deletion study of dna topoisomerase ib from leishmania donovani searching for a minimal functional heterodimer删除的研究dna拓扑异构酶ib杜氏利什曼虫寻找最小功能异质二聚体.pdfVIP

Deletion Study of DNA Topoisomerase IB from Leishmania donovani: Searching for a Minimal Functional Heterodimer ´ ´ ´ ´ ˜ ˜ Rosario Dıaz Gonzalez, Yolanda Perez Pertejo, David Ordonez, Rafael Balana-Fouce*, Rosa M. Reguera ´ ´ ´ ´ Departamento de Farmacologıa y Toxicologıa (INTOXCAL), Universidad de Leon, Leon, Spain The substantial differences between trypanosomal and leishmanial DNA topoisomerase IB concerning to their homologues in mammals have provided a new lead in the study of the structural determinants that can be effectively targeted. Leishmania donovani, the causative agent of visceral leishmaniasis, contains an unusual heterodimeric DNA topoisomerase IB. The catalytically active enzyme consists of a large subunit (LdTopIL), which contains the non-conserved N-terminal end and the phylogenetically conserved ‘‘core’’ domain, and of a small subunit (LdTopIS) which harbors the C-terminal region with the characteristic tyrosine residue in the active site. Heterologous co-expression of LdTopIL and LdTopIS genes in a topoisomerase I deficient yeast strain, reconstitutes a fully functional enzyme LdTopIL/S which can be used for structural studies. An approach by combinatorial cloning of deleted genes encoding for truncated versions of both subunits was used in order to find out structural insights involved in enzyme activity or protein-protein interaction. The role played by the non-conserved N-terminal extension of LdTopIL in both relaxation activity and CPT sensitivity has been examined co-expressing the full-length LdTopIS and a fully active LdTopIDS deletion with several deletions of LdTopIL lacking growing sequences of the N-terminal end. The sequential deletion study shows that the first 26 amino acids pl