Directed evolution to increase camptothecin sensitivity of human DNA topoisomerase I.
BACKGROUND
Human DNA topoisomerase I (top1) relaxes DNA supercoiling during basic nuclear processes. The enzyme is the main target of antitumor agents, such as camptothecins (CPT), that transform top1 into a DNA-damaging agent.


RESULTS
By directed evolution of a C-terminal portion, we selected human top1 mutants that were 22-28-fold more CPT-sensitive than wild-type top1 in Saccharomyces cerevisiae cells. The evolved enzymes showed unique mutation patterns and were more processive in plasmid relaxation assays. A top1 mutant had only two amino acid changes in the linker domain, one of which may change a linker/core domain contact surface. The mutant stimulated DNA cleavage to higher levels than the wild-type enzyme and was more sensitive to CPT in a cleavage assay. Moreover, the mutant was more CPT-sensitive than wild-type top1 in a repair-deficient yeast strain.


CONCLUSIONS
Mutations in the linker domain can affect DNA binding and CPT sensitivity of human top1. Such drug-hypersensitive topoisomerases may be useful in developing DNA cutters with high cell lethality and in new drug discovery programs.
Scaldaferro
S
S
Department of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy.
Tinelli
S
S
Borgnetto
M E
ME
Azzini
A
A
Capranico
G
G
D015153:Blotting, Western; D002166:Camptothecin / Q000494:pharmacology*; D002874:Chromosome Mapping; D004260:DNA Repair; D004264:DNA Topoisomerases, Type I / Q000235:genetics*; D019020:Directed Molecular Evolution; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015723:Gene Library; D006801:Humans; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA; D059004:Topoisomerase I Inhibitors
D004791:Enzyme Inhibitors; D059004:Topoisomerase I Inhibitors; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin