Nucleic Acids Research, 2005, Vol. 33, No. 20 6621–6634 doi:10.1093/nar/gki935 Rotation of DNA around intact strand in human topoisomerase I implies distinct mechanisms for positive and negative supercoil relaxation Levent Sari and Ioan Andricioaei* Department of Chemistry and The Program in Bioinformatics, University of Michigan, Ann Arbor, MI 48109, USA Received June 9, 2005; Revised August 1, 2005; Accepted October 12, 2005 ABSTRACT Topoisomerases are enzymes of quintessence to the upkeep of superhelical DNA, and are vital for replica- tion, transcription and recombination. An atomic- resolution model for human topoisomerase I in covalent complex with DNA is simulated using molecular dynamics with external potentials that mimic torque and bias the DNA duplex downstream of a single-strand cut to rotate around the intact strand, according to the prevailing enzymatic mech- anism. The simulations reveal the first dynamical picture of how topoisomerase accommodates large- scale motion of DNA as it changes its supercoiling state, and indicate that relaxation of positive and neg- ative supercoils are fundamentally different. To relax positive supercoils, two separate domains (the ‘lips’) of the protein open up by about 10–14 A˚ , whereas to relax negative supercoils, a continuous loop connect- ing the upper and lower parts (and which was a hinge for opening the lips) stretches about 12 A˚ while the lips remain unseparated. Normal mode analysis is addi- tionally used to characterize the functional flexibility of the protein. Remarkably, the same combination of low-frequency eigenvectors exhibit the dominant contribution for both rotation mechanisms through a see-saw motion. The simulated mechanisms sug- gest mutations to control the relaxation of either type of supercoiling selectively and advance a hypo- thesis for the debated role of the N-terminal domain in supercoil relaxation. INTRODUCTION Topoisomerases are enzymes with crucial role in maintain- ing the proper topology and physical integrity of DNA superhelical structures. They remove knots and catenanes gen- erated by unwinding at the replication fork, relieve torsional stress caused by supercoiling during DNA transcription or chromosome disentanglement, and are involved in recom- bination and repair (1–3). A precise understanding of their mechanism is important mainly for two reasons. Firstly, because topoisomerase inhibition cancels cell pro- liferation, human topo I is a major anti-cancer target (4,5). A novel class of topo I inhibitors (the camptothecin (CPT) ana- logues topotecan and CPT-11) are some of the most potent anti-cancer agents to date (6). Secondly, the chemical reaction performed by topoi- somerases induces movement of DNA segments several orders of magnitude larger than the size of the protein. For this reason, topoisomerases are prime case studies for understand- ing the triggering of large-scale mechanical motions induced by biomolecular motors, and in particular the effect that external forces or torques have in tuning such machines at the single-molecule level (7,8). Computer simulations of DNA–protein complexes under external forces (9), as virtual single-molecule experiments, are in a position suitable to suggest and explain actual single-molecule measurements on supercoiled DNA (10–15). This is particularly important since the two techniques (simu- lation and experiment) are complementary both in terms of spatial resolution and time scales. Four distinct subfamilies of topoisomerases have been identiﬁed, topo IA (16), IB (17), IIA (18), IIB (19), and sub- sequently classiﬁed according to their structure and func- tionality [recently reviewed in (3)]. Human topoisomerase I is a IB enzyme, an eukaryotic type discovered by Champoux and Dulbecco in the early 1970s (17). Topoisomerases (topos) of type I eliminate the supercoiled DNA stress by nicking one strand of the helix and passing the other strand through the nick. During strand passage, the enzyme attaches covalently to a DNA end at the nick by forming a phosphodiester bond with a tyrosine at the active site. Two models have been proposed for strand passage in type I topos: (i) the enzyme-bridging model (20) (in which the enzyme stabilizes a gate through *To whom correspondence should be addressed. Tel: +1 734 763 8013; Fax: +1 734 615 6553; Email: andricio@umich.edu The Author 2005. Published by Oxford University Press. All rights reserved. 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