Abstract
Reverse gyrase is a hyperthermophile-specific enzyme that can positively supercoil DNA concomitant with ATP hydrolysis. However, the DNA supercoiling activity is inefficient and requires an excess amount of enzyme relative to DNA. We report here several activities that reverse gyrase can efficiently mediate with a substoichiometric amount of enzyme. In the presence of a nucleotide cofactor, reverse gyrase can readily relax negative supercoils, but not the positive ones, from a plasmid DNA substrate. Reverse gyrase can completely relax positively supercoiled DNA, provided that the DNA substrate contains a single-stranded bubble. Reverse gyrase efficiently anneals complementary single-stranded circles. A substoichiometric amount of reverse gyrase can insert positive supercoils into DNA with a single-stranded bubble, in contrast to plasmid DNA substrate. We have designed a novel method based on phage-mid DNA vectors to prepare a circular DNA substrate containing a single-stranded bubble with defined length and sequence. With these bubble DNA substrates, we demonstrated that efficient positive supercoiling by reverse gyrase requires a bubble size larger than 20 nucleotides. The activities of annealing single-stranded DNA circles and positive supercoiling of bubble substrate demonstrate that reverse gyrase can function as a DNA renaturase. These biochemical activities also suggest that reverse gyrase can have an important biological function in sensing and eliminating unpaired regions in the genome of a hyperthermophilic organism.
Highlights
Transactions of genetic information in DNA invariably require unwinding and rewinding of DNA double helix, processes usually resulting in creating topological problems
The crystal structure of reverse gyrase from Archaeoglobus fulgidus revealed that the structure of the topoisomerase domain is very similar to those from bacterial type IA topoisomerases, and the helicase domain shares the structure with several DNA helicases except for some residues involved in helicase translocation [11]
Reverse Gyrase as a DNA Renaturase resistant to relaxation by a potent topoisomerase I added to the reaction, suggesting that negative supercoils are constrained, possibly because of the binding of reverse gyrase/AMPPNP to the unwound region in DNA
Summary
ANNEALING OF COMPLEMENTARY SINGLE-STRANDED CIRCLES AND POSITIVE SUPERCOILING OF A BUBBLE SUBSTRATE*□S. Type IB enzymes, present in all eukaryotes, certain eukaryotic viruses, and some bacterial species [4], form a 3Ј-phosphotyrosyl covalent adduct at the DNA cleavage site They can readily remove either positive or negative supercoils, capable of serving as an efficient swivel during DNA transactions [5]. Enzymes are ubiquitous, present in eukaryotic, eubacterial, and archaebacterial species Their enzymatic activity involves strand passage through a protein-mediated DNA gate formed with a tyrosyl linkage to a 5Ј phosphoryl group at the cleavage site [6]. These enzymes prefer DNA substrates with a single-stranded region and usually can only relax negative supercoils in a plasmid DNA. The negative supercoiling by reverse gyrase with AMPPNP is
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