The effect of amine structure on complexation with lasalocid in model membrane systems. II. Ionophore selectivity for amines in lipid bilayers and at oil/water interfaces

The ionophore antibiotic X-537A (lasalocid) transports biogenic amines across biological and artificial membranes. The major portion of amine flux (greater than 99%) occurs as a 1:1 neutral complex. The rank order of ionophore selectivity was determined for lipid bilayer membrane transport of amines based on a comparison of permeability coefficients: p-tyramine ～ β-phenylethylamine ～ amphetamine > methamphetamine > dopamine > phenylephrine ～ metanephrine > norepinephrine > epinephrine. This rank order is in agreement with results obtained from partitioning measurements which were carried out in parallel to the bilayer membrane experiments. A correlation between amine structure and binding characteristics has been developed.     

DNA-Protein Complex in Circular DNA from Phage ϕ29

THE DNA of the B. subtilis phage ?29 has been described as unpermuted linear duplex molecules¹ of molecular weight 11 × 10⁶, but the formation of circular molecules has also been indicated, suggesting the existence of cohesive ends^1,2.     

Gln54Leu of the conserved polar residue inthe interfacial coiled coil position (d) results in significant stabilization of the original structure of the COMP pentamerization domain

Summary The assembly domain of cartilage oligomeric matrix protein (COMP) forms an α-helical coiled coil homopentamer with a conserved polar glutamine in the interior (d) position. We substituted Gln⁵⁴ for apolar Leu in the recombinant fragment of the rat COMP domain. Biochemical studies and circular dichroism (CD) spectroscopy showed that the mutant, similarly to the wild-type (w.t.) peptide, forms spontaneously an α-helical pentamer. Thermal transitions of the w.t. and mutant pentamers were analyzed by CD spectroscopy and differential scanning calorimetry. The Gln⁵⁴Leu mutation increased the thermal stability of the pentamer with reduced disulfide bonds from 73°C to 104°C. The denaturation of the disulfide bonded w.t. pentamer was observed at 108°C while the mutant pentamer cannot be denatured up to 120°C (the apparatus limit). Thus, by Gln⁵⁴Leu mutation we found a way to significantly stabilize the coiled coil pentamer, making this peptide even more attractive as an oligomerization tool for various biotechnological applications.     

Cyclodepsipeptides as chemical tools for studying ionic transport through membranes

Summary This paper reports a study of the chemistry of valinomycin, enniatins and related membrane-active depsipeptides that increase alkali metal ion permeability of model and biological membranes. The antimicrobial activity of these compounds and their effect on membranes has been correlated with their cation-complexing ability. The complexing reaction has been studied by spectropolarimetric and conductimetric methods. Nuclear magnetic resonance, optical rotatory dispersion, and infrared spectrophotometric studies have revealed the coexistence of conformers of the cyclodepsipeptides in solution and have led to elucidation of the spatial structure of valinomycin, enniatin B and their K⁺ complexes. The effect of the conformational properties of the cyclodepsipeptides on their complexation efficiency and selectivity, surface-active properties and behavior towards phospholipid monolayers, bimolecular phospholipid membranes and a number of biological membrane systems has been ascertained. The studies have clearly shown the feasibility of using cyclodepsipeptides with predetermined structural and conformational parameters as chemical tools for membrane studies. it is suggested that the principle of conformation-dependent cation binding through iondipole interactions may possibly lie at the basis of the mode of action of systems governing the natural ion permeability in biological membranes.For preliminary communication,see Refs. [9, 19, 20, 27, 29].     

The Replisome-Coupled E3 Ubiquitin Ligase Rtt101Mms22 Counteracts Mrc1 Function to Tolerate Genotoxic Stress

Faithful DNA replication and repair requires the activity of cullin 4-based E3 ubiquitin ligases (CRL4), but the underlying mechanisms remain poorly understood. The budding yeast Cul4 homologue, Rtt101, in complex with the linker Mms1 and the putative substrate adaptor Mms22 promotes progression of replication forks through damaged DNA. Here we characterized the interactome of Mms22 and found that the Rtt101^Mms22 ligase associates with the replisome progression complex during S-phase via the amino-terminal WD40 domain of Ctf4. Moreover, genetic screening for suppressors of the genotoxic sensitivity of rtt101Δ cells identified a cluster of replication proteins, among them a component of the fork protection complex, Mrc1. In contrast to rtt101Δ and mms22Δ cells, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants complete DNA replication upon replication stress by facilitating the repair/restart of stalled replication forks using a Rad52-dependent mechanism. Our results suggest that the Rtt101^Mms22 E3 ligase does not induce Mrc1 degradation, but specifically counteracts Mrc1’s replicative function, possibly by modulating its interaction with the CMG (Cdc45-MCM-GINS) complex at stalled forks.     

7,8-Dihydro-8-oxo-1,N6-ethenoadenine: an exclusively Hoogsteen-paired thymine mimic in DNA that induces A→T transversions in Escherichia coli

This work investigated the structural and biological properties of DNA containing 7,8-dihydro-8-oxo-1,N⁶-ethenoadenine (oxo-ϵA), a non-natural synthetic base that combines structural features of two naturally occurring DNA lesions (7,8-dihydro-8-oxoadenine and 1,N⁶-ethenoadenine). UV-, CD-, NMR spectroscopies and molecular modeling of DNA duplexes revealed that oxo-ϵA adopts the non-canonical syn conformation (χ = 65º) and fits very well among surrounding residues without inducing major distortions in local helical architecture. The adduct remarkably mimics the natural base thymine. When considered as an adenine-derived DNA lesion, oxo-ϵA was >99% mutagenic in living cells, causing predominantly A→T transversion mutations in Escherichia coli. The adduct in a single-stranded vector was not repaired by base excision repair enzymes (MutM and MutY glycosylases) or the AlkB dioxygenase and did not detectably affect the efficacy of DNA replication in vivo. When the biological and structural data are viewed together, it is likely that the nearly exclusive syn conformation and thymine mimicry of oxo-ϵA defines the selectivity of base pairing in vitro and in vivo, resulting in lesion pairing with A during replication. The base pairing properties of oxo-ϵA, its strong fluorescence and its invisibility to enzymatic repair systems in vivo are features that are sought in novel DNA-based probes and modulators of gene expression.     

History, genetic differentiation and conservation strategies for disjunct populations of Sibiraea species from Southeastern Europe and Asia

The genetic structure of Croatian Sibirea (Sibiraea croatica), a rare and endemic tertiary relic of Croatian and Herzegovinian flora, and its relationship with sibirea from Southern Russia and Southern Siberia (Sibiraea altaiensis) was studied using amplification, restriction and sequencing of the ITS region in genomic DNA and cpDNA and their comparisons with sequences of the Rosaceae species obtained from GenBank. The restriction analysis and separation in agarose gel showed no differences in length of the digested cpDNA between or within populations. Sequencing showed only minor variability between populations. Only a minor difference of 6 bp duplication in DNA amplified with ccmp 10-R and trnM primer pair was noticed in two geographically distinct populations. No differences in the restriction pattern for the ITS region in genomic rDNA indicates that all samples of sibirea belong to the same species since the ITS region was proven to be conserved within one taxonomic species. The minor differences that were␣obtained support the hypothesis that sibirea is an old tertiary relic that shows a minor variability, confirming previous preliminary results from comparisons of the Croatian and Altaic sibireas at the morphological level. Our data suggests that Croatian sibirea from the Balkan is a disjunct population identical to the Altaic species. Due to its disjunct occurrence in Southeastern Europe, the endemic status in the Dinarics, a relic that survived the glaciations, it deserves active conservation approaches through support of traditional use of high-mountain pastures for reducing natural reforestation of sibirea ancient sites.