Improved Methods for Preparing the Telomere Tethering Complex Bqt1-Bqt2 for Structural Studies.

In eukaryotes, chromosome ends (telomeres) are tethered to the inner nuclear membrane. During the early stages of meiosis, telomeres move along the nuclear membrane and gather near the spindle-pole body, resulting in a bouquet-like arrangement of chromosomes. This chromosomal configuration appears to be widely conserved among eukaryotes, and is assumed to play an important role in the normal progression of meiosis, by mediating the proper pairing of homologous chromosomes. In fission yeast, the Bqt1-Bqt2 protein complex plays a key role in tethering the telomere to the inner nuclear membrane. However, the structural details of the complex required to clarify how telomeres are gathered near the spindle-pole body remain enigmatic. Previously, we devised a preparation procedure for the Schizosaccharomyces japonicus Bqt1-Bqt2 complex, in which a SUMO tag was fused to the N-terminus of the Bqt1 protein. This allowed us to purify the Bqt1-Bqt2 complex from the soluble fraction. In the present study, we found that a maltose-binding protein homolog, Athe_0614, served as a better fusion partner than the SUMO protein, resulting in the marked increase in the solubility of the Bqt1-Bqt2 complex. The Athe_0614 fusion partner may open up new avenues for X-ray crystallographic analyses of the structure of the Bqt1-Bqt2 complex.

Structure determination of the nucleosome core particle by selenium SAD phasing.

The eukaryotic genome is compacted inside the nucleus of the cell in the form called chromatin. The fundamental unit of chromatin is the nucleosome, which contains four types of histones (H3, H4, H2A and H2B) and approximately 150 base pairs of DNA wrapped around the histone complex. The structure of the nucleosome is highly conserved across several eukaryotic species, and molecular replacement has been the primary phasing method used to solve nucleosome structures by X-ray crystallography. However, there is currently no simple, widely applicable experimental phasing method for the nucleosome. In the present study, it is demonstrated that selenomethionine-incorporated histones H3, H2A and H2B can be reconstituted into nucleosomes and crystallized for structural determination. Unexpectedly, it was found that the nucleosome can be phased with a relatively small number of Se atoms. The structures of nucleosome core particles containing 12 and 16 Se atoms were solved by SAD phasing at 2.5 and 2.4 Šresolution, respectively. The present study demonstrates a simple method for determining nucleosome structures by experimental phasing, which may be particularly useful for noncanonical structures that cannot be solved by molecular replacement.