Caratterizzazione strutturale e funzionale della topoisomerasi IB umana e dei suoi mutanti

Human DNA topoisomerase IB (topo IN) is an essential monomeric enzyme involved in resolving the torsional stress assiciated with DNA replication, trascription and chromatin condensation. Topo IB catalyzes changes in the supehelical state of the duplex DNA by transiently breaking a single strand allowing the broken strand to move through the break. Phosphodiester bond energy is preserved during catalysis through the formation of a transient phosphotyrosine bond between the active-site tyrosine and the 3' end of the broken strand. According to X-ray crystallography and biochemical analysyses topo IB is organized into four domains: N-terminal domain,core, linker and C-terminal domain which contains the active site Tyr723. The topo IB is also an important target of the antitumor drug camptothecin for chemotherapy of uman cancers. The biochemical and structural-dynamical properties of the Asp677Gly-Val703Ile double mutant displaying a pronounced CPT sensitivity as been investigated to better understad the action mechanism of the enzyme and the interaction of CPT with the cleavable topoI-DNA complex. The results have shown that the CPT hypersensitivity of the mutations its done to a reduction of its religation rate and demonstrated the occurrence of a long range communication between domains localized far away one from the other.The inhibition efficiency on human topo IB wild type of the cEPA has been investigated analyzing the different steps of the enzyme catalytic cycle. The experiments show that cEPA has a mechanism different from CPT, inhibiting the cleavage of the enzyme on the DNA although permitting the non covalent enzyme-DNA interaction. The human topoI N-terminal domain is the only region of the enzyme with an unknown 3D structure since it is not possible to crystallize it. This domain has been overproduced into E.coli BL21,to carry out preliminary spectroscopic analysis consisting of CD, fluorescence and NMR spectra. The spectra indicate an equilibrium between the structured and unstructured regions. We are now producing the isotope labeled protein,growing the bacterial cells in minimum medium containing N15,in the attempt to gain further structural information by high-field 2D NMR spectroscopy.