Mutational analysis of the helicase-like domain of Thermotoga maritima reverse gyrase.
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.
de la Tour
Claire Bouthier
CB
Université Paris-Sud 11, UMR8621 Institut de Génétique et Microbiologie, 91405 Orsay Cedex, France. Electronic address: claire.bouthier@igmors.u-psud.fr.
Amrani
Laila
L
Université Paris-Sud 11, UMR8621 Institut de Génétique et Microbiologie, 91405 Orsay Cedex, France.
Cossard
Raynald
R
Université Paris-Sud 11, UMR8621 Institut de Génétique et Microbiologie, 91405 Orsay Cedex, France.
Neuman
Keir C
KC
Université Paris-Sud 11, UMR8621 Institut de Génétique et Microbiologie, 91405 Orsay Cedex, France.
Serre
Marie Claude
MC
Université Paris-Sud 11, UMR8621 Institut de Génétique et Microbiologie, 91405 Orsay Cedex, France.
Duguet
Michel
M
Université Paris-Sud 11, UMR8621 Institut de Génétique et Microbiologie, 91405 Orsay Cedex, France.
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D009154:Mutation; D017434:Protein Structure, Tertiary / Q000502:physiology; D020124:Thermotoga maritima / Q000201:enzymology*
D001426:Bacterial Proteins; D004278:DNA, Superhelical; D000255:Adenosine Triphosphate; D004265:DNA Helicases; C041813:DNA reverse gyrase; D004264:DNA Topoisomerases, Type I