Abstract
Eukaryotic topoisomerases I (TOP1) are ubiquitous enzymes removing DNA torsional stress. However, there is little data concerning the three-dimensional structure of TOP1 in the absence of DNA, nor how the DNA molecule can enter/exit its closed conformation. Here, we solved the structure of thermostable archaeal Caldiarchaeum subterraneum CsTOP1 in an apo-form. The enzyme displays an open conformation resulting from one substantial rotation between the capping (CAP) and the catalytic (CAT) modules. The junction between these two modules is a five-residue loop, the hinge, whose flexibility permits the opening/closing of the enzyme and the entry of DNA. We identified a highly conserved tyrosine near the hinge as mediating the transition from the open to closed conformation upon DNA binding. Directed mutagenesis confirmed the importance of the hinge flexibility, and linked the enzyme dynamics with sensitivity to camptothecin, a TOP1 inhibitor targeting the TOP1 enzyme catalytic site in the closed conformation.
Highlights
Eukaryotic topoisomerases I (TOP1) are ubiquitous enzymes removing DNA torsional stress
The eukaryotic TOP1 structure has been solved with a DNA molecule, in a closed conformation (Fig. 1b)
Each TOP1 molecule folds and there is no significant difference between SeMet CsTOP1 and native CsTOP1
Summary
Eukaryotic topoisomerases I (TOP1) are ubiquitous enzymes removing DNA torsional stress. TOP1, known as swivelase or untwisting enzyme, can relax both positive and negative supercoils by nicking DNA, enabling the rotation of the broken DNA strand around the complementary intact strand of the DNA duplex and catalyzing the closing of the nick This activity is important during transcription to remove supercoils generated behind and ahead of the transcription bubble. Its molecular and cellular biology has been extensively studied because human TOP1 protein is the target of the widely used anticancer drugs, topotecan, and irinotecan, which are derived from the alkaloid camptothecin[8,9] These drugs are known to enter the catalytic site of TOP1 while it is covalently linked to the 3′-end of DNA, inhibiting the DNA rejoining step and dissociation of TOP1 from the end of the broken DNA10. The accumulation of TOP1-covalent complexes that are converted into double-strand breaks during replication is toxic, killing preferentially cancer cells, which often overexpress TOP111
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