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The only tyrosine recombinase so far studied in archaea, the SSV1 integrase, harbors several changes in the canonical residues forming the catalytic pocket of this family of recombinases. This raised the possibility of a different mechanism for archaeal tyrosine recombinase. The residues of IntSSV tentatively involved in catalysis were modified by site-directed mutagenesis, and the properties of the corresponding mutants were studied. The results show that all of the targeted residues are important for activity, suggesting that the archaeal integrase uses a mechanism similar to that of bacterial or eukaryotic tyrosine recombinases. In addition, we show that IntSSV exhibits a type IB topoisomerase activity because it is able to relax both positive and negative supercoils. Interestingly, in vitro complementation experiments between the inactive integrase mutant Y314F and all other inactive mutants restore in all cases enzymatic activity. This suggests that, as for the yeast Flp recombinase, the active site is assembled by the interaction of the tyrosine from one monomer with the other residues from another monomer. The shared active site paradigm of the eukaryotic Flp protein may therefore be extended to the archaeal tyrosine recombinase IntSSV. [ABSTRACT FROM AUTHOR]

Reverse gyrase is a unique type IA topoisomerase that is able to introduce positive supercoils into DNA in an ATP-dependent process. ATP is bound to the helicase-like domain of the enzyme that contains most of the conserved motifs found in helicases of the SF1 and SF2 superfamilies. In this paper, we have investigated the role of the conserved helicase motifs I, II, V, VI, and Q by generating mutants of the Thermotoga maritima reverse gyrase. We show that mutations in motifs I, II, V, and VI completely eliminate the supercoiling activity of reverse gyrase and that a mutation in the Q motif significantly reduces this activity. Further analysis revealed that for most mutants, the DNA binding and cleavage properties are not significantly changed compared with the wild type enzyme, whereas their ATPase activity is impaired. These results clearly show that the helicase motifs are tightly involved in the coupling of ATP hydrolysis to the topoisomerase activity. The zinc finger motif located at the N-terminal end of reverse gyrases was also mutated. Our results indicate that this motif plays an important role in DNA binding. [ABSTRACT FROM AUTHOR]

Abstract: The DNA topoisomerases are essential for DNA replication, transcription, recombination, as well as for chromosome compaction and segregation. 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. The puzzling phylogenetic distribution of the various DNA topoisomerase families and subfamilies cannot be easily reconciled with the classical models of early evolution describing the relationships between the three cellular domains. A possible scenario is based on a Last Universal Common Ancestor (LUCA) with a RNA genome (i.e. without the need for DNA topoisomerases). Different families of DNA topoisomerases (some of them possibly of viral origin) would then have been independently introduced in the different cellular domains. We review here the main characteristics of the different families and subfamilies of DNA topoisomerases in a historical and evolutionary perspective, with the hope to stimulate further works and discussions on the origin and evolution of these fascinating enzymes. [Copyright &y& Elsevier]