Structural studies of type I topoisomerases

Abstract

Topoisomerases are ubiquitous proteins found in all three domains of life. They change the topology of DNA via transient breaks on either one or two of the DNA strands to allow passage of another single or double DNA strand through the break. Topoisomerases are classified into two types: type I enzymes cleave one DNA strand and pass either one or two DNA strands through the break before resealing it, while type II molecules cleave both DNA strands in concert and pass another double strand through the break followed by religation of the double strand break. Here we review recent work on the structure of type I enzymes. These structural studies are providing atomic details that, together with the existing wealth of biochemical and biophysical data, are bringing our understanding of the mechanism of action of these enzymes to the atomic level.

Figure 1.

Structure of a type IA topoisomerase. E. coli topoisomerase III is shown to illustrate the overall structure of a type IA topoisomerase and the typical toroidal fold observed in all members of this type. (A) Diagram showing the structure of the apo-enzyme [PDB 1D6M (7)]. In the absence of DNA, the active site, found at the intersection of domains I and III (encircled), is buried. (B) Diagram showing the structure of a complex with single-stranded DNA [PDB 1I7D (23)]. Note the movement of domains that occurs in order to accommodate DNA. In both diagrams, the four major domains of the protein are colored red, blue, purple and green for domains I, II, III and IV, respectively. The single-stranded DNA binding groove, shown circled in black, extends from domain IV to the active site. The active site residues as well as the single-stranded DNA in the complex are shown in a ball and stick representation.

Figure 2.

Diagram showing the proposed mechanism of DNA relaxation by type IA topoisomerases. The mechanism involves several transient conformational intermediates both of the protein and the DNA. The sequence of the steps and the intermediates are hypothetical and more states are likely to be involved in the cycle. Processivity by the enzyme requires that after one relaxation event the protein continues to another relaxation cycle without releasing the DNA. In the diagram, the protein is shown in grey and the DNA in red/blue. The orange dot represents the presence of the covalent protein/DNA complex. The single-stranded DNA binding groove is shown in red or yellow.

Figure 3.

Active site of E. coli topoisomerase III, a type IA topoisomerase. The diagrams show the active site region in different complexes. (A) Apo-structure, where the protein is in a closed conformation and the active site is buried between domains I and III [PDB 1D6M (7)]. (B) Intermediate structure, where the DNA starts entering the active site but it is not yet positioned properly for cleavage/religation [PDB 2O59 (20)]. (C) Fully formed active site in the wild-type enzyme, with the single-stranded DNA positioned and the active site tyrosine poised for cleavage [PDB 2O19 (20)]. The diagrams illustrate the way the active site is assembled and the interactions with single-stranded DNA. Domain I is shown in red and domain III in purple. Some of the side chains forming the active site are shown, as well as some possible hydrogen bonds. The single-stranded DNA is shown as sticks.

Figure 4.

Structures of type IB topoisomerases. Diagrams of the structures of eukaryotic and viral type IB topoisomerases. (A) Structure of human topoisomerase I in non-covalent complex with DNA [PDB 1A35 (34)]. The protein encircles the DNA by forming a clamp around it. The protein is composed of two domains: a core domain, shown in red and blue, and a C-terminal domain, shown in green. (B) Structure of variola virus topoisomerase I in covalent complex with DNA [PDB 2H7F (51)]. The viral and bacterial type IB enzymes are composed of two domains: an N-terminal domain, shown in red, and a larger, C-terminal domain, shown in blue. Note the marked similarities between the human and poxviral proteins despite their great disparity in size. In the case of human topoisomerase I, the core and C-terminal domains are joined by a linker domain, which is not present in this structure but that has been observed in other structures (44). The DNA in the complexes is shown in a ball and stick representation.

Figure 5.

Active site of type IB topoisomerases. The diagrams show the active site region in two different type IB topoisomerases. (A) Active site in the structure of a non-covalent complex of human topoisomerase I with DNA [PDB 1A35 (34)]. Note the presence of the amino acids forming the catalytic pentad, Arg488, Lys532, Arg590, His632 and Tyr723 (in this structure the tyrosine was mutated to a phenylalanine). (B) Active site in the structure of a covalent complex of variola virus topoisomerase I with DNA [PDB 2H7F (51)]. Note the equivalent pentad of amino acids in the active site and the formation of the covalent intermediate between the tyrosine and the 3′ end of the broken strand. The diagrams serve to illustrate the high similarity between the active sites of the smaller and larger type IB enzymes and also the structures of non-covalent and covalent complexes.

Figure 6.

Structure of a type IC topoisomerase. (A) Diagram showing the structure of a 61 kDa fragment of M. kandleri topoisomerase V [PDB 2CSB (72)]. The fragment comprises the topoisomerase domain and four (HhH)₂ domains, which are likely to be involved in DNA binding. The topoisomerase domain, shown in red, and the (HhH)₂ domains, shown in tones of blue, are joined by a linker helix, shown in orange. The active site, shown as ball and stick representation, is buried at the interface of the topoisomerase domain and one of the (HhH)₂ domains. To access the active site, the protein has to change conformation, probably by separating the sub-domains. (B) The putative amino acids forming the topoisomerase V active site are shown. Aside from the tyrosine, two arginines, a lysine, a histidine and a glutamate form the putative active site. Mutagenesis studies show that removal of the arginines has a marked detrimental effect on activity. Removal of the glutamate also changes the activity, although not as markedly. Removal of the histidine and lysine has a modest effect on activity (72).