The Structural Biology Research Of Rnase H2 From Staphylococcus Aureus

RNA/DNA hybrid is formed during replication and transcription of DNA. It may lead to DNA damage and potentially deleterious for genome stability. Ribonuclease H2 (RNases H2) is a ubiquitous endonuclease that exits in both prokaryotes and eukaryotes. It specifically digests the RNA moiety in an RNA/DNA hybrid by cleaving the phosphodiester bond. RNase H2 plays an important role in removal of RNA primers of Okazaki fragment and resolving transcription related R-loops. RNase H2 contains a conserved N-terminal which includes the active site that consists of four conserved carboxylates "DEDD".The catalytic activity of RNases H2 requires the coordination of two divalent metals.To study the structure and function of Staphylococcus aureus RNase H2, we solved the crystal structure of RNase H2 and further investigated the function of RNase H2 by in vitro activity assays.In parti,we expressed, purified and crystallized the full-length RNase H2 from S.aureus and then solved the crystal structure at 2.2 A resolution. Different from RNase H2 homologous in other species existing as a monomer, S. aureus RNase H2 forms a unique dimer in solution. The structural comparison shows that the overall structure of S. aureus RNase H2 is similar to its homologous with an N-terminal domain folded to the catalytic core (RNase H-fold) and a C-terminal helix-loop-helix domain. However, the active sites of two S. aureus RNase H2 monomers are buried in the interface when form a "self-inhibition"-form dimer. Further biochemical studies show that the dimeric S. aureus RNase H2 is active and capable of catalyzing different kinds of RNA/DNA hybrids. The catalytic efficiency is different with the addition of different metal ions. RNases H2 is more active in the presence of Mg2+. Our results suggest that the dimeric form of S. aureus RNase H2 may undergo a big conformational change or change to monomer to expose the active site for the catalytic reaction upon the addition of substrate.Kinetochore is a large protein network assembled at the periphery of centromeric chromatin.Kinetochore consists of inner kinetochore proteins forming a constitutive centromere associated network (CCAN),and outer kinetochore proteins forming a KMN network (composed of KNL1, Mis 12 and Ndc80 complexes), respectively. Mis 12 complex(Mis 12, Nnfl, Dsnl and Nsll) was reported to interact with the inner kinetochore protein CENP-C and form the KMN network to interact with Ndc80 complex and KNL1 complex. Mis12 complex functions as a core for the assembly of kinetochore and a bridge to link kinetochore to microtubule via its interactions with CENP-C and Ndc80 complex. However, very little is known about how Mis12 complex interact with CENP-C or Ndc80 complex.In part 2, we expressed and purified Mis 12 complex and its mutants from Schizosaccharomyces pombe and Saccharomyces cerevisiae and tested the interaction of Mis 12 complex with Ndc80 complex and Cnp3(CENP-C homolog). Additionly, we expressed and purified recombinated Arkl for crystallization and in vitro phosphorylation experiments.