title: Differential contributions of the latch in Thermotoga maritima reverse gyrase to the binding of single-stranded DNA before and after ATP hydrolysis.
abstract: Reverse gyrase catalyzes the ATP-dependent introduction of positive supercoils into DNA. Supercoiling requires the functional cooperation of its N-terminal helicase domain with the C-terminal topoisomerase domain. The helicase domain contains a superfamily 2 helicase core formed by two RecA domains, H1 and H2. We show here that a helicase domain lacking the latch, an insertion in H2, fails to close the cleft in the helicase core in response to nucleotide and DNA binding at the beginning of the catalytic cycle. In the presence of the pre-hydrolysis ATP analog ADP·BeFx, however, the closed conformer can still be formed in the absence of the latch. The helicase domain lacking the latch exhibits reduced DNA affinities. The energetic difference between the two nucleotide states involved in duplex separation is diminished, rationalizing the unwinding deficiency of reverse gyrase lacking the latch. The latch most strongly contributes to binding of single-stranded DNA in the post-hydrolysis state, before phosphate release. Our results are in line with contributions of the latch in determining the direction of strand passage, and in orienting the cleaved single-stranded DNA for re-ligation. At the same time, the latch may coordinate the re-ligation reaction with strand passage and with the nucleotide cycle.
authors: Yoandris
authors: Y
authors: Del Toro Duany
authors: Agneyo
authors: A
authors: Ganguly
authors: Dagmar
authors: D
authors: Klostermeier
mesh_terms: D000255:Adenosine Triphosphate / 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*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008958:Models, Molecular; D020124:Thermotoga maritima / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D000255:Adenosine Triphosphate; D004265:DNA Helicases; C041813:DNA reverse gyrase; D004264:DNA Topoisomerases, Type I