Influence of nucleosome structure on the three-dimensional folding of idealized minichromosomes

AbstractBackground: The closed circular, multinucleosome-bound DNA comprising a minichromosome provides one of the best known examples of chromatin organization beyond the wrapping of the double helix around the core of histone proteins. This higher level of chain folding is governed by the topology of the constituent nucleosomes and the spatial disposition of the intervening protein-free DNA linkers.Results: By simplifying the protein–DNA assembly to an alternating sequence of virtual bonds, the organization of a string of nucleosomes on the minichromosome can be treated by analogy to conventional chemical depictions of macromolecular folding in terms of the bond lengths, valence angles, and torsions of the chain. If the nucleosomes are evenly spaced and the linkers are sufficiently short, regular minichromosome structures can be identified from analytical expressions that relate the lengths and angles formed by the virtual bonds spanning the nucleosome-linker repeating units to the pitch and radius of the organized quaternary structures that they produce.Conclusions: The resulting models with 4–24 bound nucleosomes illustrate how a minichromosome can adopt the low-writhe folding motifs deduced from biochemical studies, and account for published images of the 30 nm chromatin fiber and the simian virus 40 (SV40) nucleohistone core. The marked sensitivity of global folding to the degree of protein–DNA interactions and the assumed nucleosomal shape suggest potential mechanisms for chromosome rearrangements upon histone modification

Structure, Dynamics, and Branch Migration of a DNA Holliday Junction: A Single-Molecule Fluorescence and Modeling Study

AbstractThe Holliday junction (HJ) is a central intermediate of various genetic processes, including homologous and site-specific DNA recombination and DNA replication. Elucidating the structure and dynamics of HJs provides the basis for understanding the molecular mechanisms of these genetic processes. Our previous single-molecule fluorescence studies led to a model according to which branch migration is a stepwise process consisting of consecutive migration and folding steps. These data led us to the conclusion that one hop can be more than 1 basepair (bp); moreover, we hypothesized that continuous runs over the entire sequence homology (5 bp) can occur. Direct measurements of the dependence of the fluorescence resonance energy transfer (FRET) value on the donor-acceptor (D-A) distance are required to justify this model and are the major goal of this article. To accomplish this goal, we performed single-molecule FRET experiments with a set of six immobile HJ molecules with varying numbers of bps between fluorescent dyes placed on opposite arms. The designs were made in such a way that the distances between the donor and acceptor were equal to the distances between the dyes formed upon 1-bp migration hops of a HJ having 10-bp homology. Using these designs, we confirmed our previous hypothesis that the migration of the junction can be measured with bp accuracy. Moreover, the FRET values determined for each acceptor-donor separation corresponded very well to the values for the steps on the FRET time trajectories, suggesting that each step corresponds to the migration of the branch at a defined depth. We used the dependence of the FRET value on the D-A distance to measure directly the size for each step on the FRET time trajectories. These data showed that one hop is not necessarily 1 bp. The junction is able to migrate over several bps, detected as one hop and confirming our model. The D-A distances extracted from the FRET properties of the immobile junctions formed the basis for modeling the HJ structures. The composite data fit a partially opened, side-by-side model with adjacent double-helical arms slightly kinked at the four-way junction and the junction as a whole adopting a global X-shaped form that mimics the coaxially stacked-X structure implicated in previous solution studies

The RNA backbone plays a crucial role in mediating the intrinsic stability of the GpU dinucleotide platform and the GpUpA/GpA miniduplex

The side-by-side interactions of nucleobases contribute to the organization of RNA, forming the planar building blocks of helices and mediating chain folding. Dinucleotide platforms, formed by side-by-side pairing of adjacent bases, frequently anchor helices against loops. Surprisingly, GpU steps account for over half of the dinucleotide platforms observed in RNA-containing structures. Why GpU should stand out from other dinucleotides in this respect is not clear from the single well-characterized H-bond found between the guanine N2 and the uracil O4 groups. Here, we describe how an RNA-specific H-bond between O2′(G) and O2P(U) adds to the stability of the GpU platform. Moreover, we show how this pair of oxygen atoms forms an out-of-plane backbone ‘edge’ that is specifically recognized by a non-adjacent guanine in over 90% of the cases, leading to the formation of an asymmetric miniduplex consisting of ‘complementary’ GpUpA and GpA subunits. Together, these five nucleotides constitute the conserved core of the well-known loop-E motif. The backbone-mediated intrinsic stabilities of the GpU dinucleotide platform and the GpUpA/GpA miniduplex plausibly underlie observed evolutionary constraints on base identity. We propose that they may also provide a reason for the extreme conservation of GpU observed at most 5′-splice sites