Alterations in linker flexibility suppress DNA topoisomerase I mutant-induced cell lethality.
Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of a covalent enzyme-DNA intermediate, which is reversibly stabilized by the anticancer agent camptothecin (CPT). Crystallographic studies of the 70-kDa C terminus of human Top1p bound to duplex DNA describe a monomeric protein clamp circumscribing the DNA helix. The structures, which lack the N-terminal domain, comprise the conserved clamp, an extended linker domain, and the conserved C-terminal active site Tyr domain. CPT bound to the covalent Top1p-DNA complex limits linker flexibility, allowing structural determination of this domain. We previously reported that mutation of Ala(653) to Pro in the linker increases the rate of enzyme-catalyzed DNA religation, thereby rendering Top1A653Pp resistant to CPT (Fiorani, P., Bruselles, A., Falconi, M., Chillemi, G., Desideri, A., and Benedetti P. (2003) J. Biol. Chem. 278, 43268-43275). Molecular dynamics studies suggested mutation-induced increases in linker flexibility alter Top1p catalyzed DNA religation. To address the functional consequences of linker flexibility on enzyme catalysis and drug sensitivity, we investigated the interactions of the A653P linker mutation with a self-poisoning T718A mutation within the active site of Top1p. The A653P mutation suppressed the lethal phenotype of Top1T718Ap in yeast, yet did not restore enzyme sensitivity to CPT. However, the specific activity of the double mutant was decreased in vivo and in vitro, consistent with a decrease in DNA binding. These findings support a model where changes in the flexibility or orientation of the linker alter the geometry of the active site and thereby the kinetics of DNA cleavage/religation catalyzed by Top1p.
Losasso
Carmen
C
Department of Biology, University of Padua, Padua 35131, Italy.
Cretaio
Erica
E
Department of Biology, University of Padua, Padua 35131, Italy.
Palle
Komaraiah
K
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38104.
Pattarello
Luca
L
Department of Biology, University of Padua, Padua 35131, Italy.
Bjornsti
Mary-Ann
MA
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38104.
Benedetti
Piero
P
Department of Biology, University of Padua, Padua 35131, Italy. Electronic address: pietro.benedetti@unipd.it.
D019943:Amino Acid Substitution / Q000187:drug effects / Q000235:genetics*; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D002166:Camptothecin / Q000494:pharmacology; D016923:Cell Death; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000502:physiology*; D025141:Drug Resistance, Fungal / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology; D059004:Topoisomerase I Inhibitors
D059004:Topoisomerase I Inhibitors; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin