Sequence dependence of nucleosomal DNA unwrapping

The first nucleosomes positioned around the transcription start site, +1 nucleosomes, play critical roles in transcriptional regulation. We previously exhibited that AA/TT is enriched in the entry sites of +1 nucleosomes in yeast, and AA/TT regions in nucleosomes are more susceptible to MNase. To further understand the molecular mechanisms, we performed FRET analysis of nucleosomes reconstituted with and without A-tract at the entry/exit sites. Regardless of the sequences, nucleosomes show similar amount of the wrapped state in low salt concentrations. However, we found salt-induced DNA unwrapping occurs more easily in the A-tract nucleosomes, indicating the transition energy from the wrapped to unwrapped state might be lower for the A-tract nucleosomes.第59回 日本生物物理学会年

Balance between DNA-binding affinity and specificity enables selective recognition of longer target sequences in vivo

Although genome-editing enzymes such as TALEN and CRISPR/Cas9 are being widely used, they have an essential limitation in that their relatively high molecular weight makes them unsuitable for capsulation. To develop a novel genome-editing enzyme with a smaller molecular weight, we focused on the engrailed homeodomain (EHD). We designed and constructed proteins composed of two EHDs connected by a linker to increase sequence specificity. In bacterial one-hybrid assays and EMSA analyses, the created proteins exhibited good affinity for DNA sequences consisting of two tandemly aligned EHD target sequences. However, they also bound to individual EHD targets. To avoid binding to single target sites, we introduced amino acid mutations to reduce the protein-DNA affinity of each EHD monomer and successfully created a small protein with high specificity for tandem EHD target sequences

DNA conformational transitions inferred from reevaluation of m|Fo|-D|Fc| electron density maps

Conformational flexibility of DNA plays important roles in biological processes such as transcriptional regulation and DNA packaging etc. To understand the mechanisms of these processes, it is important to analyse when, where and how DNA shows conformational variations. Recent analyses have indicated that conventional refinement methods do not always provide accurate models of crystallographic heterogeneities and that some information on polymorphism has been overlooked in previous crystallographic studies. In the present study, the m|Fo| D|Fc| electron-density maps of double-helical DNA crystal structures were calculated at a resolution equal to or better than 1.5 A ˚ and potential conformational transitions were found in 27% of DNA phosphates. Detailed analyses of the m|Fo| D|Fc| peaks indicated that some of these unassigned densities correspond to ZI $ ZII or A/B ! BI conformational transitions. A relationship was also found between ZI/ZII transitions and metal coordination in Z-DNA from the detected peaks. The present study highlights that frequent transitions of phosphate backbones occur even in crystals and that some of these transitions are affected by the local molecular environment

Local Conformational Changes in the DNA Interfaces of Proteins

<div><p>When a protein binds to DNA, a conformational change is often induced so that the protein will fit into the DNA structure. Therefore, quantitative analyses were conducted to understand the conformational changes in proteins. The results showed that conformational changes in DNA interfaces are more frequent than in non-interfaces, and DNA interfaces have more conformational variations in the DNA-free form. As expected, the former indicates that interaction with DNA has some influence on protein structure. The latter suggests that the intrinsic conformational flexibility of DNA interfaces is important for adjusting their conformation for DNA. The amino acid propensities of the conformationally changed regions in DNA interfaces indicate that hydrophilic residues are preferred over the amino acids that appear in the conformationally unchanged regions. This trend is true for disordered regions, suggesting again that intrinsic flexibility is of importance not only for DNA binding but also for interactions with other molecules. These results demonstrate that fragments destined to be DNA interfaces have an intrinsic flexibility and are composed of amino acids with the capability of binding to DNA. This information suggests that the prediction of DNA binding sites may be improved by the integration of amino acid preference for DNA and one for disordered regions.</p> </div

Conformational changes and variations of fragments.

<p>(a) Frequencies of conformational change upon DNA binding for DNA interfaces and non-interfaces. (b) Frequencies of conformational variation in the DNA-free forms for DNA interfaces and non-interfaces. Error bar indicates the 85% bootstrap confidence interval.</p

Propensities of the structural alphabets in the DNA interfaces vs. non-interfaces.

<p>(a) Conformational change upon DNA binding. (b) Conformational variation in the DNA-free form. Error bar indicates the 85% bootstrap confidence interval.</p