Epigallocatechin Gallate Affects the Structure of Chromatosomes, Nucleosomes and Their Complexes with PARP1.

The natural flavonoid epigallocatechin gallate has a wide range of biological activities, including being capable of binding to nucleic acids; however, the mechanisms of the interactions of epigallocatechin gallate with DNA organized in chromatin have not been systematically studied. In this work, the interactions of epigallocatechin gallate with chromatin in cells and with nucleosomes and chromatosomes in vitro were studied using fluorescent microscopy and single-particle Förster resonance energy transfer approaches, respectively. Epigallocatechin gallate effectively penetrates into the nuclei of living cells and binds to DNA there. The interaction of epigallocatechin gallate with nucleosomes in vitro induces a large-scale, reversible uncoiling of nucleosomal DNA that occurs without the dissociation of DNA or core histones at sub- and low-micromolar concentrations of epigallocatechin gallate. Epigallocatechin gallate does not reduce the catalytic activity of poly(ADP-ribose) polymerase 1, but causes the modulation of the structure of the enzyme-nucleosome complex. Epigallocatechin gallate significantly changes the structure of chromatosomes, but does not cause the dissociation of the linker histone. The reorganization of nucleosomes and chromatosomes through the use of epigallocatechin gallate could facilitate access to protein factors involved in DNA repair, replication and transcription to DNA and, thus, might contribute to the modulation of gene expression through the use of epigallocatechin gallate, which was reported earlier.

Hmo1 Protein Affects the Nucleosome Structure and Supports the Nucleosome Reorganization Activity of Yeast FACT.

Yeast Hmo1 is a high mobility group B (HMGB) protein that participates in the transcription of ribosomal protein genes and rDNA, and also stimulates the activities of some ATP-dependent remodelers. Hmo1 binds both DNA and nucleosomes and has been proposed to be a functional yeast analog of mammalian linker histones. We used EMSA and single particle Förster resonance energy transfer (spFRET) microscopy to characterize the effects of Hmo1 on nucleosomes alone and with the histone chaperone FACT. Hmo1 induced a significant increase in the distance between the DNA gyres across the nucleosomal core, and also caused the separation of linker segments. This was opposite to the effect of the linker histone H1, which enhanced the proximity of linkers. Similar to Nhp6, another HMGB factor, Hmo1, was able to support large-scale, ATP-independent, reversible unfolding of nucleosomes by FACT in the spFRET assay and partially support FACT function in vivo. However, unlike Hmo1, Nhp6 alone does not affect nucleosome structure. These results suggest physiological roles for Hmo1 that are distinct from Nhp6 and possibly from other HMGB factors and linker histones, such as H1.

Antiherpetic properties of acyclovir 5'-hydrogenphosphonate and the mutation analysis of herpes virus resistant strains.

In this study, we continued to study antiherpetic properties of acyclovir 5'-hydrogenphosphonate (Hp-ACV) in cell cultures and animal models. Hp-ACV was shown to inhibit the development of herpetic infection in mice induced by the HSV-1/L(2) strain. The compound suppressed replication of both ACV-sensitive HSV-1/L(2) and ACV-resistant HSV-1/L(2)/R strains in Vero cell culture. Viral population resistant to Hp-ACV (HSV-1/L(2)/R(Hp-ACV)) was developed much slower than ACV-resistant population. The analysis of Hp-ACV-resistant clones isolated from the HSV-1/L(2)/R(Hp-ACV) population demonstrated their partial cross-resistance to ACV. The mutations determining the resistance of HSV-1 clones to Hp-ACV were partly overlapped with mutations defining ACV resistance but did not always coincide. HSV-1/L(2)/R(Hp-ACV) herpes virus thymidine kinase is shortened from the C-terminus by 100 amino acid residues in length.

Development of the system ensuring a high-level expression of hepatitis C virus nonstructural NS5B and NS5A proteins.

The plasmid pET-21d-2c-5BDelta55 effectively expressing a C-terminally truncated form (NS5BDelta55) of the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) was constructed. It was derived from pET-21d-5BDelta55 plasmid and contained six mutations in the ATG-start codon region and an additional cistron upstream the target gene. The C-terminally His-tagged NS5BDelta55 protein was expressed in Rosetta(DE3) Escherichia coli strain bearing an additional pRARE plasmid encoding extra copies of rare tRNAs. The yield of the target enzyme exceeded by a factor of 29 the yield of NS5BDelta55 protein expressed from the parental pET-21d-5BDelta55 plasmid (5 mg/L). The increase in the protein yield could be explained by facilitated protein translation initiation, resulted from disruption of the stable secondary mRNA structure. The pET-21d-2c-5BDelta55 plasmid yielded one third amount of the protein when expressed in BL-21(DE3) strain, indicating that the pRARE plasmid is required for a high-level expression of NS5BDelta55 protein. The 29-fold enhancement of the protein yield was accompanied by only a 2.5-fold increase of the corresponding mRNA level. The expression of another HCV NS5A protein His-tagged at the C-terminus in the developed system yielded a similar amount of the protein (4 mg/L), whereas its N-terminally His-tagged counterpart was obtained in a 30 mg/L yield. The NS5A protein purified under denaturing conditions and renatured in solution inhibited the HCV RdRp and was a substrate for human casein kinase II.