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. 2010 Aug 4;99(3):869-78.
doi: 10.1016/j.bpj.2010.04.077.

Free energy calculations reveal rotating-ratchet mechanism for DNA supercoil relaxation by topoisomerase IB and its inhibition

Jeff Wereszczynski  1 , Ioan Andricioaei
Affiliations

Free energy calculations reveal rotating-ratchet mechanism for DNA supercoil relaxation by topoisomerase IB and its inhibition

Jeff Wereszczynski et al. Biophys J. .

Abstract

Topoisomerases maintain the proper topological state of DNA. Human topoisomerase I removes DNA supercoils by clamping a duplex DNA segment, nicking one strand at a phosphodiester bond, covalently attaching to the 3' end of the nick, and allowing the DNA downstream of the cut to rotate around the intact strand. Using molecular dynamics simulations and umbrella sampling free energy calculations, we show that the rotation of downstream DNA in the grip of the enzyme that brings about release of positive or negative supercoils occurs by thermally assisted diffusion on ratchet energy profiles. The ratchetlike free-energy-versus-rotation profile that we compute provides a model for the function of topoisomerase in which the periodic maxima along the profile modulate the rate of supercoil relaxation, while the minima provide metastable conformational states for DNA religation. The results confirm previous experimental and computational work, and suggest that relaxation of the two types of supercoils involves distinct protein pathways. Additionally, simulations performed with the ternary complex of topoisomerase, DNA, and the chemotherapeutic drug topotecan show important differences in the mechanisms for supercoil relaxation when the drug is present, accounting for the relative values of relaxation rates measured in single-molecule experiments. Good agreement is found between rate constants from tweezer experiments and those calculated from simulations. Evidence is presented for the existence of semiopen states of the protein, which facilitate rotations after the initial one, as a result of biasing the protein into a conformation more favorable to strand rotation than the closed state required for nicking of the DNA.

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Figures

Figure 1
Figure 1
Human topoisomerase I in complex with DNA. Protein is composed of four domains: the N-terminal (not resolved in crystal structures); the core domain (which is divided into three subdomains shown in red, orange, and yellow); the linker domain (shown in green); and the C-terminal domain (in blue). (A) View down the DNA axis. Protein resembles a Pac-Man with an upper cap and a lower base: subdomain I and II of the core form the upper cap, and subdomain III, together with the C-terminal domain, form the lower base of the Pac-Man. The Pac-Man-like enzyme opens its lips to remove positive supercoils and stretches its hinge when removing negative ones (see text). (B) View of the complex perpendicular to the DNA axis. A 22-basepair DNA segment is shown in gray. (C) The swiveling axis about which the DNA duplex downstream of the cut is rotated; the downstream DNA is actually running upward in this snapshot. (D) Same snapshot as in panel c, with the drug topotecan shown in its crystal-structure position, intercalating between the two DNA basepair stack flanking the nick, which displaces the rotating DNA part one flight up and imposes steric constraints during DNA rotation.
Figure 2
Figure 2
Free energy profiles along the DNA swiveling angle for the binary and ternary complexes reveal a thermally activated rotational diffusion over the barriers of a ratchetlike surface. The torque (positive or negative) tilts the surfaces to the right (clockwise rotation) or the left (anticlockwise), and drives relaxation of supercoils of the two signs. Minima on the surface (occurring after full-circle rotations) are conformations from where DNA backbone religation can occur; energy maxima modulate thermally assisted rotational diffusion rate for supercoil relaxation. The absolute value of the torque results in 5.8 kcal/mol/supercoil (corresponding to a tension of 0.2 pN; see text).
Figure 3
Figure 3
Schematic mechanism for the relaxation of DNA supercoils by human topoisomerase I which incorporates a semiopen configuration of the protein. Protein is represented in Pac-Man-like form with upper cap and lower base (in green) connected by a springlike hinge. (Yellow) DNA downstream of the cut; (red) DNA upstream of it. Note that positive supercoil relaxation is showcased here. For the relaxation of negative supercoils, the overall mechanism remains the same; however, the transition and semiopen states are replaced by configurations in which the hinge is opened as opposed to the lips, and the free energy profiles are distinct (see text).
Figure 4
Figure 4
Correlation functions of protein Cα atoms for positive and negative DNA supercoil relaxation, both for the free (top half) and inhibited (with TPT, bottom half) rotations. We observe that intercalation of TPT into the DNA helix fundamentally disrupts the protein motions that allow for efficient supercoil relaxation, and this disruption is more prevalent for negative supercoils than for positive ones.
Figure 5
Figure 5
Topoisomerase-DNA structures. (A) The initial closed conformation as observed in crystal structures. (B) A semiopen conformation observed for the relaxation of negative supercoils in which the hinge of the protein has opened to allow room for DNA rotation. (C) A semiopen conformation for the relaxation of positive supercoils in which the lips remain open after the first rotation. These semiopen conformations lower the free energy barrier for rotation compared to the initial closed conformation, thus providing a more favorable mechanism for supercoil relaxation. The semiopen states differ among themselves and from the closed state only by the overall placement of the upper cap relative to the lower base (as defined in the text); structures of the protein-composing domains are otherwise mainly unchanged.
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References

    1. Wang J.C. DNA topoisomerases. Annu. Rev. Biochem. 1996;65:635–692. - PubMed
    1. Champoux J.J. DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem. 2001;70:369–413. - PubMed
    1. Corbett K.D., Berger J.M. Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases. Annu. Rev. Biophys. Biomol. Struct. 2004;33:95–118. - PubMed
    1. Schoeffler A.J., Berger J.M. DNA topoisomerases: harnessing and constraining energy to govern chromosome topology. Q. Rev. Biophys. 2008;41:41–101. - PubMed
    1. Leppard J.B., Champoux J.J. Human DNA topoisomerase I: relaxation, roles, and damage control. Chromosoma. 2005;114:75–85. - PubMed

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