The different cleavage DNA sequence specificity explains the camptothecin resistance of the human topoisomerase I Glu418Lys mutant.
Yeast cells expressing the Glu418Lys human topoisomerase I mutant display a camptothecin resistance that slowly decreases as a function of time. Molecular characterization of the single steps of the catalytic cycle of the purified mutant indicates that it has a relaxation activity identical to the wild-type protein but a different DNA sequence specificity for the cleavage sites when compared to the wild-type enzyme, as assayed on several substrates. In particular the mutant has a low specificity for CPT sensitive cleavable sites. In fact, the mutant has, at variance of the wild-type enzyme, a reduced preference for cleavage sites having a thymine base in position -1 of the scissile strand. This preference, together with the strict requirement for a thymine base in position -1 for an efficient camptothecin binding, explains the temporary camptothecin resistance of the yeast cell expressing the mutant and points out the importance of the DNA sequence in the binding of the camptothecin drug.
Fiorani
Paola
P
CNR National Research Council, University of Rome Tor Vergata, Via Della Ricerca Scientifica, Rome 00133, Italy.
Chillemi
Giovanni
G
Losasso
Carmen
C
Castelli
Silvia
S
Desideri
Alessandro
A
D019943:Amino Acid Substitution; D001483:Base Sequence; D002166:Camptothecin / Q000494:pharmacology*; D002384:Catalysis; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D008239:Lysine / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D018698:Glutamic Acid; D004247:DNA; D004264:DNA Topoisomerases, Type I; D008239:Lysine; D002166:Camptothecin