From DNA-protein interactions to the genetic circuit design using CRISPR-dCas systems

In the last decade, the CRISPR-Cas technology has gained widespread popularity in different fields from genome editing and detecting specific DNA/RNA sequences to gene expression control. At the heart of this technology is the ability of CRISPR-Cas complexes to be programmed for targeting particular DNA loci, even when using catalytically inactive dCas-proteins. The repertoire of naturally derived and engineered dCas-proteins including fusion proteins presents a promising toolbox that can be used to construct functional synthetic genetic circuits. Rational genetic circuit design, apart from having practical relevance, is an important step towards a deeper quantitative understanding of the basic principles governing gene expression regulation and functioning of living organisms. In this minireview, we provide a succinct overview of the application of CRISPR-dCas-based systems in the emerging field of synthetic genetic circuit design. We discuss the diversity of dCas-based tools, their properties, and their application in different types of genetic circuits and outline challenges and further research directions in the field

Electron microscopy analysis of ATP-independent nucleosome unfolding by FACT

FACT is a histone chaperone that participates in nucleosome removal and reassembly during transcription and replication. We used electron microscopy to study FACT, FACT:Nhp6 and FACT:Nhp6:nucleosome complexes, and found that all complexes adopt broad ranges of configurations, indicating high flexibility. We found unexpectedly that the DNA binding protein Nhp6 also binds to the C-terminal tails of FACT subunits, inducing more open geometries of FACT even in the absence of nucleosomes. Nhp6 therefore supports nucleosome unfolding by altering both the structure of FACT and the properties of nucleosomes. Complexes formed with FACT, Nhp6, and nucleosomes also produced a broad range of structures, revealing a large number of potential intermediates along a proposed unfolding pathway. The data suggest that Nhp6 has multiple roles before and during nucleosome unfolding by FACT, and that the process proceeds through a series of energetically similar intermediate structures, ultimately leading to an extensively unfolded form

H2A-H2B Histone Dimer Plasticity and Its Functional Implications

The protein core of the nucleosome is composed of an H3-H4 histone tetramer and two H2A-H2B histone dimers. The tetramer organizes the central 60 DNA bp, while H2A-H2B dimers lock the flanking DNA segments. Being positioned at the sides of the nucleosome, H2A-H2B dimers stabilize the overall structure of the nucleosome and modulate its dynamics, such as DNA unwrapping, sliding, etc. Such modulation at the epigenetic level is achieved through post-translational modifications and the incorporation of histone variants. However, the detailed connection between the sequence of H2A-H2B histones and their structure, dynamics and implications for nucleosome functioning remains elusive. In this work, we present a detailed study of H2A-H2B dimer dynamics in the free form and in the context of nucleosomes via atomistic molecular dynamics simulations (based on X. laevis histones). We supplement simulation results by comparative analysis of information in the structural databases. Particularly, we describe a major dynamical mode corresponding to the bending movement of the longest H2A and H2B α-helices. This overall bending dynamics of the H2A-H2B dimer were found to be modulated by its interactions with DNA, H3-H4 tetramer, the presence of DNA twist-defects with nucleosomal DNA and the amino acid sequence of histones. Taken together, our results shed new light on the dynamical mechanisms of nucleosome functioning, such as nucleosome sliding, DNA-unwrapping and their epigenetic modulation

Voltage-gated ion channel modulation by lipids: Insights from molecular dynamics simulations

AbstractCells commonly use lipids to modulate the function of ion channels. The lipid content influences the amplitude of the ionic current and changes the probability of voltage-gated ion channels being in the active or in the resting states. Experimental findings inferred from a variety of techniques and molecular dynamics studies have revealed a direct interaction between the lipid headgroups and the ion channel residues, suggesting an influence on the ion channel function. On the other hand the alteration of the lipids may in principle modify the overall electrostatic environment of the channel, and hence the transmembrane potential, leading to an indirect modulation, i.e. a global effect. Here we have investigated the structural and dynamical properties of the voltage-gated potassium channel Kv1.2 embedded in bilayers with modified upper or lower leaflet compositions corresponding to realistic biological scenarios: the first relates to the effects of sphingomyelinase, an enzyme that modifies the composition of lipids of the outer membrane leaflets, and the second to the effect of the presence of a small fraction of PIP2, a highly negatively charged lipid known to modulate voltage-gated channel function. Our molecular dynamics simulations do not enable to exclude the global effect mechanism in the former case. For the latter, however, it is shown that local interactions between the ion channel and the lipid headgroups are key-elements of the modulation

Trajectories of microsecond molecular dynamics simulations of nucleosomes and nucleosome core particles

We present here raw trajectories of molecular dynamics simulations for nucleosome with linker DNA strands as well as minimalistic nucleosome core particle model. The simulations were done in explicit solvent using CHARMM36 force field. We used this data in the research article Shaytan et al., 2016 [1]. The trajectory files are supplemented by TCL scripts providing advanced visualization capabilities. Keywords: Molecular dynamics, Nucleosome, Linker DNA, Histone, Histone tail

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