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.

Na+ and K+ Ions Differently Affect Nucleosome Structure, Stability, and Interactions with Proteins.

Inorganic ions are essential factors stabilizing nucleosome structure; however, many aspects of their effects on DNA transactions in chromatin remain unknown. Here, differential effects of K+ and Na+ on the nucleosome structure, stability, and interactions with protein complex FACT (FAcilitates Chromatin Transcription), poly(ADP-ribose) polymerase 1, and RNA polymerase II were studied using primarily single-particle Förster resonance energy transfer microscopy. The maximal stabilizing effect of K+ on a nucleosome structure was observed at ca. 80­150 mM, and it decreased slightly at 40 mM and considerably at >300 mM. The stabilizing effect of Na+ is noticeably lower than that of K+ and progressively decreases at ion concentrations higher than 40 mM. At 150 mM, Na+ ions support more efficient reorganization of nucleosome structure by poly(ADP-ribose) polymerase 1 and ATP-independent uncoiling of nucleosomal DNA by FACT as compared with K+ ions. In contrast, transcription through a nucleosome is nearly insensitive to K+ or Na+ environment. Taken together, the data indicate that K+ environment is more preserving for chromatin structure during various nucleosome transactions than Na+ environment.

Mechanisms of Nucleosome Reorganization by PARP1.

Poly(ADP-ribose) polymerase 1 (PARP1) is an enzyme involved in DNA repair, chromatin organization and transcription. During transcription initiation, PARP1 interacts with gene promoters where it binds to nucleosomes, replaces linker histone H1 and participates in gene regulation. However, the mechanisms of PARP1-nucleosome interaction remain unknown. Here, using spFRET microscopy, molecular dynamics and biochemical approaches we identified several different PARP1-nucleosome complexes and two types of PARP1 binding to mononucleosomes: at DNA ends and end-independent. Two or three molecules of PARP1 can bind to a nucleosome depending on the presence of linker DNA and can induce reorganization of the entire nucleosome that is independent of catalytic activity of PARP1. Nucleosome reorganization depends upon binding of PARP1 to nucleosomal DNA, likely near the binding site of linker histone H1. The data suggest that PARP1 can induce the formation of an alternative nucleosome state that is likely involved in gene regulation and DNA repair.

PARP-1-Associated Pathological Processes: Inhibition by Natural Polyphenols.

Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme involved in processes of cell cycle regulation, DNA repair, transcription, and replication. Hyperactivity of PARP-1 induced by changes in cell homeostasis promotes development of chronic pathological processes leading to cell death during various metabolic disorders, cardiovascular and neurodegenerative diseases. In contrast, tumor growth is accompanied by a moderate activation of PARP-1 that supports survival of tumor cells due to enhancement of DNA lesion repair and resistance to therapy by DNA damaging agents. That is why PARP inhibitors (PARPi) are promising agents for the therapy of tumor and metabolic diseases. A PARPi family is rapidly growing partly due to natural polyphenols discovered among plant secondary metabolites. This review describes mechanisms of PARP-1 participation in the development of various pathologies, analyzes multiple PARP-dependent pathways of cell degeneration and death, and discusses representative plant polyphenols, which can inhibit PARP-1 directly or suppress unwanted PARP-dependent cellular processes.

Unfolding of core nucleosomes by PARP-1 revealed by spFRET microscopy.

DNA accessibility to various protein complexes is essential for various processes in the cell and is affected by nucleosome structure and dynamics. Protein factor PARP-1 (poly(ADP-ribose)polymerase 1) increases the accessibility of DNA in chromatin to repair proteins and transcriptional machinery, but the mechanism and extent of this chromatin reorganization are unknown. Here we report on the effects of PARP-1 on single nucleosomes revealed by spFRET (single-particle Förster Resonance Energy Transfer) microscopy. PARP-1 binding to a double-strand break in the vicinity of a nucleosome results in a significant increase of the distance between the adjacent gyres of nucleosomal DNA. This partial uncoiling of the entire nucleosomal DNA occurs without apparent loss of histones and is reversed after poly(ADP)-ribosylation of PARP-1. Thus PARP-1-nucleosome interactions result in reversible, partial uncoiling of the entire nucleosomal DNA.