Functional expression of a DNA-topoisomerase IB from Cryptosporidium parvum.
Cryptosporidium parvum, one of the most important causative organisms of human diarrheas during childhood, contains a monomeric DNA-topoisomerase IB (CpTopIB) in chromosome 7. Heterologous expression of CpTopIB gene in a budding yeast strain lacking this activity proves that the cryptosporidial enzyme is functional in vivo. The enzymatic activity is comprised in a single polypeptide, which contains all the structural features defining a fully active TopIB. Relaxation activity of the yeast extracts was detected only when CpTopIB ORF was expressed in a yeast expression system showing time and protein dependence under steady state kinetic conditions. The susceptibility of CpTopIB-transformed yeast to the irreversible inhibitor camptothecin and its water-soluble derivatives (topotecan and SN-38) was assessed.
Ordóñez
César
C
Departamento de Ciencias Biomédicas (INTOXCAL), Universidad de León, Campus de Vegazana s/n, 24071 León, Spain.
Alfonso
Javier
J
Balaña-Fouce
Rafael
R
Ordóñez
David
D
D000595:Amino Acid Sequence; D000818:Animals; D002166:Camptothecin / Q000494:pharmacology; D016785:Cryptosporidium parvum / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects; D016415:Sequence Alignment; D019772:Topotecan / Q000494:pharmacology
D050505:Mutant Proteins; D019772:Topotecan; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin