Structural insights on the small subunit of DNA topoisomerase I from the unicellular parasite Leishmania donovani.
Leishmania donovani, the causative organism of visceral leishmaniasis, contains a unique heterodimeric DNA topoisomerase IB (LdTop1). The catalytically active enzyme consists of a large subunit (LdTop1L), which contains the non-conserved N-terminal end and a phylogenetically conserved core domain, and of a small subunit (LdTop1S) which harbours the C-terminal region with a characteristic tyrosine residue in the active site. Heterologous co-expression of LdTop1L and LdTop1S in a topoisomerase I deficient yeast strain, reconstitutes a fully functional enzyme which can be used for structural studies. The role played by the non-conserved N-terminal extension of LdTop1S in both relaxation activity and CPT sensitivity of LdTop1 has been examined co-expressing the full-length LdTop1L with several deletions of LdTop1S lacking growing sequences of the N-terminal end. The sequential deletion study shows that the first 174 amino acids of LdTop1S are dispensable in terms of relaxation activity and DNA cleavage. It is also described that the trapping of the covalent complex between LdTop1 and DNA by CPT requires a pentapeptide between amino acid residues 175 and 179 of LdTop1S. Our results suggest the crucial role played by the N-terminal extension of the small subunit of DNA topoisomerase I.
Díaz González
Rosario
R
Departamento de Farmacología y Toxicología (INTOXCAL), Universidad de León, Campus de Vegazana s/n; 24071 León, Spain.
Pérez Pertejo
Yolanda
Y
Redondo
Carmen M
CM
Pommier
Yves
Y
Balaña-Fouce
Rafael
R
Reguera
Rosa M
RM
D000595:Amino Acid Sequence; D000818:Animals; D002166:Camptothecin / Q000494:pharmacology; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D007893:Leishmania donovani / Q000201:enzymology*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism
D021122:Protein Subunits; D015800:Protozoan Proteins; D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin