A correct chromatin structure is important for cell viability and is tightly regulated by numerous factors. Human protein complex FACT (facilitates chromatin transcription) is an essential factor involved in chromatin transcription and cancer development. Here FACT-dependent changes in the structure of single nucleosomes were studied with single-particle Förster resonance energy transfer (spFRET) microscopy using nucleosomes labeled with a donor-acceptor pair of fluorophores, which were attached to the adjacent gyres of DNA near the contact between H2A-H2B dimers. Human FACT and its version without the C-terminal domain (CTD) and the high mobility group (HMG) domain of the structure-specific recognition protein 1 (SSRP1) subunit did not change the structure of the nucleosomes, while FACT without the acidic C-terminal domains of the suppressor of Ty 16 (Spt16) and the SSRP1 subunits caused nucleosome aggregation. Proteolytic removal of histone tails significantly disturbed the nucleosome structure, inducing partial unwrapping of nucleosomal DNA. Human FACT reduced DNA unwrapping and stabilized the structure of tailless nucleosomes. CTD and/or HMG domains of SSRP1 are required for this FACT activity. In contrast, previously it has been shown that yeast FACT unfolds (reorganizes) nucleosomes using the CTD domain of SSRP1-like Pol I-binding protein 3 subunit (Pob3). Thus, yeast and human FACT complexes likely utilize the same domains for nucleosome reorganization and stabilization, respectively, and these processes are mechanistically similar.

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.

7-Methylguanine (7-MG), a natural compound that inhibits DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP-1), can be considered as a potential anticancer drug candidate. Here we describe a study of 7-MG inhibition mechanism using molecular dynamics, fluorescence anisotropy and single-particle Förster resonance energy transfer (spFRET) microscopy approaches to elucidate intermolecular interactions between 7-MG, PARP-1 and nucleosomal DNA. It is shown that 7-MG competes with substrate NAD⁺ and its binding in the PARP-1 active site is mediated by hydrogen bonds and nonpolar interactions with the Gly863, Ala898, Ser904, and Tyr907 residues. 7-MG promotes formation of the PARP-1–nucleosome complexes and suppresses DNA-dependent PARP-1 automodification. This results in nonproductive trapping of PARP-1 on nucleosomes and likely prevents the removal of genotoxic DNA lesions.

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.

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.

Previous studies have revealed the inhibitory effect of single-strand breaks in a non-template DNA strand (NT-SSB) on RNA polymerase (RNAP) transcription t