PROSITE documentation PDOC00159

Topoisomerase (Topo) IB-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].

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 (see <PDOC00333>) and IB, 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].

Type IB topoisomerases relax positive and negative supercoils via a reaction pathway that entails (i) noncovalent binding of the topoisomerase to duplex DNA, (ii) cleavage of one DNA strand with concomitant formation of a covalent DNA-(3'-phosphotyrosyl)-protein intermediate, (iii) rotation of the noncovalently held duplex segment about the phosphodiester bond in the unbroken strand opposite the protein-induced nick, and (iv) religation to the 5' OH end of the cleaved strand. The type IB enzymes can also transfer the covalently held strand to a heterologous polynucleotide to generate recombinant molecules. Type IB DNA topoisomerases are found in all eukarya, two families of eukaryotic viruses (poxviruses and mimivirus), and widespread in different bacterial phyla (Proteobacteria, Bacteroides, Actinobacteria, Chlamidia, Acidobacteria, Deinococci). Topo IB are ubiquitous in eukaryotes, where they represent the major DNA topoisomerase I activity. Their ability to relax positively supercoiled DNA allows them to play a major role both in transcription and DNA replication, by relaxing the positive supercoils that accumulate in front of moving polymerases. Two different Topo IB are present in vertebrates, one localized in the nucleus and the other in the mitochondria. Eukaryotic Topo IB are large monomeric enzymes (around 90 kDa) with a highly conserved DNA binding domain and a C-terminal catalytic domain, linked by a non conserved hydrophilic region. Topo IB sequences are also found in all currently sequenced poxviruses genomes. These viral enzymes are the smallest known DNA topoisomerases (around 30 kDa) and are homologous to the catalytic core domain of the eukaryotic enzyme. Although Topo IB is widespread in different bacterial phyla, its distribution is patchy (it is notably missing from E. coli and B. subtilis) and suggests that either this enzyme was not present in the last common ancestor of Bacteria and was later introduced by horizontal gene transfers (HGT) from sources to be identified, or that multiple independent losses occurred in different bacterial phyla [1,2,3,4,5].

The Topo IB-type catalytic domain consists of ten α helices and a three-stranded antiparallel β sheet on one surface (see <PDB:1A41>). An N-terminal lobe comprises helices 1-5 and the β sheet; a C-terminal lobe consists of helices 6-10. The long N-terminal α helix is integrated into both lobes. The two lobes make extensive interactions, and the active site residues are located at the interface between the lobes. The RKKRH/N "catalytic pentad", conserved in all members of the topo IB family, catalyzes the attack of the active site tyrosine nucleophile on the scissile phosphodiester [2,4].

In eukaryotes and poxvirus topoisomerases IB, there are a number of conserved residues in the region around the active site tyrosine, which were used to build the pattern. We also developed a profile that covers the entire Topo IB-type catalytic domain.