The use of divalent metal ions by type II topoisomerases

Abstract

Type II topoisomerases are essential enzymes that regulate DNA under- and overwinding and remove knots and tangles from the genetic material. In order to carry out their critical physiological functions, these enzymes utilize a double-stranded DNA passage mechanism that requires them to generate a transient double-stranded break. Consequently, while necessary for cell survival, type II topoisomerases also have the capacity to fragment the genome. This feature of the prokaryotic and eukaryotic enzymes, respectively, is exploited to treat a variety of bacterial infections and cancers in humans. All type II topoisomerases require divalent metal ions for catalytic function. These metal ions function in two separate active sites and are necessary for the ATPase and DNA cleavage/ligation activities of the enzymes. ATPase activity is required for the strand passage process and utilizes the metal-dependent binding and hydrolysis of ATP to drive structural rearrangements in the protein. Both the DNA cleavage and ligation activities of type II topoisomerases require divalent metal ions and appear to utilize a novel variant of the canonical two-metal-ion phosphotransferase/hydrolase mechanism to facilitate these reactions. This article will focus primarily on eukaryotic type II topoisomerases and the roles of metal ions in the catalytic functions of these enzymes.

Keywords: ATP hydrolysis; DNA cleavage; DNA ligation; Topoisomerase IIα; divalent cation; divalent metal ion; topoisomerase II poisons.

Figure 1

Catalytic cycle of topoisomerase II. The homodimeric enzyme is shown in blue, the DNA double helix that is cleaved is shown in green, and the double helix that is passed through the DNA gate is shown in yellow. The double-stranded DNA passage reaction of topoisomerase II can be separated into six discrete steps. 1) DNA binding to regions of helix-helix juxtaposition. 2) Double-stranded DNA cleavage, which requires the presence of Mg²⁺ (physiologically) or other divalent metal ions. 3) Binding of 2 ATP molecules, which promotes double-stranded DNA passage through the DNA gate generated by cleavage. Strand passage proceeds more rapidly if one of the two ATP molecules is hydrolyzed. 4) Ligation of the cleaved DNA. 5) Hydrolysis of the second ATP molecule, which allows release of the DNA through a C-terminal gate in the protein and promotes 6) enzyme turnover, which allows the enzyme to initiate a new round of catalysis.

Figure 2

Crystal structure of the ATPase domain of yeast topoisomerase II (PDB ID 1QZR) with ADPNP bound. The bound metal ion (Mg²⁺, green) is coordinated by non-bridging oxygen atoms of the phosphate groups and by an asparagine residue (Asn91 in human topoisomerase IIα ). The glutamic acid residue (Glu87 in human topoisomerase IIα ) is thought to activate a water molecule (shown as red spheres) to initiate hydrolysis of the phosphate group. Yellow dashes represent ligand interactions. The structure is shown in stereo view.

Figure 3

Amino acids postulated to coordinate divalent metal ions in the active site of nucleic acid enzymes that contain a TOPRIM domain. Amino acid sequences are shown for human topoisomerase IIα (HsTop2a), topoisomerase IIβ (HsTop2b), S. cerevisiae topoisomerase II (ScTop2), and E. coli gyrase B subunit (EcGyrB). Conserved glutamic and aspartic acid residues that are proposed to bind divalent metal ions are highlighted in blue. Sequences are from Aravind et al. and Caron and Wang.,

Figure 4

The DNA cleavage/ligation active site of topoisomerase II. Using the crystal structure of yeast topoisomerase II (PDB ID 3L4K) as a docked dimer, the key residues of the cleavage/ligation active site are shown and are labeled in red. The residues include three aspartic acid residues (labeled 1, 2, and 3 corresponding to their sequence order), one glutamic acid, one histidine, the active site tyrosine, and two arginine residues. The DNA in the crystal structure (shown in blue) is covalently bound and has been cleaved by the enzyme. The 3’-terminal group and 5’-phosphate of the cleaved DNA are denoted by pink arrows. The red spheres represent the locations of divalent metal ions that are visible in the structure (labeled M_A and M_B). The structure is shown in stereo view.

Figure 5

Proposed mechanism of DNA cleavage and ligation by topoisomerase II. The type II enzyme utilizes a novel variant of the canonical two-metal-ion mechanism employed by primases and polymerases.–, , , – Amino acids in the active site that are postulated to function in catalysis by topoisomerase IIα and topoisomerase IIβ are indicated. Metal ions (M_A²⁺ and M_B²⁺) are highlighted in red. Interactions between metal ions and nucleic acid or acidic amino acid residues are denoted in green or blue, respectively. In the proposed model, M_A²⁺ makes critical contacts with the 3’-bridging atom (red) and a non-bridging atom of the scissile phosphate. These interactions are likely to play a role in the transition chemistry and stabilizing the leaving 3’-terminal oxygen. M_B²⁺ also is required for DNA scission and contacts the non-bridging oxygen of the phosphate that connects the -1 and -2 bases upstream from the scissile bond. This metal ion appears to play a structural role in anchoring the DNA during scission. Cleavage is initiated when a general base deprotonates the active site tyrosine hydroxyl, allowing the oxyanion to attack the scissile phosphate. The base has not been identified but is believed to be a conserved histidine residue or a metal-associated water molecule. Ligation is initiated when a general acid extracts the hydrogen from the 3’-terminal hydroxyl group. The acid may be a water molecule or an unidentified amino acid in the active site of topoisomerase II. Figure is adapted from Schmidt, et al.