The Catalytic Mechanism Research Of RNase ? From Staphylococcus Aureus And The Interaction Study Of Kinetochore Protein Mis12 Complex With Related Proteins

Double-stranded RNAs (dsRNA) elements can be formed by base-pairing between complementary transcripts. They are involved in many cellular process, and produce a variety of biological effects. In addition, dsRNA is the genetic material of many viruses. In bacteria and eukaryotic cells, members of RNase ? (endoribonuclease ?) family can selectively recognize and cleave dsRNA. RNase ? are phosphodiesterase that depend on divalent metal ions. The enzymes play an important role in gene expression and regulation, host defense and genomic monitoring. As one of the key steps for maturation and decay processes of encoding RNAs and non-coding RNAs, the processing of dsRNA by RNase ? plays a significant role in the production of miRNA and siRNA. RNase ? family members share the same fold, a unique RNase ? domain, which is capable to form a dimer to bind dsRNA, and cleave phosphodiester bond of both chains, giving rise to products with 2nt 3'protruding ends.In order to understand the similarities and differences of RNase ? between S. aureus and other species, we solved the structure of S. aureus RNase ?. We found that the overall structure of Sa-RNase ? showed a high similarity to that of other bacterial RNase ?. Two RNase ? domains formed a tight dimer through hydrophobic interactions, and the key residues of the active site were highly conserved. Sa-RNase ? could hydrolyze dsRNA substrates in our biochemical studies, and its catalytic efficiency varied on different metal ions. The enzyme appeared to be more active in the presence of Mn2+ compared to Mg2+, while Ca2+ and Ni2+ inhibited its activity completely. Mutations of key residues at active site caused severe reduction on the activity of Sa-RNase ?.Kinetochore is a large protein assemblies that assembly at the centromere during mitosis. Kinetochore mediates the binding of chromosomes to mitotic spindles, promoting an accurate separation of sister chromatids. It mainly includes the inner protein-network called CCAN and the outer protein-network called KMN. KMN network is composed of Kn11 protein, Mis 12 complex and Ndc80 complex. Mis 12 complex is a bridge that connects inner kinetochore with outer kinetochore. Mis 12 complex is recruited to kinetochores by interaction with CENP-C, and it further recruits other outer kinetochore components and promotes the connections of kinetochore with microtubules by interaction with Knll and Ndc80 complex. To date, although there are many studies on the outer kinetochore, how the interaction occurred between Mis 12 complex and Ndc80 complex within the KMN network is still unclear. In meiosis I, it is termed mono-orientation that kinetochores on both sister chromatids bind to microtubules emanating from the same spindle pole. In Saccharomyces cerevisiae, mono-orientation mainly depends on a four component protein complex referred to as monopolin complex. Monopolin complex binds to kinetochores during meiosis I and prevents bipolar attachment. However, its binding site on the kinetochores is unknown and the binding mechanism has not been established. Recent studies have shown that the interaction of monopolin subunit Csml with MIND complex subunit Dsnl, is essential for both the association of monopolin with kinetochores and monopolar attachment of sister kinetochores during meiosis ?.We expressed and purified Human Mis12C subunit Dsnl/Nsll truncations and Human/fission yeast Ndc80bonsai complex in high purity. By in vitro pull-down experiment, we tried to figure out in both human and fission yeast how the Mis 12 complex interact with Ndc80 complex. In addition, we expressed and purified Saccharomyces cerevisiae Csm1 and Dsn1 proteins, and verified the interaction between them by biochemical experiments in vitro. Crystallization and crystal optimization were also set up of Sc-Csml with Dsnl peptide.