PROSITE documentation PDOC00333

Topoisomerase (Topo) IA-type active site signature and catalytic domain profile

DNA topoisomerases (Topos) are enzymes that solve topological problems associated with important processes such as DNA replication, transcription, recombination and chromatin remodeling by introducing transient single or double stranded breaks in the DNA and releasing accumulated strain. They may have appeared early during the formation of the modern DNA world. Several families and subfamilies of the two types of DNA topoisomerases (I and II) have been described in the three cellular domains of life (Archaea, Bacteria and Eukarya), as well as in viruses infecting eukaryotes or bacteria. The main families of DNA topoisomerases, Topo IA, Topo IB, Topo IC (Topo V), Topo IIA and Topo IIB (Topo VI) are not homologous, indicating that they originated independently. However, some of them share homologous modules or subunits that were probably recruited independently to produce different topoisomerase activities [1,2,3,4,5,6].

Type I topoisomerases are enzymes that effect topological changes on DNA by transiently cleaving one DNA strand at a time. All Topo I use the same general chemistry to break the phosphodiester bond. A tyrosyl group of the enzyme attacks a phosphodiester bond on DNA and remains covalently attached to one side of the break while releasing a free hydroxylated strand. The attack of the phosphotyrosine bond by the hydroxyl end of the free strand restores a phosphodiester bond and releases the enzyme for the next catalytic cycle. As the energy of the phosphodiester bond is conserved in the protein-DNA covalent intermediate, the cleavage-religation step does not require ATP hydrolysis. Type I topoisomerases were further divided into two families, IA and IB (see <PDOC00159>), on the basis of the polarity of strand cleavage. Topo IA form a transient 5'-phospho-tyrosine covalent intermediate and release a free 3'-OH strand whereas Topo IB form a 3'-phospho-tyrosine covalent intermediate and release a free 5'-OH strand. The division between Topo IA and Topo IB is supported by the absence of sequence or structural similarity between these two enzyme families, thus indicating independent origin [1,2,3,4,5,6].

Topo IA are widespread in all domains of life, from bacteria to archaea and higher eukaryotes. They share a common mechanism known as "enzyme-bridged strand passage". Topo IA catalyse the cleavage of a single-strand DNA, forming a transient 5'-phosphotyrosine covalent complex. The other DNA end is not free to rotate but rather bound to the enzyme, thus allowing the passage of another DNA strand through the break prior to rejoining of the DNA ends. The overall reaction cycle is Mg(2+) dependent and results in the modification of DNA topology strictly by steps of one. All Topo IA promote a partial relaxation of negatively, but not positively supercoiled DNA, most likely because these enzymes require an exposed single-strand region within the substrate DNA. In addition to DNA relaxation, Topo IA can catalyse the knotting, unknotting and interlinking of single-strand DNA circles as well as the knotting, unknotting and catenation/decatenation of nicked double-strand DNAs. The Topo IA family is divided into five subfamilies corresponding to (i) bacterial topoisomerase I (Topo I), (ii) bacterial Topo III, (iii) eukaryal Topo III, (iv) archaeal Type I DNA topoisomerases (annotated either as Topo I or Topo III), and (v) reverse gyrase present in hyperthermophilic archaea and bacteria [1,2,3,4,5,6].

The type IA topoisomerase core region is divided into four subdomains (I or TOPRIM (see <PDOC50880>), II, III and IV), where subdomains II and IV result from two separated regions in the protein primary sequence, which come together in the three-dimensional structure. The Topo IA-type catalytic domain consisting of subdomains III, II and IV exhibits a typical toroidal shape with a central hole able to accommodate single-strand or double-strand DNA. Subomain II forms over half of the torus and achieves its curvature through several β strands. Subdomain III is mainly helical and the last subdomain, IV, forms part of the main body (see <PDB:1D6M>). There are two potential flexible regions making the toroidal structure free to open: the first between subdomains II and IV, the second between subdomains II and III. The active site is at the interface between domain I (TOPRIM domain, see <PDOC50880>) and domain III, while the single-stranded DNA to be cleaved is accommodated in the groove at the interface of the TOPRIM domain and subdomain IV. The catalytic tyrosine on which the trans-esterification reaction relies is located in subdomain III. It introduces a nucleophilic attack on a phosphate group of the nucleotide chain, creating a transient phosphodiester bond with the broken DNA strand [1,2,3,4,5,6].

There are a number of conserved residues in the region around the active site tyrosine; we used this region as a signature pattern. We also developed a profile for the Topo IA-type catalytic domain that covers subdomains II to IV.