Proc. Natl. Acad. Sci. USA Vol. 93, pp. 106-110, January 1996 Biochemistry A two-subunit type I DNA topoisomerase (reverse gyrase) from an extreme hyperthermophile REGIS KRAH*, SERGEI A. KOZYAVKIN*t, ALEXEI I. SLESAREVt, AND MARTIN GELLERT*§ *Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892; and tMolecular Biology Institute and Department of Biology, University of California, Los Angeles, CA 90024 Contributed by Martin Gellert, September 6, 1995 ABSTRACT A recently described reverse gyrase from the helicases (15-17). These helicases are nucleic acid-dependent hyperthermophilic methanogen Methanopyrus kandleri is the ATPases, as is reverse gyrase (18, 19). It was proposed that the only known example of a heterodimeric type I topoisomerase. helicase-like ATPase domain uses ATP to translocate along The enzyme is made up of a 42-kDa subunit which covalently the DNA, generating a transient wave of positive and negative interacts with DNA (RgyA) and a 138-kDa subunit which supercoiling (15, 20, 21), while the topoisomerase domain binds ATP (RgyB). We have now cloned and sequenced the selectively removes negative supercoils, producing a super- genes for both subunits of this enzyme. Surprisingly, the coiled molecule. It is important to note that helicase activity universally conserved type I topoisomerase domain [Lima, has not yet been demonstrated for reverse gyrase and that ATP C. D., Wang, J. C. & Mondragon, A. (1994) Nature (London) hydrolysis may also be more directly linked to the mechanism 367, 138-146] which has been found as a contiguous polypep- of DNA strand passage. tide in the prokaryotes and eukaryotes is shared between the Recently, a reverse gyrase was described from the hyper- protomers. The subdomain with the active-site tyrosine is thermophilic methanogen Methanopyrus kandleri (18). Unlike entirely within RgyA, whereas the subdomain implicated in other type 1-5' topoisomerases, this enzyme was found to be a noncovalent binding of the cleaved DNA strand is contained heterodimer composed of a smaller subunit with an apparent entirely in RgyB. The appearance of this unique structure in molecular mass of 50 kDa (RgyA) that formed a covalent a highly conserved enzyme family supports the hypothesis that complex with DNA, and a larger subunit with an apparent the methanogens branched from other prokaryotes and eu- molecular weight of 150 kDa (RgyB) that was involved in the karyotes very early in evolution. hydrolysis ofATP. To further study this protein, we cloned and sequenced the genes encoding the subunits of this enzyme.1 A DNA topoisomerases are found in all organisms. They are remarkable feature of the sequence is that the domain which required for replication, decatenation, and unknotting of DNA characterizes all type I-5' topoisomerases, and is found as a in cells (1-3), and by controlling the torsional stress of DNA single polypeptide chain in all other enzymes of this class, is they can also affect the efficiency of gene expression and here divided between the two protomers. various recombination processes. All topoisomerases can be classified into three general subgroups that are distinguished MATERIALS AND METHODS by common biochemical properties and similarity in primary structure. These are the type 1-5' topoisomerases, the type I-3' Amino-Terminal Sequencing of Proteins and Development topoisomerases, and the type II topoisomerases. of DNA Probes. Microsequencing of the amino termini of Escherichia coli topoisomerase I, the first topoisomerase RgyA and RgyB was performed on an Applied Biosystems discovered (4), is the prototype of the type 1-5' topoisomer- 471A protein sequencer. Five picomoles ofM. kandleri reverse ases. Enzymes of this class have been found in eubacteria, gyrase (18) was separated into the A and B subunits by archaea, and eukaryotes (4-9). They share a number of SDS/PAGE. The proteins were blotted to Immobilon_psq common properties: they change the linking number of DNA transfer membrane (Millipore), and subjected to 20 cycles of by steps of 1, require a single-stranded region in the DNA Edman degradation (22). The amino-terminal amino acid substrate, are active only in the presence of a divalent metal sequence of RgyA was determined to be MNATLRIRNRP- cation, and form a covalent bond to DNA through a 5' VAE. Determination of the amino-terminal sequence of RgyB phosphoryl group during the cleavage reaction. required the comparison of two separate sequencing reactions, In the mid 1980s, a novel type I-5' topoisomerase, reverse and the sequence was found to be VLXRAXXMVPKGF (X gyrase, was isolated from extremely hyperthermophilic ar- indicates unreadable positions). The valine in the first position chaea (6, 7, 10, 11). This enzyme was able to positively indicates that the amino terminus was modified by proteolytic supercoil DNA in the presence ofATP or dATP. Although the cleavage. The observed signal for each cycle during the physiological role of this enzyme remains obscure, positive microsequencing was much weaker than expected, suggesting supercoiling would help to stabilize the DNA duplex against that the amino terminus in a significant fraction of the protein denaturation at the extreme temperatures at which these molecules is blocked. A blocked amino terminus has also been organisms grow (12, 13). Reverse gyrase has been found in all found in reverse gyrase from S. acidocaldarius (15). major branches of hyperthermophilic archaea as well as in PCR amplification with degenerate oligonucleotide primers hyperthermophilic eubacteria and is thus considered a molec- was used to generate DNA probes for rgyA and rgyB. Knowing ular marker for hyperthermophilic prokaryotes (12, 14). that the amino acid sequence of S. acidocaldarius RgyS had The cloning and sequencing of a reverse gyrase from Sulfolobus acidocaldarius (RgyS) revealed a monomeric pro- Abbreviation: ORF, open reading frame. tein which fused the universally conserved type 1-5' topo- tPresent address: Third Wave Technologies, Madison, WI 53711-5399. isomerase domain to a domain reminiscent of nucleic acid §To whom reprint requests should be addressed at: National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Laboratory of Molecular Biology, Building 5, Room 241, The publication costs of this article were defrayed in part by page charge 9000 Rockville Pike, Bethesda, MD 20892-0540. payment. This article must therefore be hereby marked "advertisement" in 1The sequences reported in this paper have been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank database (accession nos. U41058 and U41059). 106 Downloaded by guest on September 30, 2021 Biochemistry: Krah et al. Proc. Natl. Acad. Sci. USA 93 (1996) 107 revealed a protein that fused a helicase-like ATPase domain to RESULTS AND DISCUSSION the type 1-5' topoisomerase domain (15), we used alignments of helicases and topoisomerases with RgyS to locate conserved Cloning and Sequence Analysis of rgyB. Sequence analysis amino acid sequences and direct the design of these primers. revealed an open reading frame (ORF) encoding a protein of For rgyB, a probe complementary to the amino terminus 1221 aa, with a predicted molecular mass of 138 kDa. This is (probe 1) and a second probe (probe 2) based on highly in good agreement with the molecular mass of 150 kDa conserved motifs found in the type 1-5' DNA topoisomerases determined by SDS/PAGE (18). The coding sequence of the were developed. For probe 1, a series of degenerate PCR ORF agrees completely with the protein microsequencing primers based on the amino acid sequence MVPKG taken data: there is an ATG codon for methionine directly 5' of the from the amino terminus of RgyB were paired with a series of codon for the first valine. Strong similarity with S. acidocal- darius RgyS near aa 52 of degenerate PCR primers based on the sequence flanking and begins RgyB (Fig. 1) and includes a conserved zinc near including a conserved motif, the DEAH box of nucleic acid tetracysteine finger motif the amino termini of both enzymes. helicases (16, 17) (see Fig. 1). The second probe for rgyB was RgyB has the series of amino acid motifs found in the nucleic developed by using PCR primers based on the highly con- acid helicases which have also been identified in RgyS (Fig. 1) served motifs TEGEKI and VQTPVL found in the type I-5' (16, 17). Throughout the ATPase domain, RgyB and RgyS are DNA topoisomerases (see Fig. 2A). PCR yielded amplification 30% identical and 57% similar. Protein motifs common to products from both sets of that were cloned and primer pairs helicases are overlined and underlined in Fig. 1, and within and The usefulness of 1 for was sequenced. probe rgyB confirmed near these regions RgyB and RgyS share the greatest identity by the presence of a phenylalanine codon adjacent to the 5' (64%). However, between these regions RgyB has 14 inser- primer, in agreement with the sequence analysis of the amino terminus. Adjacent to the 3' primer, amino acid sequences with similarity to RgyS were found (15). For probe 2, amino acid 1 MVLKRAADMVPKGFRDLVEPILDDCADLEELADRVVETEMEPDEVRRRDVG 51 rgyB sequences conserved in type I-5' topoisomerases were found. 52 NTDSNEPVAIFGSSCVLCGGDCSSVRLTSRIGICERCLPVDTE..TLREV 99 rgyB Probe 1 was used to screen 5000 plaques from an amplified M. ..1: 1..1: 1111.1 11 :1 1111 .:: kandleri A library. The DNA from 6 plaques that hybridized to 2 QSLSDIPPSIYLFSCPNCGRSISTYRLL . LGSVCNICLEEDKEYKNIGDL 50 rgyS this probe was purified and probed with probe 2. All 6 A clones 100 LKEARKRHGYVGEALLMFILVERYSPDRVEEFFRRYVWPELFTEIVDRVF 149 rgyB were found to hybridize to probe 2 and were found to share 51 IKDIEKQGN .......... LIKLKDIQRVLDDYESFVS ..........VF 80 rgyS common restriction fragments that hybridized to both probes. 150 DRATGFRLYSAQRVWTRRLVKGCSFSILAPTGTGKTSWGSLVAAVFGHAG 199 rgyB For rgyA, a single probe was developed on the basis of 81 RRLLGFPPFGPQKSWIYRLLSGESFAIIAPPGLGKTTFGLISSIYLYLRG 130 rgyS sequence taken from the amino terminus (MNATLRI) and 200 the sequence of conserved motifs near the active-site tyrosine RRVYYLVPTTTLVRQVENR...