Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin, its 3-N analog and their metallocomplexes with duplex DNA

Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin (TOEPyP(4)), its 3-N analog (TOEPyP(3)) and their Co, Cu, Ni, Zn metallocomplexes with duplex DNA have been investigated by uv/visible absorbance and circular dichrosim spectroscopies. Results reveal the interactions of these complexes with duplex DNA are of two types. (1) External binding of duplex DNA by metalloporphyrins containing Zn and Co, and (2) Binding of duplex DNA both externally and internally (by intercalation) by porphyrins not containing metals, and metalloporphyrins containing Cu and Ni. Results indicate that (4N-oxyethylpyridyl) porphyrins intercalate more preferably in the structure of duplex DNA and have weaker external binding than 3N-porphyrins.     

Interactions of Meso-tetra-(4-N-oxyethylpyridyl) Porphyrin,Its 3-N Analog and Their Metallocomplexes with Duplex DNA

Abstract

Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin (TOEPyP(4)), its 3-N analog (TOEPyP(3)) and their Co, Cu, Ni, Zn metallocomplexes with duplex DNA have been investigated by uv/visible absorbance and circular dichrosim spectroscopies. Results reveal the interactions of these complexes with duplex DNA are of two types. (1) External binding of duplex DNA by metalloporphyrins containing Zn and Co, and (2) Binding of duplex DNA both externally and internally (by intercalation) by porphyrins not containing metals, and metalloporphyrins containing Cu and Ni. Results indicate that (4N-oxyethylpyridyl) porphyrins intercalate more preferably in the structure of duplex DNA and have weaker external binding than 3N-porphyrins.     

The effect of poly(ADP-ribose) on interactions of DNA with histones H1, H3 and H4

The competition between poly(ADP-ribose) and DNA for binding of the histones H1, H3 and H4 was studied, using a membrane filter-binding test. Poly(ADP-ribose) differently affected the interaction between DNA and the individual histones. While poly(ADP-ribose) effectively competed with DNA for binding of histone H4, it equally competed with DNA for binding of histone H3 and only inefficiently competed with DNA for binding of histone H1. Moreover, preformed complexes were correspondingly affected by the addition of competing polynucleotides, thereby also indicating the reversibility of complex formation. The competition capacity of DNA for histone H4 binding did not depend on DNA size. Competition experiments with poly(A) also indicated that poly(ADP-ribose) preferentially affected DNA-histone H4 interaction. The significance of the differing binding properties is discussed with regard to the possible molecular function of poly(ADP-ribose), especially with regard to its potential effect on nucleosome structure.     

A putative role of Drep1 in apoptotic DNA fragmentation system in fly is mediated by direct interaction with Drep2 and Drep4

DNA fragmentation is common phenomenon for apoptotic cell death. DNA fragmentation factor, called DFF40 (CAD: mouse homologue), is a main nuclease for apoptotic DNA fragmentation. Nuclease activity of DFF40 is normally inhibited by DFF45 by tight interaction via CIDE domain without apoptotic stimuli. Once effector caspase is activated during apoptosis signaling, it cleave DFF45, allowing DFF40 to enter the nucleus and cleave chromosomal DNA. Unlike mammalian system, apoptotic DNA fragmentation in the fly might be controlled by four DFF-related proteins, known as Drep1, Drep2, Drep3 and Drep4. Although the function of Drep1 and Drep4 is well known as DFF45 and DFF40 homologues, respectively, the function of Drep2 and Drep3 is still unclear. DFF-related proteins contain a conserved CIDE domain of ~90 amino acid residues that is involved in protein–protein interaction. Here, we showed that Drep1 directly bind to Drep2 as well as Drep4 via CIDE domain. In addition, we found that the interaction of Drep2 and Drep4 to Drep1 was not competitive indicating that Drep2 and Drep4 bind different place of Drep1. All together, we suggest that Drep1 might be involved in apoptotic DNA fragmentation of fly system by direct interaction with Drep2 as well as Drep4.     

Interaction of double-stranded T4 DNA with cationic gel of poly(diallyldimethylammonium chloride)

Interaction between duplex T4 DNA and a slightly cross-linked cationic gel of poly(diallyldimethylammonium chloride) in aqueous media was studied by fluorescent microscopy. While short DNA chains such as plasmid DNAs penetrate into the gel and form a phase of polyelectrolyte complex with the cationic network, the genomic giant DNA chains of T4 phages form complexes only on local areas of the gel surface. The DNA/gel complex exhibited different characteristic morphologies depending on the conditions for preparing the complex, such as the DNA concentration, flux of the solution, and surface geometry of the gel: (1) In the interaction with the flat surface of film-type gel, compact round objects, which reflected a condensed state of single DNA chains, were observed. (2) In the interaction with partly dried gel, a characteristic pattern similar to propagating waves was formed on the gel surface. (3) When flux is generated for a concentrated DNA solution, long oriented fiberlike structures were formed, which consisted of ensembles of chains. The interaction with small pieces of mechanically decomposed gel leads to complete covering of their surface by the DNA.     

Tetraplex structure of fission yeast telomeric DNA and unfolding of the tetraplex on the interaction with telomeric DNA binding protein Pot1

To understand the regulation mechanism of fission yeast telomeric DNA, we analysed the structural properties of Gn: d(GnTTAC) (n=2-6) and 4Gn: d(GnTTAC)4 (n=3 and 4), and their interaction with the single-stranded telomeric DNA binding domain of telomere-binding protein Pot1 (Pot1DBD). G4, G5 and G6 formed a parallel tetraplex in contrast with no tetraplex formation by G2 and G3. Also, 4G4 adopted only an antiparallel tetraplex in spite of a mixture of parallel and antiparallel tetraplexes of 4G3. The variety of tetraplex structures was governed by the number of consecutive guanines in a single copy and the number of repeats. The antiparallel tetraplex of 4G4 became unfolded upon the interaction with Pot1DBD. The interaction with mutant Pot1DBD proteins revealed that the ability to unfold the antiparallel tetraplex was strongly correlated with the specific binding affinity for the single-stranded telomeric DNA. The result suggests that the decrease in the free single strand upon the complex formation with Pot1DBD may shift the equilibrium from the tetraplex to the single strand, which may cause the tetraplex unfolding. Considering that the antiparallel tetraplex inhibits telomerase-mediated telomere elongation, we conclude that the ability of Pot1 to unfold the antiparallel tetraplex is required for telomerase-mediated telomere regulation.     

Molecular mechanisms of chemical mutagenesis: 9-aminoacridine inhibits DNA replication in vitro by destabilizing the DNA growing point and interacting with the DNA polymerase

9-Aminoacridine was found to inhibit dNTP incorporation into DNA homopolymer duplexes by phage T4 DNA polymerase in vitro. Systematic variation of the molar ratio of 9-aminoacridine to DNA, to DNA polymerase, and to DNA precursors demonstrated that this inhibition at 9-aminoacridine concentrations below 10 microM was mainly due to interaction of 9-aminoacridine with the DNA and suggested that the basis for the preferential inhibition of incorrect precursor incorporation was destabilization of the DNA growing point. Consistent with destabilization, 9-aminoacridine stimulated the hydrolysis of correctly base paired DNA by the 3'-5' exonuclease activity of phage T4 DNA polymerase. This is the first indication to my knowledge that an intercalating dye destabilizes the DNA growing point, whereas it raises the overall Tm of the DNA. At 9-aminoacridine concentrations above 10 microM overall incorporation of dNTPs was inhibited by 9-aminoacridine interaction with the DNA polymerase. A possible explanation for the induction of both deletion and addition frameshift mutations by 9-aminoacridine during DNA biosynthesis is discussed in light of growing-point destabilization.     

Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV

Nonhomologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells. A critical step in this process is DNA ligation, involving the Xrcc4-DNA ligase IV complex. DNA end processing is often a prerequisite for ligation, but the coordination of these events is poorly understood. We show that polynucleotide kinase (PNK), with its ability to process ionizing radiation-induced 5'-OH and 3'-phosphate DNA termini, functions in NHEJ via an FHA-dependent interaction with CK2-phosphorylated Xrcc4. Analysis of the PNK FHA-Xrcc4 interaction revealed that the PNK FHA domain binds phosphopeptides with a unique selectivity among FHA domains. Disruption of the Xrcc4-PNK interaction in vivo is associated with increased radiosensitivity and slower repair kinetics of DSBs, in conjunction with a diminished efficiency of DNA end joining in vitro. Therefore, these results suggest a new role for Xrcc4 in the coordination of DNA end processing with DNA ligation.     

Preparation of photoreactive oligonucleotide duplexes and their application for photoaffinity modification of DNA-binding proteins]

To introduce photoreactive dNTP residues to the 3'-end of a mononucleotide gap, base-substituted photoreactive deoxynucleoside triphosphate derivatives, (5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-trans-3-aminopropenyl-1]- and 5-(N-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]- trans-3-aminopropenyl-1)-2'-deoxyuridine 5'-triphosphates, were used as substrates in the DNA polymerase beta-catalyzed reaction. The resulting nick, containing a modified base at the 3'-end, was sealed by T4 phage DNA ligase. This approach enables the preparation of DNA duplexes bearing photoreactive groups at predetermined position(s) of the nucleotide chain. Using the generated photoreactive DNA duplexes, the photoaffinity modifications of DNA polymerase beta and human replicative protein A (hRPA) were carried out. It was shown that DNA polymerase beta and hRPA subunits were modified with the photoreactive double-stranded DNA considerably less effectively than by the nicked DNA. In the case of double-stranded DNA, the hRPA p70 subunit was preferentially labeled, implying a crucial role of this subunit in the protein-DNA interaction.     

Bundling and aggregation of DNA by cationic dendrimers

Dendrimers are unique synthetic macromolecules of nanometer dimensions with a highly branched structure and globular shape. Among dendrimers, polyamidoamine (PAMAM) have received most attention as potential transfection agents for gene delivery, because these macromolecules bind DNA at physiological pH. The aim of this study was to examine the interaction of calf-thymus DNA with several dendrimers of different compositions, such as mPEG-PAMAM (G3), mPEG-PAMAM (G4), and PAMAM (G4) at physiological conditions, using constant DNA concentration and various dendrimer contents. FTIR, UV-visible, and CD spectroscopic methods, as well as atomic force microscopy (AFM), were used to analyze the macromolecule binding mode, the binding constant, and the effects of dendrimer complexation on DNA stability, aggregation, condensation, and conformation. Structural analysis showed a strong dendrimer-DNA interaction via major and minor grooves and the backbone phosphate group with overall binding constants of K(mPEG-G3) = 1.5 (±0.5) × 10(3) M(-1), K(mPEG-G4) = 3.4 (±0.80) × 10(3) M(-1), and K(PAMAM-G4) = 8.2 (±0.90) × 10(4) M(-1). The order of stability of polymer-DNA complexation is PAMAM-G4 > mPEG-G4 > mPEG-G3. Both hydrophilic and hydrophobic interactions were observed for dendrimer-DNA complexes. DNA remained in the B-family structure, while biopolymer particle formation and condensation occurred at high dendrimer concentrations.     

Molecular interaction and synergistic activation of a promoter by Six,Eya, and Dach proteins mediated through CREB binding protein

Drosophila sine oculis, eyes absent, and dachshund are essential for compound eye formation and form a gene network with direct protein interaction and genetic regulation. The vertebrate homologues of these genes, Six, Eya, and Dach, also form a similar genetic network during muscle formation. To elucidate the molecular mechanism underlying the network among Six, Eya, and Dach, we examined the molecular interactions among the encoded proteins. Eya interacted directly with Six but never with Dach. Dach transactivated a multimerized GAL4 reporter gene by coproduction of GAL4-Eya fusion proteins. Transactivation by Eya and Dach was repressed by overexpression of VP16 or E1A but not by E1A mutation, which is defective for CREB binding protein (CBP) binding. Recruitment of CBP to the immobilized chromatin DNA template was dependent on FLAG-Dach and GAL4-Eya3. These results indicate that CBP is a mediator of the interaction between Eya and Dach. Contrary to our expectations, Dach binds to chromatin DNA by itself, not being tethered by GAL4-Eya3. Dach also binds to naked DNA with lower affinity. The conserved DD1 domain is responsible for binding to DNA. Transactivation was also observed by coproduction of GAL4-Six, Eya, and Dach, indicating that Eya and Dach synergy is relevant when Eya is tethered to DNA through Six protein. Our results demonstrated that synergy is mediated through direct interaction of Six-Eya and through the interaction of Eya-Dach with CBP and explain the molecular basis for the genetic interactions among Six, Eya, and Dach. This work provides fundamental information on the role and the mechanism of action of this gene cassette in tissue differentiation and organogenesis.     

Dual apoptotic DNA fragmentation system in the fly: Drep2 is a novel nuclease of which activity is inhibited by Drep3

DNA fragmentation is the hallmark of apoptotic cells and mainly mediated by the DNA fragmentation factor DFF40(CAD)/DFF45(ICAD). DFF40 is a novel nuclease, whereas DFF45 is an inhibitor that can suppress the nuclease activity. Apoptotic DNA fragmentation in the fly is controlled by four DFF-related proteins, known as Drep1, 2, 3 and 4. However, the functions of Drep2 and Drep3 are totally unknown. Here, we found that Drep2 is a novel nuclease whose activity is inhibited by Drep3 through a tight interaction with the CIDE domain. Our results suggest that the fly has dual apoptotic DNA fragmentation systems: Drep1: Drep4 and Drep2: Drep3 complexes.Structured summary of protein interactionsDrep2 CIDE and Drep-3 CIDE bind by blue native page (View interaction)Drep2 CIDE and Drep-3 CIDE bind by molecular sieving (View interaction)     

Investigation of the interaction between DNA and cobalt ferrite nanoparticles by FTIR spectroscopy

The interaction of DNA with nanoparticles of cobalt ferrite powder prepared by the mechano-chemical method was studied. It was shown that CoFe₂O₄ nanoparticles efficiently bind DNA in aqueous solutions (Tris-HCl), forming a bionanocomposite. The adsorption capacity of CoFe₂O₄ nanoparticles for DNA was evaluated to be 5.25 × 10⁻³ mol/m². The desorption of DNA from the surface of the particles was analyzed while changing the pH, the ionic strength, and the chemical content of the medium. The DNA-CoFe₂O₄ nanocomposite was investigated by FTIR spectroscopy. The block of the data allowed one to consider the mechanism of the interaction between a polynucleotide and CoFe₂O₄ nanoparticles and to make the assumption that the binding occurred due to the coordination interaction of the phosphate groups and heterocyclic bases of DNA (oxygen atoms of thymine and guanine) with metal ions on the particle surface. The analysis of the IR spectra showed that binding can lead to the partial destabilization of the DNA structure, with the B conformation of a polynucleotide being preserved.     

Site-directed polyclonal antibodies inhibit binding of activated estrogen receptor to DNA

We have generated three polyclonal antisera to the DNA-binding domain of the human estrogen receptor (hER). Antiserum AT2A was generated against a peptide spanning amino acids 231-245 of hER, while antisera AT3A and AT3B were generated against a peptide spanning amino acids 247-261 of hER. The interaction of these three antisera with ER has been characterized by sucrose density gradient analysis. The antisera bound to the unactivated (8S), salt-activated (4S), and heat transformed (5S) ER complex. All the antisera appeared to be site-specific since binding of salt-activated ER to the antisera was blocked by the presence of excess free synthetic peptides. Antisera AT3A and AT3B inhibited the binding to DNA of the KCl-activated 4S ER and the heat-transformed 5S ER. Although antiserum AT2A bound to ER, it did not inhibit DNA binding of activated ER complexes. The ability of antisera AT3A and AT3B to inhibit ER binding to DNA was concentration dependent. Once bound to the DNA, ER complexes were not significantly affected by incubation with the antisera, suggesting that binding of DNA to ER inhibits antibody ER interaction and renders that domain inaccessible to the antibodies. These results demonstrate that site-directed antibodies to ER inhibit binding of activated ER complexes to DNA in vitro.     

Comparison of the primary structure of reconstructed minimal nucleosomes and nucleosomes isolated from the chromatin

We have reconstructed nucleosomes from a histone octamer (H2A, H2B, H3, H4)2 and DNA 146 b.p. or 2-3 thousands b.p. in length. Comparison by means of DNA-histone cross-links of the primary organization of minimal nucleosomes obtained by reconstruction or isolated from chromatin of chicken erythrocyte nuclei has demonstrated a high similarity in histone location on their DNAs. Simultaneously, there have been observed some variations in the character of interaction for all core histones with DNA on nucleosomes. Thus, the cross-link of histone H4 with DNA of a core particle at H4 sites (65), unlike H4(55) and H4(88) sites, significantly depends on the superstructure of chromatin, ionic strength of solution and the presence of denaturating agents. All these differences are expected to probe the existence of conformational isomers for core particles. (Bracketed is the distance from the histone interaction site with the DNA of the core particle to the DNA 5'-terminus.)     

Loss of H3 K79 Trimethylation Leads to Suppression of Rtt107-dependent DNA Damage Sensitivity through the Translesion Synthesis Pathway

Genomic integrity is maintained by the coordinated interaction of many DNA damage response pathways, including checkpoints, DNA repair processes, and cell cycle restart. In Saccharomyces cerevisiae, the BRCA1 C-terminal domain-containing protein Rtt107/Esc4 is required for restart of DNA replication after successful repair of DNA damage and for cellular resistance to DNA-damaging agents. Rtt107 and its interaction partner Slx4 are phosphorylated during the initial phase of DNA damage response by the checkpoint kinases Mec1 and Tel1. Because the natural chromatin template plays an important role during the DNA damage response, we tested whether chromatin modifications affected the requirement for Rtt107 and Slx4 during DNA damage repair. Here, we report that the sensitivity to DNA-damaging agents of rtt107Δ and slx4Δ mutants was rescued by inactivation of the chromatin regulatory pathway leading to H3 K79 trimethylation. Further analysis revealed that lack of Dot1, the H3 K79 methyltransferase, led to activation of the translesion synthesis pathway, thereby allowing the survival in the presence of DNA damage. The DNA damage-induced phosphorylation of Rtt107 and Slx4, which was mutually dependent, was not restored in the absence of Dot1. The antagonistic relationship between Rtt107 and Dot1 was specific for DNA damage-induced phenotypes, whereas the genomic instability caused by loss of Rtt107 was not rescued. These data revealed a multifaceted functional relationship between Rtt107 and Dot1 in the DNA damage response and maintenance of genome integrity.     

Interaction between SV40 DNA and camptothecin, an antitumor alkaloid

Camptothecin specifically interacted with closed superhelical circular SV40 DNA during incubation in 1.0 M NaCl at 37 degrees C and induced an alkali-labile linkage in the E strand. No interaction occurred in the reaction mixture containing 0.1 M NaCl, or at 4 degrees C. As camptothecin did not affect linear SV40 DNA, the superhelical structure of DNA appeared to be essential. The site of the alkali-labile linkage induced in SV40 DNA I through interaction with camptothecin was near the origin of replication on the basis of the results of experiments with restriction enzymes. Neither sulfhydryl reagents nor EDTA affected the interaction between camptothecin and SV40 DNA I, so the action of camptothecin is different from those of antitumor antibiotics, bleomycin or neocarzinostatin. Analysis of the s20,0w value of SV40 DNA I after the interaction with camptothecin and the sedimentation profiles of DNA after heating in the reaction mixture indicated that the interaction between camptothecin and SV40 DNA I was different from those of intercalating or alkylating agents such as ethidium bromide and methylmethanesulfonate. Replacement of the OH group at C-20 in the E ring of camptothecin by H-, CH3-, and Cl- resulted in the reduction, in this order, of the potency for interaction with SV40 DNA I to induce an alkali-labile linkage.     

Fission yeast telomeric DNA binding protein Pot1 has the ability to unfold tetraplex structure of telomeric DNA

To understand the regulation mechanism of fission yeast telomeric DNA, we analyzed the structural properties of 4Gn: d(G(n)TTAC)(4) (n = 3, 4) and their interaction with the single-stranded telomeric DNA binding domain of telomere-binding protein Pot1 (Pot1DBD). 4G4 adopted only an antiparallel tetraplex in spite of a mixture of parallel and antiparallel tetraplexes of 4G3. The antiparallel tetraplex of 4G4 became unfolded upon the interaction with Pot1DBD. Considering that the antiparallel tetraplex inhibits telomerase-mediated telomere elongation, we conclude that the ability of Pot1 to unfold the antiparallel tetraplex is required for telomerase-mediated telomere regulation.