Reverse gyrase: a helicase-like domain and a type I topoisomerase in the same polypeptide.

Abstract

Reverse gyrase is a type I DNA topoisomerase able to positively supercoil DNA and is found in thermophiic archaebacteria and eubacteria. The gene coding for this protein was cloned from Sulfolobus acidocaldarius DSM 639. Analysis of the 1247-amino acid sequence and comparison of it with available sequence data suggest that reverse gyrase is constituted of two distinct domains: (i) a C-terminal domain of -630 amino acids clearly related to eubacterial topoisomerase I (Escherichia coli topA and topB gene products) and to Saccharomyces cerevisiae top3; (ii) an N-terminal domain without any similarity to other known topoisomerases but containing several helicase motifs, including an ATP-binding site. These results are consistent with those from our previous mechanistic studies of reverse gyrase and suggest a model in which positive supercoiling is driven by the concerted action of helicase and topoisomerase in the same polypeptide: this constitutes an example of a composite gene formed by a helicase domain and a topoisomerase domain.It is today well established that topoisomerases play a crucial role in DNA structure and function (1, 2). These enzymes seem to act in two ways: (i) they are able to solve the topological problems intrinsic to the DNA double helix during replication, transcription, recombination, or chromatin condensation and decondensation; (ii) some topoisomerases have exploited the circular or pseudocircular (chromatin loops) structure of the genetic material to introduce stress into DNA by means of supercoiling. The enzymes specialized in the production of superhelical turns in a circular DNA were named "gyrases" (3, 4). Among these, the best characterized enzyme is the eubacterial gyrase, a type II topoisomerase that uses the energy of ATP hydrolysis to maintain the in vivo level of negative supercoiling of the bacterial chromosome (5). Several years ago, another kind of DNA supercoiling activity was discovered in hyperthermophilic archaebacteria (6) and was called "reverse gyrase." We have shown that reverse gyrase is widely distributed in various archaebacterial families and also in thermophilic eubacteria (7,8). This enzyme has the specific ability to catalyze in vitro positive supercoiling of a closed circular DNA at high temperature (9). Surprisingly, reverse gyrase turned out to be a type I topoisomerase (9) and is the only topoisomerase I that depends on ATP and can catalyze DNA supercoiling. The biological function of reverse gyrase is still unclear, but we found that positive supercoiling occurs in vivo (10). The enzyme from Sulfolobus acidocaldarius is composed of a single polypeptide of apparent molecular mass of -130,000 (11). Mechanistic studies (12) indicated that reverse gyrase transiently cleaves a single DNA strand, forming a covalent link with the 5' end of the broken strand, as described for eubacterial topoisomerase I (13). In addition, we have shown that stoichiometric binding of the enzyme changes the DNA conformation, presumably by unwinding the double helix (12). We ...

The complete genome of the crenarchaeon Sulfolobus solfataricus P2

“…Also encoded are four topoisomerases: one ATP-independent type I, homologous to TopA (Sso0907), and two ATP-dependent reverse gyrases (Sso0420 and Sso0963). One of the latter is closely related to TopR of S. acidocaldarius (46), whereas the other is nearly identical to TopR of S. shibatae (47). The genome contains only one type II topoisomerase of the TopoVI family (Sso0968 and Sso0969) (48).…”

Reverse gyrase from hyperthermophiles: probable transfer of a thermoadaptation trait from Archaea to Bacteria

“…Mechanistic studies showed that it is transiently linked to the DNA by a 5Ј phosphotyrosyl bond (22,24), classifying it in the type I 5Ј topoisomerase family as proposed by Roca (38). Sequence analysis further showed that it is a single polypeptide containing putative helicase and topoisomerase domains located in the amino-and carboxy-terminal, respectively, parts of the protein (9). The helicase domain exhibits motifs found in DNA and RNA helicases, and the topoisomerase domain exhibits a significant similarity with the 5Ј topoisomerase I (protein ) from Escherichia coli.…”

The mechanism of ATP-dependent RNA unwinding by DEAD box proteins

“…In numerous DEAD box proteins, the helicase core provides the basic DEAD box protein functions, and large N-and/or C-terminal extensions (Figure 1) modulate the activity of the helicase core by conferring substrate specificity or by mediating contacts with interacting proteins (Schmid and Linder 1992; see 'Modulation of the helicase core activity by interacting partners and flanking domains'). In addition, the helicase core appears as a module in several enzymes involved in nucleic acid processing, such as DNA topoisomerases (Confalonieri et al, 1993;Rodriguez and Stock, 2002), restriction enzymes (Gorbalenya and Koonin, 1991;Szczelkun, 2000), chromatin remodeling enzymes (Flaus and Owen-Hughes, 2001), or Dicer (Ma et al, 2008).…”