Annotation of the results obtained in 2018
Using spFRET microscopy and integrative modeling techniques, studies have been conducted to study the flexibility of DNA, a factor that plays an important role in communication processes in chromatin. Experimental data obtained using spFRET microscopy suggest existence of a new curved conformation of the yeast centromere DNA fragment in solution. The curved conformation of the yeast centromeric DNA can play a key role in performing its biological function — interaction with kinetochore proteins and distribution of chromosomes to daughter cells after division. It was also established that, depending on the nucleotide sequence, the flexibility of double-stranded oligonucleotides (around 100 bp in length) can vary at least 2.3 fold. The introduction of a single-strand break in DNA increases the flexibility of both rigid and flexible oligonucleotides ~ 2.5 fold, and the interaction with cisplatin - ~ 5 fold. Experimental and theoretical studies have been conducted to study the effects of variations in DNA sequence and histone composition on the structure and dynamics of nucleosomes. It was shown that the presence of a linker DNA region on only one side of the nucleosome leads to destabilization of its structure in comparison with the structure of nucleosomes with two linkers or without them. It has been shown that unwrapping of up to 35 bp DNA from the histone octamer in solutions in the presence of various ion concentrations can occur reversibly without the loss of histones. This unwrapping is accompanied by the formation of two new conformational states of nucleosomes. It was established that in the complex of nucleosomes with histone H1.0 from X. laevis the linker DNA fragments assume a single conformation, and the structuring effect of histone H1.0 spreads along the linker DNA to a distance of at least 25 bp from the core nucleosome. It was shown that the removal of the N-terminal tail of the histone H2B causes destabilization of the structure of the linker DNA of the nucleosome. This indicates the important role of interactions of the “tails” of core histones with linker DNA fragments that results in the linker DNA proximity necessary for the correct packaging of nucleosomes into the chromatin fibers. Using the methods of integrative modeling based on spFRET microscopy data, the models were constructed describing the conformation of the linker regions of nucleosomal DNA in the presence and in the absence of two variants of human linker histone H1.5. The effect of different H1.5 domains on the conformation of linker DNA has been characterized. The differences in the DNA deformation energy for two models of nucleosome complexes with histone H1 were calculated: (1) H1 is bound at a dyad axis and (2) H1 is bound in the position that is shifted off the dyad axis. All possible sequences of linker DNA fragments interacting with histone H1 were evaluated to determine the sequences that are favorable for DNA deformation. DNA sequences that facilitate alternative modes of histone H1 binding (on dyad and off dyad) were identified. The hypothesis on the mechanism of nucleosome unfolding by yeast factor yFACT has been experimentally confirmed. In particular, it was shown that the unfolding is accompanied by: (1) destabilization of the contacts between nucleosomal DNA and the histone H2A/H2B dimers; (2) both H2A/H2B dimers remain bound to either yFACT or a histone tetramer H3/H4 after nucleosome unfolding. The functional domains of Spt16-Pob3 subunits of yFACT and a nucleosome were determined that are involved in interactions between these supramolecular complexes. In particular, it was shown that yFACT with deleted C-termini of Spt16 and Pob3 cannot unfold a nucleosome, and M62E mutation in histone H2B of the histone octamer impairs the interaction between yFACT and a nucleosome. Based on the data obtained, a detailed model of yFACT-unfolded nucleosome complex was developed. Structures of two key intermediate elongation complexes (EC) were determined using DNA footprinting assays: EC(+39) that is essential for nucleosome survival during transcription and EC(-9) controlling the height of the nucleosomal barrier to transcription. The roles of various domains of human factor FACT (hFACT) in transcription through chromatin and reorganization of nucleosomes were determined. The mechanism of inhibition of FACT-dependent transcription of chromatin by anticancer drugs curaxins was determined in vivo and in vitro: curaxins cause a redistribution of hFACT from transcribed genes to other genome regions, and thus inhibit transcription. It is shown that reorganization of a nucleosome caused by hFACT in the presence of curaxins is symmetric and based on the "all-or-none" principle, i.e. it is similar to the reorganization mediated by yFACT. New data on the mechanism of RNA polymerase pausing in intranucleosomal loops were obtained. The mechanism of action of protein factor FACT and the effect nucleotide sequence on the pausing were determined. An original method to study RNA polymerase elongation complexes with a nucleosome using electron microscopy was developed. This method includes a two-stage purification process followed by concentration of the complexes on a heparin resin and affinity grid bearing an immobilized lipid monolayer. Based on the data of cryoelectron microscopy and molecular modeling, a three-dimensional model of the elongation complex EC(+39) was calculated, which revealed the presence of a Ø-loop (zero-size loop) formed by nucleosomal DNA, and enabled one to clarify the structural features of this complex. Using transmission electron microscopy and Fourier transformation, the structure of the two-dimensional crystals of the DNA-binding histone-like Dps protein with DNA was visualized. It was found that the orientation of Dps in crystals depends on the length of the interacting DNA.

 

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Annotation of the results obtained in 2017
To study the flexibility and bending in short fragments of double stranded DNA we have developed experimental protocols and created a set of biotinylated, fluorescently-labeled DNA–constructs that varied in length and a nucleotide sequence. These constructs allow the study of DNA flexibility through their ability to circularize that can be detected by spFRET microscopy. Using computer modeling we created molecular models of short circular DNA fragments of various length and sequence. Energies of DNA cyclization were calculated and algorithms to estimate spontaneous cyclization probabilities of short DNA segments were developed. A software was developed that can analyze the photon arrival times in the single photon counting mode during spFRET experiments. This approach allows one to study structural dynamics of single supramolecular complexes with sub-millisecond resolution during their diffusion through the focus of a laser beam under a microscope (5-8 ms). This new approach of spFRET data analysis in combination with the molecular modeling methods can be applied to analysis of the structural dynamics of the nucleosomes and their complexes with various protein factors. Two different types of binding of the globules of linker histones H1 (off-dyad) and H5(on-dyad) with nucleosomes under identical conditions in solution were confirmed using spFRET microscopy. Taking into account the data of spFRET microscopy, the models of complexes of nucleosomes with globular domains of linker histones H1 and H5 were developed and conformation of linker DNA was predicted for these complexes. Using bioinformatics approaches, the types of complex (on-dyad or off-dyad) between a nucleosome and different known variants of a linker histone were predicted. The model of a complex between a nucleosome and human histone H1.0 was created using homology modeling. Computer modeling methods were used to select the optimal positions of the Cy5 label on the H2A histone in combination with the Cy3 label on the nucleosomal DNA at positions +13 and +57 b.p. (relative to the nucleosome boundary). This labeling strategy will allow us to discriminate between several hypothetical mechanisms of nucleosome unwrapping by the protein complex FACT. Synthesis of mononucleosomes fluorescently labeled at the selected positions is in progress. Using spFRET microscopy it was shown that fivefold molar excess of Nhp6 subunit over Spt16/Pob3 subunit is needed for complete ATP-independent nucleosomal DNA uncoiling by FACT. The requirement for Nhp6 excess indicates that the binding of several Nhp6 molecules with a nucleosome is a prerequisite of nucleosome uncoiling by FACT. In turn, the formation of FACT from Spt16/Pob3 and Nhp6 subunits may occur either directly on a nucleosome, which already bound Nhp6, or in solution before the binding of FACT to a nucleosome. DNA footprinting assays were successfully used to determine the structure of elongation complex EC(+24) formed at the position of +24 bp relative to the nucleosome boundary, when a single strand break (SSB) is present in a non-template strand at the position +12. Atomistic models of EC(+24) with or without SSB were created on the basis of footprinting and cryo-electronic microscopy data using computational approaches. The main differences between the intermediates of transcription in intact and damaged chromatin were determined. Regulatory barrier arising in vivo at the boundary of +1 nucleosome during transcription by RNA polymerase II was reproduced using a mononucleosome-contaning experimental system. The data suggest that DNA-histone interactions in the +1 nucleosome form the main part of this regulatory barrier in vivo. Facilitating effect of yeast FACT subunits during transcription through nucleosome by RNA polymerase 2 (RNAP2) was evaluated experimentally. Nhp6 subunit and Spt16-Pob3 complex predominantly affect pausing of RNAP2 at + (11 ÷ 15) bp and + (45 ÷ 48) bp positions, respectively, reducing the barrier for transcription at the corresponding positions. It was determined that Spt16 and Pob3 C-terminal regions and M-domain of Spt16 participate in the interaction of yFACT with a nucleosome during transcription. The data obtained suggest the simultaneous interaction of the C-termini of the Spt16 and Pob3 subunits with two different targets (most likely with both H2A/H2B dimers) in the nucleosome. It was shown that interaction of the N-terminal moiety of histone H2B with yFACT plays an important role during activation of transcription through chromatin by RNAP2. Using spFRET microscopy, it was found that neither the curaxin CBL0137 nor human hFACT alone caused significant structural changes in the nucleosomal DNA. At the same time, CBL0137 and hFACT together cause significant uncoiling of the nucleosomal DNA, reversible for most of the nucleosomes undergoing structural changes. Such structural rearrangement may accompany the curaxin-dependent binding of hFACT to heterochromatin, previously found in vivo. The detailed mechanism of RNAP pausing in intranucleosomal DNA loops was determined. The effects of protein factors GreB and FACT and nucleotide sequence on this pausing were established. Using transmission electron microscopy with negative staining of samples, structures of elongated complexes stalled at position +39 b.p. from the nucleosomal boundary (EC(+39)) and complexes of nucleosomes with yeast FACT were studied. After concentration of EC(+39) on the affine lipid monolayer, transfer of complexes onto a substrate-grid and negative staining, more than 5,000 individual projection images of EС(+39) were collected. The structure of EC (+39) obtained as a result of a preliminary reconstruction has two domains, and the voluminous sizes of the domains are comparable to the sizes of RNA polymerase and a nucleosome (known from x-ray crystallography data). The resulting structure suggests the formation of the previously predicted intra-nuclesome DNA loop of a small size. The FACT-nucleosome complex was purified using a native polyacrylamide gel. Several structures of the FACT-nucleosome complex have been identified, which presumably correspond to various stages of the interaction between FACT and the nucleosome. The obtained structures suggest that the structure of nucleosomes is significantly changed in the FACT-nucleosome complex.

 

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