PKC SUMOylation inhibits the binding of 14–3–3τ to GluK2

Phosphorylation and SUMOylation of the kainate receptor (KAR) subunit GluK2 have been shown to regulate KAR surface expression, trafficking and synaptic plasticity. In addition, our previous study has shown that a phosphorylation-dependent interaction of 14–3–3τ and GluK2a-containing receptors contributes to the slow decay kinetics of native KAR-EPSCs. However, it is unknown whether SUMOylation participates in the regulation of the interaction between 14–3–3τ and GluK2a-containing receptors. Here we report that SUMOylation of PKC, but not GluK2, represses the binding of 14–3–3τ to GluK2a via decreasing the phosphorylation level of GluK2a. These results suggest that PKC SUMOylation is an important regulator of the 14–3–3 and GluK2a protein complex and may contribute to regulate the decay kinetics of KAR-EPSCs.     

Dynamics of drug-DNA interactions: a comparative temperature jump study of ellipticinium and 9-hydroxy ellipticinium

The temperature-jump method has been used to compare the binding of 2-N methyl ellipticinium (NME) and 2-N methyl 9 hydroxy ellipticinium (NMHE) to three natural DNA's of different AT/GC composition. The relaxation signals, analyzed by the Padé-Laplace method, are characterized by two distinct relaxation times, tau 1 and tau 2, respectively in the 1-4 ms and 20-80 ms range. In the case of the NMHE/DNA interaction, the slower relaxation time tau 2 depends on the DNA composition, as follows: tau 2 (Micrococcus lysodeikticus) greater than tau 2 (Calf thymus) greater than tau 2 (Clostridium perfringens). Contrary to NMHE, NME which does not possess an OH group at the C-9 position, shows no relaxation time dependence upon DNA base composition. The observation of two relaxation times indicates that the binding equilibria are associated with at least two distinct drug/DNA complexes (probably arising from two distinct DNA binding sites). Three kinetic models, involving the formation of a weak intermediate ionic complex, are given to explain the binding reaction between these cationic drugs and the DNA. They allow the determination of the four rate constants associated with the two binding steps and lead to equilibrium association constants in agreement with those obtained from spectroscopic studies. The validity of the models is discussed and it is shown that the best kinetic scheme, for either NMHE or NME, could be that in which the ionic step is not a prerequiste to intercalation. The kinetic results show that the residence time of 9 hydroxy ellipticinium is markedly increased in GC rich DNA's and this could be related to the higher in vitro and in vivo cytotoxic properties of 9 hydroxy substituted ellipticines.     

Kinetic manifestations of slow conformation rearrangements of lactate dehydrogenase. An experiment and a mathematical model

Non-steady-state kinetics of lactate dehydrogenase (LDH) catalyzed reaction was investigated for a wide time interval (from 100 msec to 1-3 min) by using stopped-flow methods. A two-stage character of LDH reaction, slow changes like a lag-period on kinetic curves at pH 8.0, flexions on kinetic curves after pre-mixing LDH with NAD+ and pyruvate have been revealed. The graph theory for mathematical analysis of experimental data was applied, which has been developed for the non-steady-state kinetics. An enzyme model of the two-conformer LDH structure was used. The reaction scheme with a preferential inhibition of one of the conformers (pH 8.0) is suggested. The obtained values of kinetic constants prove that transitions between LDH conformers must be slow.     

The N-terminal region of the bacterial DNA polymerase PolC features a pair of domains, both distantly related to domain V of the DNA polymerase III τ subunit

PolC is one of two essential replicative DNA polymerases in Bacillus subtilis and other Gram-positive bacteria. The 3D structure of PolC has recently been solved, yet it lacks the N-terminal region. For this PolC region of ～ 230 residues, both the structure and function are unknown. In the present study, using sensitive homology detection and comparative protein structure modeling, we identified, in this enigmatic region, two consecutive globular domains, PolC-NI and PolC-NII, which are followed by an apparently unstructured linker. Unexpectedly, we found that both domains are related to domain V of the τ subunit, which is part of the bacterial DNA polymerase III holoenzyme. Despite their common homology to τ, PolC-NI and PolC-NII exhibit very little sequence similarity to each other. This observation argues against simple tandem duplication within PolC as the origin of the two-domain structure. Using the derived structural models, we analyzed residue conservation and the surface properties of both PolC N-terminal domains. We detected a surface patch of positive electrostatic potential in PolC-NI and a hydrophobic surface patch in PolC-NII, suggesting their possible involvement in nucleic acid and protein binding, respectively. PolC is known to interact with the τ subunit, however, the region responsible for this interaction is unknown. We propose that the PolC N-terminus is involved in mediating the PolC-τ interaction and possibly also in binding DNA.     

Optimal conditions and specificity of interaction of a distinct class of nonhistone chromosomal proteins with DNA.

A subclass of nonhistone chromatin proteins with high DNA affinity has been isolated from rat liver. The interaction of the isolated proteins with DNA in vitro was characterized utilizing a nitrocellulose filter binding technique. The temperature, time, concentration, ionic strength, and pH dependence were characterized. Optimal interaction was observed at 0.19 M naCl, pH 7.5 with a protein to DNA ratio of 13 (w/w). Equilibrium and kinetic competition experiments indicated that these proteins interact optimally with A-T rich and single-stranded DNA. The data also suggest that these proteins might affect the helixcoil transiton of DNA.     

Effect of the molecular structure of anthracycline compounds on their intercalation into DNA

Kinetics of interaction of rubomycin, carminomycin, and their C9-analogues with deoxyribonucleic acid have been studied by the stop - flow method. Possible reasons of nonexponentiality of the kinetic curves are discussed. The rate constants of direct and reverse intercalation of the drugs between DNA base pairs were determined and relatively small differences found for various drugs. These data and of equilibrium binding studies of interaction of the antibiotics and their analogs with DNA led to conclusion that C9-modification of anthracyclines has little effect on the binding process. That was confirmed by quantum-mechanic calculations of potential energy of interaction between various anthracycline aglicones and DNA base-pairs.     

A minimal kinetic model for a viral DNA packaging machine

Terminase enzymes are common to both eukaryotic and prokaryotic double-stranded DNA viruses. These enzymes possess ATPase and nuclease activities that work in concert to "package" a viral genome into an empty procapsid, and it is likely that terminase enzymes from disparate viruses utilize a common packaging mechanism. Bacteriophage lambda terminase possesses a site-specific nuclease activity, a so-called helicase activity, a DNA translocase activity, and multiple ATPase catalytic sites that function to package viral DNA. Allosteric interactions between the multiple catalytic sites have been reported. This study probes these catalytic interactions using enzyme kinetic, photoaffinity labeling, and vanadate inhibition studies. The ensemble of data forms the basis for a minimal kinetic model for lambda terminase. The model incorporates an ADP-driven conformational reorganization of the terminase subunits assembled on viral DNA, which is central to the activation of a catalytically competent packaging machine. The proposed model provides a unifying mechanism for allosteric interaction between the multiple catalytic sites of the holoenzyme and explains much of the kinetic data in the literature. Given that similar packaging mechanisms have been proposed for viruses as dissimilar as lambda and the herpes viruses, the model may find general utility in our global understanding of the enzymology of virus assembly.     

Calculation of DNA molecule packing in crystals by the method of atom-atom potentials I. B-form of DNA]

The van-der-Waals interaction of DNA molecules in the B-form has been studied by the method of atom-atom potentials. This study was carried out to clarify to what degree does this interaction determinate packing of DNA molecules in a crystal lattice. Interaction energy of two parallel molecules has been calculated as a function of 4 parameters (interaxial distance, axial shift, screw rotation of molecules). Energy as a function of these parameters is shown to have two minima, one of them exactly corresponding to mutual arrangement of DNA molecules in the lattice determined from X-ray data.     

Energy Landscape for DNA Rotation and Sliding through a Phage Portal

Molecular motors involved in the packaging of DNA in tailed viruses are among the strongest known. The mechanism by which the motors operate has long been speculated to involve a coupling between rotation of the portal pore (the gate through which DNA passes upon its packaging or ejection), and translation of DNA. Recent experimental evidence rules out portal rotation with a substantial degree of certainty. We have created an atomistic model for the interaction between DNA and the portal of the bacteriophage SPP1, on the basis of cryo-electron microscopy images and of a recently solved crystal structure. A free energy surface describing the interaction is calculated using molecular dynamics simulations, and found to be inconsistent with a mechanism in which portal rotation drives DNA import. The low-energy pathways on the surface are used to advance a hypothesis on DNA import compatible with all available experiments. Additionally, temperature-dependent kinetic data are used to validate computed barriers to DNA ejection.     

Surface plasmon resonance study of the molecular recognition between polymerase and DNA containing various mismatches and conformational changes of DNA-protein complexes

Although surface plasmon resonance (SPR) biosensor technique has been used to study protein-protein interactions and to detect conformational changes of proteins, it has not been shown whether the SPR biosensor can be used to study a complex kinetic system such as the protein-DNA binding, which sometimes involves several binding steps as well as dynamic conformational changes of the complexes. In this study, we have used SPR biosensor and T7 polymerase as the model system to study the interactions of the polymerase with a series of DNA template-primer duplexes containing different number of mismatches and GC contents at various positions near the primer 3'-end. In general, the binding constants measured by the SPR are several magnitudes smaller than those determined in solution, indicating the limitation of the surface-based technique for measuring solution-based interactions. However, the distinct polymerase binding profiles obtained for DNA duplexes differed by as low as a single mismatch suggest that the SPR data can be used for relative comparison purpose among a set of experiments carried out under identical conditions. The successful fitting of the binding profiles using the established translocation model also demonstrated that SPR can be used to monitor conformational changes, as well as to derive relative kinetic values, within a complicated DNA-protein interaction system. The results also demonstrated that SPR biosensor may be used as a sensitive technique for studying molecular recognition events, such as single-base discrimination involved in protein-DNA interactions.     

Interaction of the EcoRV restriction endonuclease with the deoxyadenosine and thymidine bases in its recognition hexamer d(GATATC)

A set of dA and T analogues suitable for the study of protein DNA interactions have been incorporated into the central d(ATAT) sequence within d(GACGATATCGTC). The individual analogues have one potential protein contact (either a hydrogen-bonding group or a CH3 group capable of a van der Waals interaction) deleted. In general, the modified bases do not perturb the overall structure of the dodecamer, enabling results obtained to be simply interpreted in terms of loss of protein DNA contacts. We have used the modified oligodeoxynucleotide set to study the recognition of DNA by the EcoRV restriction endonuclease [recognition sequence d(GATATC)]. The kcat and Km values for the set have been determined, and a comparison with results seen with the parent oligodeoxynucleotide (containing no modified bases) has been carried out. Three classes of results are seen. First, some analogues lead to no change in kinetic parameters, meaning no enzyme contact at the altered site. Second (this is seen for most of the modified oligodeoxynucleotides), a drop in the kcat/Km ratio relative to the parent is observed. This comes mainly from a decrease in kcat, implying that the endonuclease uses the interaction under study to lower the transition-state barrier rather than to bind the substrate. Analyses of these results show that the drop in kcat/Km is what would be expected for the simple loss of a hydrogen bond or a CH3 contact between the enzyme and the oligodeoxynucleotide. This implies a contact of these types at these sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction features of adenine DNA glycosylase MutY from <Emphasis Type="Italic">E. coli</Emphasis> with DNA substrates

Kinetic characteristics of specific recognition of damaged base by the DNA glycosylase MutY in model DNA substrates, containing oxoG/A-, G/A-, oxoG/C- and F/G pairs in the central position, were investigated. Conformational changes of the MutY enzyme during the recognition of the damaged base in DNA have been recorded by the change in the fluorescence intensity of tryptophan residues using the stopped-flow technique in real time. DNA duplexes containing a fluorescein residue were used for the registration of DNA conformational changes. Analysis of the kinetic curves allowed us to determine the values of rate constants for the kinetic stages of the interaction. It was shown that nonspecific contacts between the DNA-binding site of the enzyme and the DNA duplex are formed at the first stage of the interaction. It was found that the discrimination of Gua and oxoGua bases occurs at the second stage of the MutY interaction with the DNA duplex. The data obtained for the oxoG/C-substrate showed that the recognition of the base located opposite oxoGua also occurs at this stage.     

Study of the electrostatic effects of mutations on the surface of dehaloperoxidase-hemoglobin A

Point mutations of dehaloperoxidase-hemoglobin A (DHP A) that affect the surface charge have been prepared to study the interaction between DHP A with its substrate 2,4,6-trichlorophenol (TCP). Kinetic studies of these surface mutations showed a correlation, in which the more positively charged mutants have increased catalytic efficiency compared with wild type DHP A. As a result, the hypothesis of this study is that there is a global electrostatic interaction between DHP A and TCP. The electrostatic nature of substrate binding was further confirmed by the result that kinetic assays of DHP A were affected by ionic strength. Furthermore, isoelectric focusing (IEF) gel study showed that the pI-6.8 for DHP A, which indicates that DHP A has a slight negative charge pH 7, consistent with the kinetic observations.     

Mechanism of the inhibition of milk xanthine oxidase activity by metal ions: a transient kinetic study

The nature and mechanism of the inhibition of the oxidoreductase activity of milk xanthine oxidase (XO) by Cu(2+), Hg(2+) and Ag(+) ions has been studied by steady state and stopped flow transient kinetic measurements. The results show that the nature of the inhibition is noncompetitive. The inhibition constants for Cu(2+) and Hg(2+) are in the micromolar and that for Ag(+) is in the nanomolar range. This suggests that the metal ions have strong affinity towards XO. pH dependence studies of the inhibition indicate that at least two ionisable groups of XO are involved in the binding of these metal ions. The effect of the interaction of the metal ions on the reductive and oxidative half reactions of XO has been investigated, and it is observed that the kinetic parameters of the reductive half reaction are not affected by these metal ions. However, the interaction of these metal ions with XO significantly affects the kinetic parameters of the oxidative half reaction. It is suggested that this may be the main cause for the inhibition of XO activity by the metal ions.     

DNA length-dependent cooperative interactions in the binding of Ku to DNA

Despite its central role in the nonhomologous DNA end joining process, we still have an incomplete picture of the interaction between Ku and DNA. Here we describe both kinetic (surface plasmon resonance or SPR) and equilibrium (electrophoretic mobility shift assay or EMSA) studies of Ku binding to linear double-stranded DNA. Ku interaction with 1-site DNA is noncooperative, as expected. Electrophoretic mobility shift assays indicate cooperativity in the binding of Ku molecules to DNA long enough for two Ku molecules to bind (2-site DNA). For the kinetic studies, we use surface plasmon resonance in which one end of the DNA molecules is linked to a surface while the other end is free to interact with Ku. We find that one Ku molecule dissociates from 1-site DNA with simple Langmuir (i.e., independent) kinetics. However, two Ku molecules associate and dissociate from 2-site DNA with a time course that cannot be described as a simple Langmuir interaction. On 3- and 4-site DNA, EMSA and SPR studies do not reveal any cooperativity, suggesting that the middle Ku does not exhibit cooperative interaction with the two Ku molecules bound at the DNA ends. These results indicate that Ku molecules can demonstrate cooperative interaction, and this is influenced by their positions along the DNA.     

Binding of ligands to a one-dimensional heterogeneous lattice. III. Kinetic model and relaxation study of the interaction of tilorone with DNA and polynucleotides

A temperature-jump relaxation study of the interaction of tilorone with different polynucleotides and DNA has been performed. A single relaxation time, attributed to the intercalation step, has been observed in the case of poly[d(A-T)]·poly[d(A-T)], poly[d(A-C)]·poly[d(G-T)], poly[d(G-C)]·poly[d(G-C)], and poly(dG)·poly(dC). No intercalation into poly(dA)·poly(dT) occurs, and the interaction with poly(dG)·poly(dC) is different from what is observed with the other intercalating homopolymers. Refinement of the binding model is suggested from the analysis of the kinetic data. The relaxation curves obtained with DNA are well simulated based on a binding mechanism where DNA is considered a heterogeneous lattice and each type of site behaves as if it were located in the corresponding homopolymer. Poly(dA)·poly(dT) shows a unique behavior: studies of the effects of concentration and temperature indicate that tilorone acts as a probe of a process involving the polynucleotide alone. This process appears to be related to the dynamic structure of the nucleic acid and is detectable only when the bound dye is not intercalated.     

FRET-based analysis of protein-nucleic acid interactions by genetically incorporating a fluorescent amino acid

Protein–nucleic acid interaction is an important process in many biological phenomena. In this study, a fluorescence resonance energy transfer (FRET)-based protein–DNA binding assay has been developed, in which a fluorescent amino acid is genetically incorporated into a DNA-binding protein. A coumarin-containing amino acid was incorporated into a DNA-binding protein, and the mutant protein specifically produced a FRET signal upon binding to its cognate DNA labeled with a fluorophore. The protein–DNA binding affinity was then measured under equilibrium conditions. This method is advantageous for studying protein-nucleic acid interactions, because it is performed under equilibrium conditions, technically easy, and applicable to any nucleic acid-binding protein.     

Poly(ADP-ribose) polymerase interacts with a novel human ubiquitin conjugating enzyme: hUbc9

Poly(ADP-ribose) polymerase (PARP) has been suggested to play a regulatory role in vivo, in DNA replication and/or DNA repair based mainly on its capacity to bind to DNA strand breaks. This interaction is modulated through auto poly(ADP-ribosylation). However, the biological function of PARP may also involve interactions with proteins such as topoisomerase I or DNA polymerase , which may or may not be themselves ADP-ribosylated. Using the yeast two-hybrid method search for other proteins interacting with PARP, we have isolated a full-length cDNA clone coding for a protein of 158 amino acid residues. This amino acid sequence is 66 and 56% identical to yeast ubiquitin-conjugating enzymes Hus5 and Ubc9 of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Moreover, we have demonstrated that the expressed protein complements a S. cerevisiae yeast strain deficient for Ubc9. The protein encoded by the isolated cDNA is thus a new human counterpart of the ubiquitin-conjugating enzyme family and has been called hUbc9. The hubc9 gene locus has been assigned to the chromosomal location 16p13.2-p13.3. By means of two-hybrid analysis it was discovered that hUbc9 interacts with the automodification domain of PARP. This interaction was further confirmed using GST (glutathione-S-transferase) tagged fusion proteins: (i) in vivo, by transfecting cos7 cells with hUbc9 cloned in an eukaryotic expression vector, and (ii) in vitro, by mixing purified PARP with hUbc9 purified and expressed in bacteria. The possible significance and function of this interaction is discussed while taking into account the possible intracellular role of hUbc9.