Base Pair Precision Research Articles

Photon Cascade with Clip‐On Fluorophores

Bird on a wire: a recent study by Rant and Burley showed that it is possible to transfer excited-state energy along double-stranded DNA by arranging fluorophore relays with base-pair precision through Dervan-polyamides in a cascade-like process. The resulting assembly acts as the largest DNA-based photonic wire yet.

Unzipping Single DNA Molecules to Study the Structure of the Yeast Centromeric Nucleosome

Each centromere provides the essential locus for microtubule attachment on each chromosome during mitosis and meiosis in all eukaryotes. The centromeric nucleosome, the fundamental unit that makes up all centromeres, is distinct from conventional nucleosomes due to its lack of canonical H3 histones in place of a centromeric H3 histone variant (CenH3). Histone variants potentially change the structural properties of chromatin significantly and, therefore, impact chromosomal processes such as chromatid separation, pairing, etc. The structure of centromeric nucleosomes is widely debated, with main competing models proposed. In an effort to resolve the controversy, we have applied a unique single molecule technique that employs an optical trap to probe the force applied to unzip a single DNA molecule as its double strand is converted into two single strands. This produces a sequential map of the location of individual histone-DNA interactions with near base pair precision and accuracy. We have analyzed and compared the unzipping signatures of assembled recombinant canonical and centromeric yeast (Cse4) histones. Our results reveal features that are unique to centromeric structures in vitro and have implications for functional behavior in vivo.

Architectural specificity in chromatin structure at the TATA box in vivo: nucleosome displacement upon beta-phaseolin gene activation.

Extensive studies of the beta-phaseolin (phas) gene in transgenic tobacco have shown that it is highly active during seed embryogenesis but is completely silent in leaf and other vegetative tissues. In vivo footprinting revealed that the lack of even basal transcriptional activity in vegetative tissues is associated with the presence of a nucleosome that is rotationally positioned with base pair precision over three phased TATA boxes present in the phas promoter. Positioning is sequence-dependent because an identical rotational setting is obtained upon nucleosome reconstitution in vitro. A comparison of DNase I and dimethyl sulfate footprints in vivo and in vitro strongly suggests that this repressive chromatin architecture is remodeled concomitant with gene activation in the developing seed. This leads to the disruption of histone-mediated DNA wrapping and the assembly of the TATA boxes into a transcriptionally competent nucleoprotein complex.

Single Base-pair Precision and Structural Rigidity in a Small IHF-induced DNA Loop

The prokaryotic integration host factor (IHF) is a DNA-binding protein that binds to specific DNA sites as a heterodimer. Genetic and mutational analyses have previously identified asymmetric protein-DNA contacts by the individual subunits. By exploiting the unique sequence and positional context of one IHF binding site, H′ in Lambda attachment sites ( attsites), we have identified a symmetry element of binding and have localized the functional bend center to the center of this symmetry. A shift of the H′ bend center by a single base-pair to the right or to the left within the very tight loop formed with Lambda integrase (int) and IHF in att-site “intrasomes” severely reduces recombination. This suggests that a precise, but wrongly positioned, DNA bend within a loop constant length negatively influences the juxtaposition or “phasing” of the core-type and arm-type Int binding sites by differentially affecting the length of each leg of the loop. Furthermore, ten base-pair insertions within this loop that should not interfere with correct helical phasing are sensed in a position-dependent manner. Distal insertions abolish recombination, whereas proximal or double insertions (in both legs of the loop) are well tolerated.

Nucleosomes are positioned with base pair precision adjacent to the alpha 2 operator in Saccharomyces cerevisiae.

Analysis of the chromatin structure of minichromosomes containing the binding site for the yeast alpha 2 repressor protein by indirect end-labeling has previously indicated that nucleosomes are stably positioned over sequences adjacent to the alpha 2 operator in the presence of the repressor. Development of a primer extension assay for nucleosome position now allows a more detailed examination of the location of these nucleosomes relative to the operator sequence, and indicates that nucleosomes are precisely and stably positioned both translationally and rotationally over sequences adjoining the operator. In addition, this assay enables analysis of the chromatin structure of single copy, genomic sequences. Chromatin structures determined for two genes regulated by alpha 2, STE6 and BAR1, are consistent with nucleosomes precisely positioned downstream of the operator sequence, incorporating promoter elements, in alpha cells but not in a-cells. The location of these nucleosomes relative to the operator sequence is highly analogous to that observed in the minichromosome. The stability of the nucleosomes adjacent to the operator together with the precision of their location suggests that they may play a role in repression of a specific gene expression by alpha 2. Further, the primer extension assay allows a comparison of the structure of these positioned nucleosomes formed in vivo to that previously described for core particles reconstituted in vitro.