinations of B and C DNA oligonucleotides on A generated ternary complexes each composed of two Watson-Crick helices displaying different kinetic properties. The target-analyte B-C duplexes were formed by sequentially injecting three increasing concentrations of the analytes C during the dissociation phase of the target B from the anchor A. The sensorgrams for the target-analyte complexes dissociating from the functionalized surface were successfully fitted by the SCK method while the target dissociated from the anchor, i.e. on a decaying surface. Within the range of applicability of the method which is driven by the rate of dissociation of the target from the anchor, the rate and equilibrium constants characteristic of these target-analyte duplexes of the ternary complexes did not depend on how fast the targets dissociated from the immobilized DNA anchor. In addition the results agreed very well with those obtained when such duplexes were analysed directly as bimolecular complexes, i.e. when the target, modified with a biotin, was directly immobilized onto a streptavidin sensor chip surface rather than captured by an anchor. Therefore the method we named SCKODS (Single-Cycle Kinetics On a Decaying Surface) can also be used to investigate complexes formed during a dissociation phase, in a ternary complex context. The SCKODS method can be combined with the SCK one to fully characterize the two bimolecular complexes of a ternary complex.     

Molecular dynamics simulations show altered secondary structure of clawless in binary complex with DNA providing insights into aristaless-clawless-DNA ternary complex formation

Aristaless (Al) and clawless (Cll) homeodomains that are involved in leg development in Drosophila melanogaster are known to bind cooperatively to 5′-(T/C)TAATTAA(T/A)(T/A)G-3′ DNA sequence, but the mechanism of their binding to DNA is unknown. Molecular dynamics (MD) studies have been carried out on binary, ternary, and reconstructed protein–DNA complexes involving Al, Cll, and DNA along with binding free energy analysis of these complexes. Analysis of MD trajectories of Cll–3A01, binary complex reveals that C-terminal end of helixIII of Cll, unwind in the absence of Al and remains so in reconstructed ternary complex, Cll–3A01–Al. In addition, this change in secondary structure of Cll does not allow it to form protein–protein interactions with Al in the ternary reconstructed complex. However, secondary structure of Cll and its interactions are maintained in other reconstructed ternary complex, Al–3A01–Cll where Cll binds to Al–3A01, binary complex to form ternary complex. These interactions as observed during MD simulations compare well with those observed in ternary crystal structure. Thus, this study highlights the role of helixIII of Cll and protein–protein interactions while proposing likely mechanism of recognition in ternary complex, Al–Cll–DNA.     

MSH-MLH complexes formed at a DNA mismatch are disrupted by the PCNA sliding clamp

In the yeast Saccharomyces cerevisiae, mismatch repair (MMR) is initiated by the binding of heterodimeric MutS homolog (MSH) complexes to mismatches that include single nucleotide and loop insertion/deletion mispairs. In in vitro experiments, the mismatch binding specificity of the MSH2-MSH6 heterodimer is eliminated if ATP is present. However, addition of the MutL homolog complex MLH1-PMS1 to binding reactions containing MSH2-MSH6, ATP, and mismatched substrate results in the formation of a stable ternary complex. The stability of this complex suggests that it represents an intermediate in MMR that is subsequently acted upon by other MMR factors. In support of this idea, we found that the replication processivity factor proliferating cell nuclear antigen (PCNA), which plays a critical role in MMR at step(s) prior to DNA resynthesis, disrupted preformed ternary complexes. These observations, in conjunction with experiments performed with streptavidin end-blocked mismatch substrates, suggested that PCNA interacts with an MSH-MLH complex formed on DNA mispairs.