Role of Histone Tails and Single Strand DNA Breaks in Nucleosomal Arrest of RNA Polymerase.

Transcription through nucleosomes by RNA polymerases (RNAP) is accompanied by formation of small intranucleosomal DNA loops (i-loops). The i-loops form more efficiently in the presence of single-strand breaks or gaps in a non-template DNA strand (NT-SSBs) and induce arrest of transcribing RNAP, thus allowing detection of NT-SSBs by the enzyme. Here we examined the role of histone tails and extranucleosomal NT-SSBs in i-loop formation and arrest of RNAP during transcription of promoter-proximal region of nucleosomal DNA. NT-SSBs present in linker DNA induce arrest of RNAP +1 to +15 bp in the nucleosome, suggesting formation of the i-loops; the arrest is more efficient in the presence of the histone tails. Consistently, DNA footprinting reveals formation of an i-loop after stalling RNAP at the position +2 and backtracking to position +1. The data suggest that histone tails and NT-SSBs present in linker DNA strongly facilitate formation of the i-loops during transcription through the promoter-proximal region of nucleosomal DNA.

Structure of an Intranucleosomal DNA Loop That Senses DNA Damage during Transcription.

Transcription through chromatin by RNA polymerase II (Pol II) is accompanied by the formation of small intranucleosomal DNA loops containing the enzyme (i-loops) that are involved in survival of core histones on the DNA and arrest of Pol II during the transcription of damaged DNA. However, the structures of i-loops have not been determined. Here, the structures of the intermediates formed during transcription through a nucleosome containing intact or damaged DNA were studied using biochemical approaches and electron microscopy. After RNA polymerase reaches position +24 from the nucleosomal boundary, the enzyme can backtrack to position +20, where DNA behind the enzyme recoils on the surface of the histone octamer, forming an i-loop that locks Pol II in the arrested state. Since the i-loop is formed more efficiently in the presence of SSBs positioned behind the transcribing enzyme, the loop could play a role in the transcription-coupled repair of DNA damage hidden in the chromatin structure.

Production of Bacterial Cellulose Aerogels With Improved Physico-Mechanical Properties and Antibacterial Effect.

Aerogels have gained significant interest in recent decades because of their unique properties such as high porosity, low density, high surface area, and excellent heat and noise insulation. However, their high cost and low mechanical strength limit their practical application. We developed appropriate conditions to produce aerogels with controlled density, high mechanical strength, and thermal characteristics from bacterial cellulose (BC) synthesized by the strain Komagataeibacter sucrofermentans H-110. Aerogels produced using TEMPO oxidized BC (OBC) exhibited high mechanical strength and lower shrinkage than those from native bacterial cellulose (NBC). Compared to the NBC, the use of TEMPO-oxidized BC with oxidation degrees (OD) of 1.44 and 3.04% led to the reduction of shrinkage of the aerogels from 41.02 to 17.08%. The strength of the aerogel produced from the TEMPO-oxidized BC with an oxidation degree of 1.44% was twice that of the aerogel produced from NBC. The addition of Mg2+ at concentrations of 20 and 40 mM during the preparation of the aerogels increased the strength of the aerogels by 4.9 times. The combined use of TEMPO-oxidized BC and Mg2+ allowed pore size reduction from 1,375 to 197.4 µm on the outer part of the aerogels, thereby decreasing the thermal conductivity coefficient from 0.036 to 0.0176 W/(m•K). Furthermore, novel biocomposites prepared from the aerogels based on NBC and OBC and sodium fusidate, which have high antibiotic activity against Staphylococcus aureus, were obtained. Owing to their antibacterial properties, these aerogels can be used as functional biomaterials in a wide range of applications such as in tissue engineering and fabrication of wound dressing materials.

A Study of the Physical and Mechanical Properties of Aerogels Obtained from Bacterial Cellulose.

Aerogels with a density of 4.2-22.8 kg/m3 were obtained from bacterial cellulose synthesized under static and dynamic cultivation conditions on a molasses medium. The strength properties and porous structure of the aerogels strongly depended on their density. With an aerogel density of 22.8 kg/m3, the modulus of elasticity at 80% compression of the sample was 0.1 MPa. The decrease in the density of aerogels led to an increase in the pore sizes ranging from 20 to 1000 µm and a decrease in the modulus of elasticity. These characteristics were more pronounced in aerogels obtained from bacterial cellulose under static cultivation conditions. The aerogels had a low coefficient of thermal conductivity (0.0257 W m-1 °C-1), which is comparable to the thermal conductivity of air, and moderate thermal stability because the degradation processes of the aerogels began at 237 °C. The aerogels obtained from bacterial cellulose had high sound absorption coefficients in the frequency range of 200-5000 Hz, which makes it possible to use the aerogels as heat- and sound-insulating materials.

Structure of transcribed chromatin is a sensor of DNA damage.

Early detection and repair of damaged DNA is essential for cell functioning and survival. Although multiple cellular systems are involved in the repair of single-strand DNA breaks (SSBs), it remains unknown how SSBs present in the nontemplate strand (NT-SSBs) of DNA organized in chromatin are detected. The effect of NT-SSBs on transcription through chromatin by RNA polymerase II was studied. NT-SSBs localized in the promoter-proximal region of nucleosomal DNA and hidden in the nucleosome structure can induce a nearly quantitative arrest of RNA polymerase downstream of the break, whereas more promoter-distal SSBs moderately facilitate transcription. The location of the arrest sites on nucleosomal DNA suggests that formation of small intranucleosomal DNA loops causes the arrest. This mechanism likely involves relief of unconstrained DNA supercoiling accumulated during transcription through chromatin by NT-SSBs. These data suggest the existence of a novel chromatin-specific mechanism that allows the detection of NT-SSBs by the transcribing enzyme.

Effects of inactivation of the PPN1 gene on exopolyphosphatases, inorganic polyphosphates and function of mitochondria in the yeast Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae with inactivated endopolyphosphatase gene PPN1 did not grow on lactate and ethanol, and stopped growth on glucose earlier than the parent strain. Their mitochondria were defective in respiration functions and in metabolism of inorganic polyphosphates. The PPN1 mutants lacked exopolyphosphatase activity and possessed a double level of inorganic polyphosphates in mitochondria. The average chain length of mitochondrial polyphosphates at the stationary growth stage on glucose was about 15-20 and about 130-180 phosphate residues in the parent strain and PPN1 mutants, respectively. Inactivation of the PPX1 gene encoding exopolyphosphatase had no effect on respiration functions and on polyphosphate level and chain length in mitochondria.

Inorganic polyphosphate in mitochondria of Saccharomyces cerevisiae at phosphate limitation and phosphate excess.

Isolated mitochondria of Saccharomyces cerevisiae cells grown on glucose possess acid-soluble inorganic polyphosphate (polyP). Its level strongly depends on phosphate (P(i)) concentration in the culture medium. The polyP level in mitochondria showed 11-fold decrease under 0.8 mM P(i) as compared with 19.3 mM P(i). When spheroplasts isolated from P(i)-starved cells were incubated in the P(i)-complete medium, they accumulated polyP and exhibited a phosphate overplus effect. Under phosphate overplus the polyP level in mitochondria was two times higher than in the complete medium without preliminary P(i) starvation. The average chain length of polyP in mitochondria was of <15 phosphate residues at 19.3 mM P(i) in the culture medium and increased at phosphate overplus. Deoxyglucose inhibited polyP accumulation in spheroplasts, but had no effect on polyP accumulation in mitochondria. Uncouplers (FCCP, dinitrophenol) and ionophores (monensin, nigericin) inhibited polyP accumulation in mitochondria more efficiently than in spheroplasts. Fast hydrolysis of polyP was observed after sonication of isolated mitochondria. Probably, the accumulation of polyP in mitochondria depended on the proton-motive force of their membranes.