Telomeric chromatin structure.

Eukaryotic DNA is packaged into nucleosomes, which further condenses into chromosomes. The telomeres, which form the protective end-capping of chromosomes, play a pivotal role in ageing and cancer. Recently, significant advances have been made in understanding the nucleosomal and telomeric chromatin structure at the molecular level. In addition, recent studies shed light on the nucleosomal organisation at telomeres revealing its ultrastructural organisation, the atomic structure at the nucleosome level, its dynamic properties, and higher-order packaging of telomeric chromatin. Considerable advances have furthermore been made in understanding the structure, function and organisation of shelterin, telomerase and CST complexes. Here we discuss these recent advances in the organisation of telomeric nucleosomes and chromatin and highlight progress in the structural understanding of shelterin, telomerase and CST complexes.

Columnar structure of human telomeric chromatin.

Telomeres, the ends of eukaryotic chromosomes, play pivotal parts in ageing and cancer and are targets of DNA damage and the DNA damage response1-5. Little is known about the structure of telomeric chromatin at the molecular level. Here we used negative stain electron microscopy and single-molecule magnetic tweezers to characterize 3-kbp-long telomeric chromatin fibres. We also obtained the cryogenic electron microscopy structure of the condensed telomeric tetranucleosome and its dinucleosome unit. The structure displayed close stacking of nucleosomes with a columnar arrangement, and an unusually short nucleosome repeat  length that comprised about 132 bp DNA wound in a continuous superhelix around histone octamers. This columnar structure is primarily stabilized by the H2A carboxy-terminal and histone amino-terminal tails in a synergistic manner. The columnar conformation results in exposure of the DNA helix, which may make it susceptible to both DNA damage and the DNA damage response. The conformation also exists in an alternative open state, in which one nucleosome is unstacked and flipped out, which exposes the acidic patch of the histone surface. The structural features revealed in this work suggest mechanisms by which protein factors involved in telomere maintenance can access telomeric chromatin in its compact form.

Molecular dynamics simulations demonstrate the regulation of DNA-DNA attraction by H4 histone tail acetylations and mutations.

The positively charged N-terminal histone tails play a crucial role in chromatin compaction and are important modulators of DNA transcription, recombination, and repair. The detailed mechanism of the interaction of histone tails with DNA remains elusive. To model the unspecific interaction of histone tails with DNA, all-atom molecular dynamics (MD) simulations were carried out for systems of four DNA 22-mers in the presence of 20 or 16 short fragments of the H4 histone tail (variations of the 16-23 a. a. KRHRKVLR sequence, as well as the unmodified fragment a. a.13-20, GGAKRHRK). This setup with high DNA concentration, explicit presence of DNA-DNA contacts, presence of unstructured cationic peptides (histone tails) and K(+) mimics the conditions of eukaryotic chromatin. A detailed account of the DNA interactions with the histone tail fragments, K(+) and water is presented. Furthermore, DNA structure and dynamics and its interplay with the histone tail fragments binding are analysed. The charged side chains of the lysines and arginines play major roles in the tail-mediated DNA-DNA attraction by forming bridges and by coordinating to the phosphate groups and to the electronegative sites in the minor groove. Binding of all species to DNA is dynamic. The structure of the unmodified fully-charged H4 16-23 a.a. fragment KRHRKVLR is dominated by a stretched conformation. The H4 tail a. a. fragment GGAKRHRK as well as the H4 Lys16 acetylated fragment are highly flexible. The present work allows capturing typical features of the histone tail-counterion-DNA structure, interaction and dynamics.

Principles of electrostatic interactions and self-assembly in lipid/peptide/DNA systems: applications to gene delivery.

Recently, great progress has been achieved in development of a wide variety of formulations for gene delivery in vitro and in vivo, which include lipids, peptides and DNA (LPD). Additionally, application of natural histone-DNA complexes (chromatin) in combination with transfection lipids has been suggested as a potential route for gene delivery (chromofection). However, the thermodynamic mechanisms responsible for formation of the ternary lipid-peptide-DNA supramolecular structures have rarely been analyzed. Using recent experimental studies on LPD complexes (including mixtures of chromatin with cationic lipids) and general polyelectrolyte theory, we review and analyze the major determinants defining the internal structure, particle composition and size, surface charge and ultimately, transfection properties of the LPD formulations.

Structure and internal organization of overcharged cationic-lipid/peptide/DNA self-assembly complexes.

The combination of cationic lipids with cationic peptides and DNA vectors can produce synergistic effects in gene delivery to eukaryotic cells. Binary complexes of cationic lipids with DNA are well-studied whereas little information is available about the structure of the ternary lipid/peptide/DNA (LPD) complexes and mechanisms defining DNA protection and delivery. Here we use synchrotron small angle X-ray scattering and dynamic light scattering zeta-potential measurements to determine structure and the net charge of supramolecular aggregates of complexes in mixtures of plasmid DNA, cationic liposomes formed from DOTAP, plus a linear cationic epsilon-oligolysine with the pendant alpha-amino acids Leu-Tyr-Arg (LYR), epsilon-(LYR)K10. These ternary complexes display multilamellar structures with relatively constant separation between DOTAP bilayers, accommodating a hydrated monolayer of parallel DNA rods. The DNA-DNA distance in the complexes varies as a function of the net positive to negative (lipid + peptide)/DNA charge ratio. An explanation for the observed dependence of DNA-DNA distance on charge ratio was proposed based on general polyelectrolyte properties of non-stoichiometric polycation-DNA mixtures.

Design and biophysical characterization of novel polycationic epsilon-peptides for DNA compaction and delivery.

Design and solid-phase synthesis of novel and chemically defined linear and branched -oligo( l-lysines) (denoted -K n, where n is the number of lysine residues) and their alpha-substituted homologues (epsilon-(R)K10, epsilon-(Y)K10, epsilon-(L)K10, epsilon-(YR)K10, and epsilon-(LYR)K10) for DNA compaction and delivery are reported. The ability to condense viral (T2 and T4) and plasmid DNA as well as the size of -peptide DNA complexes under different conditions was investigated with static and dynamic light scattering, isothermal titration calorimetry, and fluorescence microscopy. Nanoparticle diameters varied from 100 to 150 and 375 to 550 nm for plasmid and T4 DNA peptide complexes, respectively. Smaller sizes were observed for oligo(L-lysines) compared to alpha-poly( L-lysine). The linear -oligo-lysines are less toxic and epsilon-(LYR)K10 showed higher transfection efficiency in HeLa cells than corresponding controls. The results also demonstrate that with a branched design having pendent groups of short alpha-oligopeptides, improved transfection can be achieved. This study supports the hypothesis that available alpha-oligolysine derived systems would potentially have more favorable delivery properties if they are based instead on epsilon-oligo( L-lysines). The flexible design and unambiguous synthesis that enables variation of pendent groups holds promise for optimization of such -peptides to achieve improved DNA compaction and delivery.