A novel and unified two-metal mechanism for DNA cleavage by type II and IA topoisomerases

Abstract

Type II topoisomerases are required for the management of DNA tangles and supercoils, and are targets of clinical antibiotics and anti-cancer agents. These enzymes catalyse the ATP-dependent passage of one DNA duplex (the transport or T-segment) through a transient, double-stranded break in another (the gate or G-segment), navigating DNA through the protein using a set of dissociable internal interfaces, or 'gates'. For more than 20 years, it has been established that a pair of dimer-related tyrosines, together with divalent cations, catalyse G-segment cleavage. Recent efforts have proposed that strand scission relies on a 'two-metal mechanism', a ubiquitous biochemical strategy that supports vital cellular processes ranging from DNA synthesis to RNA self-splicing. Here we present the structure of the DNA-binding and cleavage core of Saccharomyces cerevisiae topoisomerase II covalently linked to DNA through its active-site tyrosine at 2.5A resolution, revealing for the first time the organization of a cleavage-competent type II topoisomerase configuration. Unexpectedly, metal-soaking experiments indicate that cleavage is catalysed by a novel variation of the classic two-metal approach. Comparative analyses extend this scheme to explain how distantly-related type IA topoisomerases cleave single-stranded DNA, unifying the cleavage mechanisms for these two essential enzyme families. The structure also highlights a hitherto undiscovered allosteric relay that actuates a molecular 'trapdoor' to prevent subunit dissociation during cleavage. This connection illustrates how an indispensable chromosome-disentangling machine auto-regulates DNA breakage to prevent the aberrant formation of mutagenic and cytotoxic genomic lesions.

Fig. 1. Structure of a topo II/DNA cleavage complex

a. S. cerevisiae topo II domain arrangement. Functional regions are coloured and labelled. Active-site residues are red; “trapdoor” residues green. b. DNA substrate. One strand (top) contains a 3′-bridging phosphorothiolate between the −1/+1 positions (red, boxed). Two complementary strands (bottom) adjoin at a nick (red arrowhead). Blue diamonds indicate where DNA bend points. The terminal two base-pairs (grey) are disordered. c. The cleavage complex. One topo II monomer is coloured as in (a). 2F_o-F_c density (1σ contour, orange) is shown around the DNA. d. The 5′-phosphotyrosine link modelled into a composite simulated-annealing omit map (1 σ contour).

Fig. 2. A cleavage-competent active site

Close-up showing DNA (yellow), catalytic amino acids (cyan – TOPRIM, green – WHD) and metal coordination (black spheres). A Zn-anomalous difference map is shown as purple mesh (5σ contour). The two modelled zinc ions are spaced 3.5 Å apart. Hydrogen bonds and metal interactions are displayed as dashed lines. Both nonbridging phosphotyrosyl oxygens are liganded, one by metal A and the other by Arg781. Mg²⁺ ions seen in a non-covalent topo II/DNA complex and in the DNA-free topo VI A-subunit are shown as blue and red spheres, respectively.

Fig. 3. DNA cleavage by type IA and II topoisomerases

a. Proposed cleavage mechanism. The general base (B:) and acid (HA) are unknown, but may be metal-associated waters. Metal A and Arg781 stabilize the transition state; metal B and His736 anchor the (−1) phosphate. Amino acids (blue – TOPRIM, green – WHD) are labelled based on yeast topo II, except for a lysine residue present in type IA (but not type II) topoisomerases (grey, E. coli topo I/III numbering). b. Type IA/II topoisomerase active site superposition. A DNA-bound (and uncleaved) topo III structure (grey) is overlaid on topo II complex (cyan/green). Catalytic residues are labelled (yeast topo II/E. coli topo III numbering).

Fig. 4. Cleavage-dependent control of C-gate dynamics

a. Superposition of noncovalent (grey) and cleavage (orange) complexes between topo II and DNA reveal how C-gate opening and closure is linked to active site status. The connection from the active-site tyrosines to the coiled-coil arms is coloured green/magenta. For clarity, the TOPRIM domains are hidden. b. Close-up of positional shifts. (Upper) Upward movement of the active-site tyrosine upon becoming attached to the DNA. (Lower) Concomitant inward movement of the coiled-coil joint through a conserved salt bridge network.