no. the 5, of s. u. 1991 rc) i11 thermophilic extremely ...the journal of biological chemistry rc)...

THE JOURNAL OF BIOLOGICAL CHEMISTRY rc) 1991 by The Amencan Society for Biochemistry and Molecular Biology, Inc

Vol. 266, No. 19, Issue of July 5 , PP . 12321-12328,1991 Printed in U. S. A.

DNA Topoisomerase I11 from Extremely Thermophilic Archaebacteria ATP-INDEPENDENT TYPE I TOPOISOMERASE FROM DESULFUROCOCCUS AMYLOLYTICUS DRIVES EXTENSIVE UNWINDING OF CLOSED CIRCULAR DNA AT HIGH TEMPERATURE*

(Received for publication, February 15, 1991)

Alexei I. SlesarevS$V, Dmitrii A. ZaitzevS, Vladimir M. KopylovS, Karl 0. Stetterg, and Sergei A. Kozyavkin**ll From the $Institute of Molecular Genetics, Union of Soviet Socialist Republics Academy of Sciences, 46 Kurchatov Square, Moscow 123182, Union of Soviet Socialist Republics, the SLehrstuhl f u r Mikrobiologie, Universitat Regensburg, Universitatsstrasse 31, 0-8400 Regensburg, Federal Republic of Germany, and the **R. E. Kauetsky Institute for Oncology Problems, Ukrainian Academy of Sciences, 45 Vasilkovskaya Street, Kiev 252127, Union of Soviet Socialist Republics

A second type I topoisomerase was purified from the extremely thermophilic archaebacterium Desulfuro- coccus amylolyticus. In contrast to the previously described reverse gyrase from this organism, the novel enzyme designated as Dam topoisomerase I11 is an ATP-independent relaxing topoisomerase. It is a monomer with M,. 108,000, as determined by electrophoresis under denaturing conditions and by size exclusion chromatography. Dam topoisomerase 111, like other bacterial type I topoisomerases, absolutely requires Mg”+ for activity and is specific for single-stranded DNA. At 60-80 “C, it relaxes negatively but not positively supercoiled DNA and is inhibited by single- stranded M13 DNA. At 95 “C, the enzyme unwinds both positively and negatively supercoiled substrates and produces extensively unwound form I* and I** DNA. The peculiarities of DNA topoisomerization at high temperatures are discussed.

Topoisomerases catalyze the concerted breakage and re- union of DNA strands (for reviews, see Refs. 1-8). Two types of topoisomerases are distinguished by how they catalyze topological reactions: type I enzymes break and rejoin one DNA strand at a time and change the linking number (Lk) in steps of 1; type I1 enzymes make a transient double-stranded break and alter the Lk in steps of 2.

DNA topoisomerases, especially type I enzymes, have recently received increased attention due to the discovery of a number of new topoisomerases. It has been recognized that both eubacterial and eukaryotic genomes can harbor several genes for different topoisomerases of the same type (9-16). At present, the family of type I topoisomerases comprises eubacterial topoisomerases I (1-5), Escherichia coli topoisomerase I11 (17-19), the type I topoisomerase from chloroplasts that closely resembles eubacterial enzymes (20), nuclear topoisomerases I and closely related enzymes isolated from

* This work was supported in part by funds of the Leibniz Preis (to K. 0. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ll Recipient of the Alexander von Humboldt Fellowship. To whom correspondence should be addressed Lehrstuhl fur Mikrobiologie, Universitat Regensburg, Universitatsstrasse 31, D-8400 Regensburg, Federal Republic of Germany. Tel.: 49-941-9433180; Fax: 49-941- 9432403.

11 Present address: Laboratory of Molecular Biology, NIDDK, Na- tional Institutes of Health, Bldg. 2, Rm. 304, Bethesda, MD 20892.

mitochondrial and viral sources (7), and reverse gyrase of extremely thermophilic archaebacteria (21-24). The putative products of the yeast TOP3 and HPRl genes also appear to be type I topoisomerases (10-13), although confirmation of these activities require further biochemical characterization.

With respect to the mechanism of catalysis and substrate specificity, type I DNA topoisomerases can be divided into two groups. The first consists of bacterial enzymes that absolutely require magnesium for activity and form a transient covalent complex with the 5’-phosphoryl termini. They are also specific for single-stranded DNA; this results in their substrate preference to negatively rather than positively supercoiled DNA (1-6, 25-29). The second group includes eukaryotic topoisomerases I that are able to function in the absence of a divalent cation, bind covalently to the 3’-phosphoryl termini, and operate on duplex DNAs (1-8, 30). The group I topoisomerases do not constitute an homogeneous family but differ in some respects. Eco topoisomerase I and topoisomerase I11 differ in cleavage site specificity, temperature optimum, and kinetics of decatenation and relaxation of negatively supercoiled DNA (17-19, 31-34). The reverse gyrase of extremely thermophilic archaebacteria is an ATP- dependent type I-group I topoisomerase capable of driving positive supercoiling of DNA (21-29). It differs in this respect from both topoisomerase I and topoisomerase 111. We discussed previously, however, that reverse gyrase, owing to its specific properties, possibly takes part in controlling DNA duplex stability at high temperatures like Eco topoisomerase I does a t moderate temperatures (27, 28). The functional analogy between reverse gyrase and eubacterial topoisomerases I is supported by the finding that the sequence specificity of DNA cleavage is similar for these enzymes (28).

In this paper, we report the purification and characterization of a novel ATP-independent type I topoisomerase from the extremely thermophilic archaebacterium Desulfurococcus amylolyticus. We previously reported the isolation of reverse gyrase, an ATP-dependent type I topoisomerase, from this organism (24). Following the designation of two type I topoisomerases of E . coli as topoisomerase I and topoisomerase 111, the novel archaebacterial enzyme has been designated as topoisomerase I11 (we also propose to designate the reverse gyrase as archaebacterial topoisomerase I). We do not know, however, whether archaebacterial topoisomerase 111 is related to the Eco topoisomerase I11 or topoisomerase I lineages, or constitutes yet another lineage.

EXPERIMENTAL PROCEDURES

strain 2-533 (DSM 3822) has already been described (35, 36). 0.5- D. amylolyticus Strain and Growth Conditions-D. amylolyticus

12321

12322 A Novel Archaebacterial Topoisomerase

liter cultures were grown as previously described (24). For large scale preparation, cells were grown with stirring (130 rpm) in a 300-liter enamel-protected fermentor (HTE Bioengineering, Wald, Switzer- land), pressurized with N2/C02 (20230) (200 kilopascals). The anaerobic conditions were created according to Balch and Wolfe (37), and the medium of Brock et al. (38) was supplemented with 0.02% yeast extract, 0.2% peptone, and 0.2% So. The cells were rapidly cooled in the early exponential growth phase and harvested with a separator (Westfalia, Germany). The cells were stored a t -70 "C.

Materials-Reverse gyrase from D. amylolyticus (Dam reverse gyrase) was purified to homogeneity as previously described (24). Eukar- yotic topoisomerase I was from Serva. The protein markers were from Sigma, from Serva (human albumin and transferrin), and from Phar- macia LKB Biotechnology Inc. (aldolase). Immunoglobulin H chains were prepared from human IgG (Sigma) as described (39). The RNA polymerase from Thermoplasma acidophilum was a gift from Dr. R. Beabealashvilli (All-Union Cardiologic Scientific Center, Academy of Medical Sciences of the Union of Soviet Socialist Republics, Moscow). Bovine serum albumin and E. coli tRNA were from Boehringer Mannheim. Phenyl-Sepharose, heparin-Sepharose, DEAE-Sepha- rose, and DEAE-Sephacel were purchased from Pharmacia LKB. The phosphocellulose P-11 was from Whatman. Reagents for polyacrylamide gel electrophoresis were from Pharmacia LKB. Agarose (medium electroendosmosis, type 11) was from Sigma and low melting agarose was from Bio-Rad. Spermidine/3HCI, dithiothreitol, and ATP were purchased from Serva, 2-mercaptoethanol was from Carl Roth KG (Germany), glycerol was from Fluka, and ethylene glycol was from Shostkinskii Chimkombinat (U.S.S.R.). Protease inhibitors PMSF,' TPCK, TLCK (40), pepstatin A, and leupeptin were from Serva; soybean trypsin inhibitor and benzamidine were from Sigma.

DNA-The pBR322 DNA was obtained from NPO Ferment (Vil- nius, Lithuania). The Col E l and pUC19 DNAs were prepared by standard procedures (41). The pAT48 DNA, which contains a 48- base pair long AT sequence inserted at the Sma-I site of pUC19 (42), was a gift from Dr. I. Panyutin (N. D. Zelinsky Institute of Organic Chemistry, U.S.S.R. Academy of Sciences, Moscow). M13 DNA was a gift from D. Laptev (Institute of Molecular Genetics, U.S.S.R. Academy of Sciences, Moscow). Relaxed DNAs were obtained through incubation with eukaryotic topoisomerase I.

Methods of Protein Characterization-Protein concentrations were determined by the method of Bradford (43), using bovine serum albumin as a reference.

The protein composition of the fractions was analyzed in an SDS- pore gradient polyacrylamide gel according to the recommendations of Pharmacia LKB, using the Multiphor system for horizontal electrophoresis. The gels were stained with silver according to the method of Merril et al. (44).

High pressure liquid chromatography of Dam topoisomerase I11 was performed as follows. An aliquot of the final fraction was injected into a 250 X 4.6-mm reverse-phase AK-34 column (manufactured by the Institute of Chemical Physics, U.S.S.R. Academy of Sciences, Moscow). A gradient of 0-30% acetonitrile, 0.1% trifluoroacetic acid was applied a t a flow rate of 1 ml/min. Size exclusion chromatography of Dam topoisomerase I11 with marker proteins was performed on a Shodex WS-802.5 column (300 X 8 mm). Proteins were eluted with 0.7 M NaCl in 50 mM Tris/HCl (pH 7.3) a t a flow rate of 0.5 ml/min.

Tris/HCl (pH 8.0 a t 25 "C), 5 mM MgC12, 1 mM dithiothreitol, 0.7 Topoisomerase Assay-Reaction mixtures (10 pl) contained 50 mM

mM spermidine/3HCl, 30 pg/ml bovine serum albumin, 5% glycerol (standard buffer). The concentrations of other salts and amounts of DNA and enzyme are indicated in the figure legends as are the time and temperature of incubation. Where indicated, Dam topoisomerase I11 was incubated with DNA in buffer K: 30 mM Tris/HCl, pH 7.9, 135 mM NaC1, 5 mM MgCI2, 10 pg/ml bovine serum albumin. Reac- tions were terminated by rapidly cooling the mixtures to 0 or 25 "C and adding 0.2 volume of 5% SDS, 50 mM EDTA, 50% glycerol, 0.05% bromphenol blue. Dam reverse gyrase was assayed as described (24). For the topoisomerase assay in crude extract, a series of dilutions of the cleared lysate were incubated in standard buffer, without MgCI,, but containing 100 pg/ml tRNA. After incubation at 80 "C for 2 min, reaction mixtures were cooled to room temperature. The DNA and MgC1, were then added separately and incubation continued for 10 min. The reactions were stopped as above.

' The abbreviations used are: PMSF, phenylmethylsulfonyl fluo- ride; TLCK, ~-l-chloro-3-tosylamido-7-amino-2-heptanone/HCl; TPCK, ~-l-chloro-3-tosylamido-4-phenyl-2-butanone; SDS, sodium dodecyl sulfate.

Gel electrophoresis (in 1% agarose unless otherwise stated) was carried out essentially as described (24). The gels were stained with 1 pg/ml ethidium bromide for 30 min, rinsed with water, and photo- graphed under UV illumination (254 or 305 nm). The gels with chloroquine were soaked in water for 2 h before the staining.

Relaxation of a Topoisomer with a Unique Linking Number-A single, negatively supercoiled topoisomer of pUC19 DNA was obtained in the following way: pUC19 was subjected to electrophoresis (in several lanes) in a 1% low melting agarose gel in the presence of 4 pg/ml chloroquine for 16 h at 2 V/cm. One lane of the gel was cut off and stained with ethidium bromide, and the rest of the gel was soaked in 1 liter of T E buffer (IO mM Tris/HCl, pH 8.0, 0.1 mM EDTA) for 3 h. The piece of gel corresponding to the unique topoisomer was identified by comparison with the stained lane, excised, and soaked in 1 ml of T E buffer for 1 h. The gel fragment was then taken from the buffer, placed in an Eppendorf tube, and melted by heating. The unique topoisomer was incubated with the enzyme in buffer K + 0.75% agarose (total volume 20 pl) a t 80 "C for 15 min. The reaction was stopped as above, and the melted mixture was loaded into a well of a 2% agarose gel and electrophoresed as above.

Purification of Dam Topoisomerase III-The isolation of Dam topoisomerase 111 as a co-purified relaxing activity in the course of Dam reverse gyrase isolation has already been described (24). A modified procedure was used for large scale purification. All steps were carried out a t 0-4 "C. The cell pellet (20 g) was thawed and suspended in 80 ml of buffer A (100 mM Tris/HCl, pH 8.0 (at 25 "C), 10 mM 2-mercaptoethanol, 1 mM EDTA, 0.7 M NaC1,0.4 mM PMSF, 50 pg/ml TPCK, 50 pg/ml TLCK, 50 pg/ml leupeptin, 50 pg/ml pepstatin A, 10 pg/ml soybean trypsin inhibitor, 1 mM benzamidine). Cell extract, polymin P, and ammonium sulfate fractions were prepared essentially as previously described (24). The protein pellet after ammonium sulfate precipitation was dissolved in 70 ml of buffer B (50 mM sodium phosphate, pH 7.4, 0.5 mM EDTA, 2 mM 2-mercaptoethanol, 0.4 mM PMSF, 50 pg/ml leupeptin, 10 pg/ml pepstatin A) containing 0.8 M (NH&S04 and 1 M NaC1. After centrifugation, this fraction was applied to a phenyl-Sepharose column (2.6 X 20 cm). This step was carried out as in Ref. 45, with modifications. The column was washed with 1 liter of buffer B containing 0.8 M (NH,),SO,, 1 M NaCl at a flow rate 50 ml/h and then with 700 ml of buffer B containing 250 mM NaCl. I t was further washed with 500 ml of buffer B, containing 250 mM NaCl and 30% ethylene glycol a t a flow rate of 30 ml/h and developed with a linear gradient of 30- 60% ethylene glycol (2 X 360 ml in buffer B containing 250 mM NaCI). Active fractions were pooled (500 ml) and dialyzed against buffer C (50 mM Tris/HCl, pH 8.0, 0.5 mM EDTA, 2 mM 2-mercaptoethanol, 100 mM NaC1, 0.4 mM PMSF, 50 pg/ml leupeptin, 10 pg/ ml pepstatin A). After ammonium sulfate precipitation (65% saturation), the pellet was dissolved in 20 ml of buffer C + 10% glycerol and dialyzed against the same buffer. This fraction was loaded onto a DEAE-Sepharose column (1.2 X 9 cm). The column was then washed with 30 ml of buffer C + 10% glycerol. Active fractions, which appeared in the flow-through and wash, were pooled and dialyzed against buffer D (20 mM sodium phosphate, pH 7.4, 100 mM NaC1, 2 mM 2-mercaptoethanol, 0.5 mM EDTA, 0.4 mM PMSF, 50 pg/ml leupeptin, 1 pg/ml pepstatin A) containing 10% glycerol. This fraction was applied to a phosphocellulose column (2.6 X 8 cm). The column was washed with the same buffer and eluted with a 500-ml gradient of NaCl (0.1-0.8 M) in buffer D + 10% glycerol. The active fractions were pooled, dialyzed against buffer E (50 mM Tris/HCl, pH 8.0, 0.5 mM dithiothreitol, 10% glycerol) containing 200 mM NaCl and loaded onto a heparin-Sepharose column ( 5 ml). The column was washed with buffer E + 200 mM NaCl and eluted with a 40-ml gradient of NaCl (0.2-0.8 M) in buffer E. The relaxing activity was pooled and dialyzed against buffer E + 200 mM NaCI. This fraction was added to a slurry of equilibrated heparin-Sepharose (0.5 ml) in a Nalgene centrifugation tube and gently mixed by the tilting action of a gyratory mixer for 2 h. The mixture was packed into a column, and the enzyme was concentrated by elution with 1 ml of buffer E + 0.7 M NaCl and stored a t -20 "C. One unit of activity was defined as the amount of enzyme required to relax 50% of form I pBR322 DNA (0.1 pg) in standard buffer after 10 min at 80 "C.

RESULTS

Purification of Dam Topoisomerase 111-We have not SUC- ceeded previously in testing both positive supercoiling and relaxing activities in the crude extracts because of interfering

A Novel Archuebacterial Topoisomerase 12323

endonucleases (24). This problem was mentioned also by other authors (19,22,45). Here, we used a modified assay procedure as described under "Experimental Procedures," which includes the preincubation of the lysates with tRNA but without Mg'+ and DNA. After the preincubation, added covalently closed DNA is not degraded by nucleases. This allows accurate determination of both the ATP-independent relaxing and ATP-dependent positive supercoiling activities. Fig. 1 illus- trates the assay of topoisomerases in the crude extract by their action on negatively supercoiled DNA. Two different topoisomer distributions are generated in the absence of ATP and at a moderate concentration of NaCl (lanes 2-7), and with 1 mM ATP and at a high NaCl concentration (lanes 15- 20). The topoisomer distribution in lanes 2-6 is highly specific for the novel relaxing topoisomerase, whereas the appearance of positively supercoiled topoisomers in lunes 15-1 7 is specific for reverse gyrase. Note that the topoisomer distributions remain specific for each enzyme after their separation and throughout all steps of purification.

The novel topoisomerase was found in the course of Dam reverse gyrase purification (24) but was not properly charac- terized. The relaxing activity was associated with one poly- peptide of 108 f 5 kDa (Fig. 2). The enzyme was co-purified with reverse gyrase over DEAE-Sephacel and phosphocellulose columns, and only heparin-Sepharose chromatography

&$lzsE- , I O 20 40 80 160320. - 10 20 40 80 160320 10 20 40 80 160320;

NaCl 150 mM 250 mM

ATP 1mM

. .

FIG. 1. Detection of ATP-independent relaxing and ATP- dependent reverse gyrase activities in soluble extracts of D. amylolyticus. 1 p1 of an appropriate dilution of the cleared lysate (15 mg/ml) was incubated with 0.1 pg of pBR322 DNA at 80 "C for 10 min in standard buffer, as described under "Experimental Proce- dures." The products were analyzed by 1% agarose gel electrophoresis.

135- 108-

80- 69-

50 -

-Dam RG -DamTopom

.El

N

FIG. 2. Dam topoisomerase I11 was found as a relaxing activity co-purified with reverse gyrase. The panel shows the total protein SDS-polyacrylamide gel electrophoresis pattern from each chromatographic step of the purification, as well as the peak fractions of' topoisomerase activities from heparin-Sepharose. The location of protein standards is indicated on the ordinate (kilodaltons). The positions of reverse gyrase and relaxing enzyme are indicated by Dam RG and Dam Top0 III, respectively. For details see Ref. 24.

TABLE I Purification of relanine DNA towoisomerase from D. amvlolvticus

~~~ ~

Fraction Step Total Total Specific y. "o'. protein activity activity leld

rnl rng units unitslrng Ot I Crude extract 80 1200 2.6 X lo6 2.1 X 10' 100 I1 Polymin P 78 1175 2.5 x lo6 2.1 X 10" 9 i 111 Ammonium sulfate 84 630 3.4 X loG 5.3 X 10:' 129 IV Phenyl-Sepharose 25 131 3.2 X IO6 2.4 X 10' 123 V DEAE-Sepharose 45 101 1.4 X loG 1.4 X 10' 55 VI Phosphocellulose 58 1.7 1.2 X 1 0 6.7 X 10' 4.5 VI1 Heparin-Sepharose 12 0.06 4.8 X 10' 8.0 X 10; 1.8

separated the two activities. To overcome this difficulty (the co-purification of two topoisomerases up to heparin chromatography), the isolation scheme was modified so that phenyl- Sepharose was inserted as a first chromatographic step. A summary of the purification of Dam topoisomerase I11 is presented in Table I. Multiple protease inhibitors were in- cluded in each step of the topoisomerase I11 isolation up to phosphocellulose chromatography. Phenyl-Sepharose separated the topoisomerase activity into two peaks. The ATP- independent activity began to elute, upon developing with a 30-60% ethylene glycol gradient, a t 35% and formed a broad trailing peak up to 45%. The second peak, associated with reverse gyrase, eluted a t 50% ethylene glycol, as reported earlier for reverse gyrase from Sulfolobus acidocaldarius (Sac reverse gyrase) (45). At this stage, about 10-fold purification of the enzyme was achieved without loss of activity. However, the electrophoretic pattern of proteins remained complex (Fig. 3A) . DEAE-chromatography did not give a significant purification (Fig. 3A, Table I). In contrast, the use of a phosphocellulose column resulted in the elimination of the overwhelm- ing majority of the loaded proteins (Fig. 3A) . Unfortunately, this step was coupled with large losses of topoisomerase I11 activity (we should also note that the greatest part of reverse gyrase activity was lost at this step (45);2 we do not know the reasons for this). The bound proteins were eluted from the phosphocellulose column with a gradient of 0.1-0.8 M NaCl and topoisomerase I11 activity eluted at 0.55-0.6 M NaCl. The final step in the purification was chromatography of fraction VI over a heparin-Sepharose column, which separated the remaining proteins and gave a nearly homogeneous preparation of the enzyme. The relaxing activity was eluted as a single peak at about 0.6 M NaCl and was associated with the single protein peak, corresponding to -105 kDa. This was revealed by size exclusion chromatography of the resulting fraction (Fig. 3B) . Thus, SDS-polyacrylamide gel electrophoresis and size exclusion chromatography in nondenaturing conditions give the same molecular weight within experimental error.

Hydrophobic interaction chromatography revealed two peaks (Fig. 3C). We do not know yet whether one of them is a contamination protein or is a modified form of the same protein that is distinguished by its hydrophobicity. In the literature, it was reported that reverse-phase high pressure liquid chromatography can cause abnormal elution behavior of proteins; single polypeptides can elute as multiple peaks due to partial chemical modification such as oxidation, ester- ification, or deamidation of amino acid residues (46, 47). Unfortunately, this chromatography deactivates the enzyme.

Ionic Requirements for Dam Topoisomerase III Activity- Dam topoisomerase I11 relaxes negatively supercoiled DNA in an ATP-independent manner and absolutely requires Mg' for catalytic activity (Fig. 4, lanes 1-4; Fig. 6, lunes 3, 7, and

A. Slesarev, unpublished results.


45- w I 29- 24-

14-

158

C Retention tim

1

(IC 9c I

- - - - - -AeN ~Ob)contO.l% TFA

OD 280

I

FIG. 3. A, SDS-pore gradient polyacrylamide gel electrophoresis ( T = 4-22.596) of proteins for some purification steps used in the course of large scale Darn topoisomerase I11 isolation. The amount of protein loaded per lane was 10 pg of crude extract, 10 pg of phenyl- Sepharose load, 5 pg of DEAE-Sepharose load, 5 pg of phosphocellulose P-11 load, and 2 pg of heparin-Sepharose load. R, size exclusion chromatography of fraction VI1 on a Shodax WS-802.5 column (300 X 8 mm). Proteins were eluted with 0.7 M NaCl in 50 mM Tris/HCl buffer (pH 7.3) a t a flow rate of 0.5 ml/min. Aldolase (158 kDa), p- galactosidase from E. coli (subunit, 116 kDa), and phosphorylase b from rabbit muscle (subunit, 97.4 kDa) were added as internal markers. All unmarked peaks were from aldolase’s sample. AUFS, absorb- ance units for scale. Topo, topoisomerase. C, Chromatogram of elution of 25 pl of fraction VI1 containing 0.8 pg of protein from an AK-34 reverse phase column (250 X 4.6 mm). The gradient used was 0-3096 acetonitrile ( A c N ) , 0.1% trifluoroacetic acid ( T F A ) a t a flow rate of 1 ml/min.

9). Note that Dam topoisomerase I11 is able to relax negatively supercoiled DNA at equal concentrations of MgC12 and EDTA, at a high enzyme/DNA ratio (Fig. 6, lane 10).

ATP produces no effect of Dum topoisomerase 111-mediated reactions (Fig. 4, lanes 1 and 2; Fig. 5, lane 15), whereas the reverse gyrase strongly depends on ATP (Fig. 4, lanes 5 and 6 ) (22, 24). NaCl above 170 mM inhibits the topoisomerase I11 activity (Fig. 5, lunes 2-9). KC1 has essentially the same effect as NaCl (Fig. 5, lanes 10-14). Interestingly, the most favorable monovalent cation concentrations for Dum reverse gyrase, 170-250 mM NaCl (24), are inhibitory for Dum topoisomerase 111. This difference should be used if one wants to determine accurately both the topoisomerase 111-associated

0 1 0 0

TopoIII R G

FIG. 4. ATP-independent relaxation of negatively supercoiled pBR322 DNA by Dum topoisomerase 111. 30 ng of topoisomerase 111 (Top0 I l l , lanes 1-4) or 10 ng of reverse p r a s e (KG, lanes 5 and 6 ) was incubated with 0.12 pg of pBR322 in standard buffer a t 80 “C for 10 min. Lane 7, untreated pBR322 DNA. FI, form I; FII , form 11.

FIG. 5. Na’ and K’ ion requirements for optimal Dam topoisomerase 111 activity. Reaction mixtures containing 8 ny (lanes 1-14) or 3 0 ng (Iane 1 5 ) of topoisomerase 111 and 0.12 pg of pRR322 DNA were incubated in standard buffer a t 80 “C for 15 min. NaCl was added a t 20, 50, 80, 120, 170, 220, 270, 320, and 420 nM to the assay mixtures (lanes 1-9); KC1 was added a t 20,40,80,120, and 220 mM (lanes 10-14). In addition, each sample in lanes 10-14 contained 200 mM NaCI. Lane 15, standard buffer containing 70 mM NaC1, 100 mM KCI, and 1 mM ATP, Lane 16, relaxed pBR322 DNA.

gl“ 3- 4

5 6 7 8 9 to

Mgz* + - + + - - + EDTA - + + - + + +

FIG. 6. Action of Dam topoisomerase I11 on a single topoisomer. By the method described under “Experimental Procedures,” the topoisomer “-14” (lanes 5-7, -30 ngllane) and the topoisomer “-15” (lanes 8-10, -30 ngllane) were isolated from the pUC19 topoisomer population shown in lane I (0.3 pg). Lanes 2-4, native pUC19 (0.3 pg). Reactions with 30 ng of topoisomerase I11 were in buffer K (lanes 2 and 6), in buffer K without MgCl? and with 5 mM EDTA (Lanes 3, 7, and 9 ) , or in buffer K with 5 mM EDTA (lanes 4 and IO). Reaction mixtures were incubated a t 80 “C for 15 min. Gel electrophoresis was in 2% agarose with 4 pg/ml chloroquine.

activity and the positive supercoiling by reverse gyrase in crude extracts, since a t lower NaCl concentrations topoisomerase I11 will interfere with reverse gyrase.

Dam Topoisomerase III Is A Single-stranded DNA-specific Type I Topoisomerase-The novel Dam topoisomerase was shown to be a type I enzyme by incubating it with a single topoisomer of pUC19 DNA. Fig. 6 clearly shows that the enzyme increases the Lk of the unique topoisomer in steps of 1 (lanes 6 and 10). The same result was obtained at 75 “C (not shown). Therefore, Dum topoisomerase I11 is a type I topoisomerase. Note that the reactions took place in buffer that contained melted agarose (see “Experimental Proce- dures”), but this viscous medium did not affect the topoisomerization process.

Dum topoisomerase I11 appears to have a preference for

A Novel Archaebacterial Topoisomerase 12325

single-stranded DNA over double-stranded DNA, since the relaxation of the negatively supercoiled pBR322 was almost completely inhibited by the addition of single-stranded M13 DNA (Fig. 7). In addition, the enzyme had no effect on positively supercoiled pBR322 even a t a high protein/DNA ratio (-10) (Fig. 8). By contrast, reverse gyrase at the same conditions and protein/DNA ratio -6 winds DNA to a high positive superhelix density (Ref. 24 and Fig. 8d).

Temperature "Switches Over" Dam Topoisomeraie III Ac- tivity from the Relaxation of Negatively Supercoiled DNA to Extensive Unwinding of the Duplex-To determine the temperature range for the relaxation of negatively supercoiled DNA, we incubated form I pBR322 DNA with the enzyme at different temperatures. 1% agarose gel electrophoresis revealed relaxed topoisomers only after incubations a t 70 and 80 "C (Fig. 9) and did not reveal them after incubation outside this temperature range (Fig. 9A, lanes 2-4,6-8,14-16,18, and 19). At higher temperatures, after prolonged incubation with topoisomerase 111, form I pBR322 was converted to a form that migrated faster than untreated form I (Fig. 9A, lanes 17 and 19). This result indicates that the enzyme may be active above 80 "C, but the topoisomerization products may differ from the simply relaxed double helical circular DNA.

To examine the temperature range where the enzyme is able to catalyze the change in Lk and to determine the structure of the DNA species with abnormal electrophoretic mobility, we used pUC19 and pAT48 DNAs and separated the topoisomerization products in the presence of different amounts of chloroquine. Fig. 10 shows that from 60-70 "C up

0 0 1 0.2 0 4 08 1 6 M13IpBR322

FIG. 7. Single-stranded M13 DNA inhibits the relaxation of pBR322 DNA by Dum topoisomerase 111. Standard reaction mixtures with 140 mM NaCl containing 50 ng of topoisomerase 111, 0.12 pg of pBR322 DNA, and different amounts of M13 DNA were incubated a t 80 "C for 15 min. Imzes 1 and 8, untreated pBR322 and M13 DNAs, respectively. FI, form I; FII , form 11.

FIG. 8. Two-dimensional electrophoresis of positively supercoiled pBR322 DNA after treatment with topoisomerase 111 or with reverse gyrase. The second dimension (from left to riEht) was in the presence of 2 pg/ml chloroquine. The relaxed preparation of pRR322 (0.12 pg) containing topoisomers from 0 to +4 was incubated without enzyme ( a ) , with 20 ng of topoisomerase 111 ( b ) , with 40 ng of topoisomerase 111 (c), or with 50 ng of reverse gyrase ( d ) in standard buffer containing 160 mM NaCl at 80 "C for 15 min. ATP to 1 mM was added to lane d. Cooling the mixture from the incubation temperature (80 "C) to the electrophoresis temperature (20 "C) causes the number of supercoils in each topoisomer to decrease by -9 as calculated by taking the temperature dependence of the helix rotation angle to be -0.012 degrees/"C (69,70). Therefore, all topoisomers in lanes a-d had -9 additional positive supercoils under the incubation conditions.

A 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9

-1:

FIG. 9. A, temperature dependence of relaxation of pBR322 DNA by Darn topoisomerase 111. 30 ng of topoisomerase 111 (topo I I I ) and 0.12 pg of pBR322 were incubated in standard buffer containing 120 mM NaCI. FI, form I; FII , form 11. R, time course of relaxation of pBR322 by topoisomerase 111 a t 80 "C. The reaction mixture composition was as in A. Lane 1, native pBR322.

FIG. 10. Relaxation of negatively supercoiled pUC19 and pAT48 DNAs by Dum topoisomerase 111 at different temperatures. A, reaction mixtures containing 70 ng of topoisomerase 111 and 0.3 pg of pUC19 were incubated in standard buffer with 160 mM NaCl for 15 min. The band in lane 7 that corresponds to the remains of unwound form I is indicated by the arrow. Gel electrophoresis was in 2% agarose with 4.5 pg/ml chloroquine. R, reaction mixtures containing 17 ng of topoisomerase 111 and 0.2 pg of pUC19 or pAT48 were incubated a t different temperatures in buffer K for 15 min. Gel electrophoresis was in 1% agarose with 25 pg/ml chloroquine.

to 80 "C Dam topoisomerase I11 acts like the eubacterial relaxing enzymes do a t moderate temperatures and removes some or all negative supercoils (Fig. 10A, lane 2; B, lanes 3- 18). However, as temperature increases from 80 to 86 "C, topoisomerase I11 converts form I DNA to a more unwound closed circular form (Fig. 10A, lanes 2-4; B, lanes 17-24). At 90 "C, the linking number has been reduced by the enzyme to such an extent that topoisomers are not separated by gel electrophoresis even in the presence of chloroquine (Fig. 10A, lane 5 ) . Moreover, in spite of the saturation of the mobility of topoisomers during the shift from 85 to 90 "C (Fig. 10A, lanes 4 and 5 ) , a further shift to 95 "C leads to the appearance of a faster migrating form of pUC19 DNA (Fig. 10A, lanes 5 and 6) . The same effect was detected for pBR322 (Fig. 9A lanes 17 and 19) and has been described previously for extensively unwound DNA generated by other methods (48-57). At higher temperatures, an extensively unwound DNA is generated (Fig. 10A, lanes 5 and 6) . Complete separation of the strands might occur a t 99 "C (Fig. 10A, lane 7) , although we did not check this directly. Thus, a temperature threshold is

!:


observed in the reaction of topoisomerization by Dam topoisomerase I11 that must be passed to change the direction of the reaction from the increase to the decrease of the linking number. We found that this threshold is about 82 "C and corresponds to the beginning of the melting of pUC19 DNA (58).

Another observation worthy of note is that pUC19 and pAT48 behave differently in relaxation by Dam topoisomerase 111. The enzyme begins to relax PAT 48 DNA a t about 60 "C and pUC19 a t about 70 "C (Fig. lOB), and the relaxation proceeds more efficiently with pAT48 than with pUC19 (Fig. 10B, lanes 7-18). These data are in agreement with the fact that the 48-base pair long AT insertion in pAT48 begins to melt a t about 70 "C, whereas pUC19 DNA starts to melt around 80 "C (data for linear DNAs in 1 x SSC buffer (0.15 M NaCl, 0.015 M trisodium citrate (58)). Thus, the existence of the lower melting region in pAT48 in comparison with pUC19 stimulates the relaxing activity of single-stranded DNA-specific Dam topoisomerase I11 on this template. In contrast to the relaxation of negative superhelicity of pUC19 and pAT48, the extensive unwinding of both DNAs is initiated at the same temperature, 82 "C (where they begin to melt considerably), and proceeds with comparable efficiency (Fig. 10B, lanes 19-24).

Time Course of Relaxation of Negatively Supercoiled DNAs by Dam Topoisomerase III-We observed the time course of practically complete relaxation of negatively (but not positively) supercoiled DNAs a t a temperature just below the melting range of linear DNAs (80 "C, Figs. 9B and 11).

At lower temperatures, the enzyme fails to relax all negative supercoils from DNA (Figs. 10B and 11; see also the legend to Fig. 8). At these temperatures the mode of topoisomerization is distributive. The rate of relaxation by the enzyme drops with the decrease in the number of negative supercoils. For example, Dam topoisomerase I11 a t 80 "C introduces first about 10 turns into pAT48 DNA in 3 min, whereas the last turn is introduced in 20 min (Fig. 11B, lanes 8-10). The linking number of Col E l DNA is increased by the enzyme a t

70 75 8n m un'c

FIG. 11. Time course of relaxation of negatively supercoiled DNAs by Dum topoisomerase I11 at different temperatures in buffer K. A , reaction mixtures contained 17 ng of topoisomerase I11 and 0.33 pg of Col El . Lane 1, untreated Col El. R, reaction mixtures contained 17 ng of topoisomerase I11 and 0.15 pg of pAT48 or 0.3 pg of pUC19. Lanes I and 1 I contained, respectively, untreated pAT48 and pUC19 DNAs.

80 "C by 2 in 7 min, whereas the next unit step in Lk needs a further 20 min (Fig. 11A, lanes 5-7). In addition, as one can see from Fig. 11, the rate of topoisomerization at a constant superhelix density drops with the decrease in temperature. Thus, the time course of relaxation of negatively supercoiled DNA by Dam topoisomerase I11 is similar to that of relaxing type I DNA topoisomerases from mesophilic eubacteria when they act distributively (1, 6, 31).

Action of Dam Topoisomerase III on Positively Supercoiled DNA; Generation of Extensively Unwound Form I* and Form I** D N A Species-It was reported previously that single- stranded-DNA-specific type I topoisomerases from eubacteria are able to relax positively supercoiled DNA in two cases: at moderately high temperature and high enzyme/DNA ratio (59) or when a single-stranded bubble is inserted into DNA (60). Archaebacterial reverse gyrase, which belongs to the same group of enzymes, is able to use as substrate both positively supercoiled DNA with a single-stranded bubble and DNA without the bubble a t a high enzyme/DNA ratio (22, 24, 27). DNA relaxation by a topoisomerase in the melting range of linear DNA has not been investigated before. We have shown in the previous section that Dam topoisomerase I11 produces in this range extensively unwound topoisomers (Figs. 9A and 10). Another feature of high temperatures is the ability of even moderately positively supercoiled topoisomers to melt to some extent. Thus, there is no need to insert special single-stranded loops in DNA for activation of the enzyme.

Fig. 12B (lanes 5-8) shows the time course of linking reduction of positively supercoiled pUC19 by Dam topoisomerase I11 a t 95 "C (i.e. at the end of melting range of linear DNA (58)). In spite of the low enzyme/DNA ratio and the presence of negatively supercoiled topoisomers from the very beginning (Fig. 12A), Dam topoisomerase I11 is active on positive topoisomers. Note that the most positive topoisomer in the preparation has superhelix density +0.04 a t 95 "C (see the legend to Fig. 8). Thus, the melting of positively supercoiled topoisomers is enough for the enzyme activation at

" I

-FII

-FI -FI* -Floe

EO" 85" g d ' I 3 10 30 IO

FIG. 12. Action of Dam topoisomerase I11 on the composite preparation of pUCl9 DNA contained relaxed and native DNA.A, two-dimensional polyacrylamide-agarose gel electrophoresis of the composite preparation of pUC19 DNA contained 80% pUC19 relaxed by nicking-closing enzyme a t 25 "C and 20% of native pUC19. Marked are the positions of some topoisomers (see Ref. 71 for details). R, Dam topoisomerase 111 (17 ng) was incubated with the composite preparation of pUC19 (0.6 pg) in buffer K. Lane I, untreated pUCl9 DNA. Note that the relaxed topoisomers of the composite preparation were positively supercoiled under the incubation conditions (see the legend to Fig. 8). We followed Ref. 54 when labeling bands below FI as FI* and FI**, F, form.

A Novel Archaebacterial Topoisomerase 12327

95 "C, whereas the absence of melted regions does not permit the enzyme to relax positive supercoiling at 80 "C. These and other data (Figs. 7 and 8) prove the specificity of Dam topoisomerase I11 for single-stranded DNA.

Two new bands below the band corresponding to the form I appeared after the action of the enzyme on the composite preparation of pUC19 DNA at 95 "C (Fig. 12B, lanes 7 and 8) . The only example of the separation of extensively unwound DNA by gel electrophoresis in two bands below native form I DNA was described in Ref. 54. These species were named form I* and form I**, and it was shown that the faster migrating form I** pBR322 is more unwound than the form I*. We designated the bands below FI in Fig. 12B in the same way. However, we should indicate that the reasons for the change of mobility, as well as the relationships between the Lk and the mobility, remain unknown for highly unwound topoisomers.

The time course of topoisomerization differ from that described earlier (Figs. 9B and 11). The topoisomer distribution becomes broad, and extensively unwound I* and I** forms of DNA appear before disappearance of the initial positive topoisomers (Fig. 12B, lanes 5-8). It appears that topoisomers with practically all possible linking numbers are present in the sample after 30 min of incubation (lane 8) . These data indicate that the rate of topoisomerization does not substan- tially depend on Lk, as was the case at lower temperatures (Figs. 9B and 11). More detailed characterization of this process awaits new procedures for separation of topoisomers with small Lk.

Fig. 12B shows also that Dam topoisomerase I11 is not active on positively supercoiled DNA at 85 "C (lane 3 ) , i.e. at the beginning of linear DNA melting (58). In the middle of the linear DNA melting range, the enzyme converts moderately positively supercoiled topoisomers (numbers -3 and -2 in Fig. 12) to form I, whereas more positively supercoiled topoisomers remain intact (90 "C, Fig. 12B, lane 4 ) . Thus, the larger the positive superhelix density of substrate DNA, the higher the temperature necessary to activate the enzyme.

DISCUSSION

Dam Topoisomerase 111 Is a Novel Type I-Group I D N A Topoisomerase-We have purified to near homogeneity a novel DNA topoisomerase from the extremely thermophilic anaerobic archaebacterium. D. amylolyticus. The enzyme has a M , 108,000 f 5,000, as determined by electrophoresis under denaturing conditions, and is a monomer at 0.7 M NaCl, as judged by size exclusion chromatography. I t changes the linking number in steps of 1 and is specific to single-stranded DNA (Figs. 6-8 and 12). Dam topoisomerase I11 can work in either direction; at 60-80 "C it relaxes negatively but not positively supercoiled DNA (i.e. it increases the linking number of DNA (Figs. 8 and lo)), and at 82-99 "C the enzyme unwinds both positively and negatively supercoiled substrates (i.e. it decreases the L k ) and produces extensively unwound form I* and I** DNA (Figs. 10 and 12). Mg2+ is absolutely required for the topoisomerization reactions, whereas ATP produces no effect; the optimal concentration of NaCl or KC1 for relaxation at 80 "C is about 150 mM. The enzyme is active in the pressence of agarose. Thus, Dam topoisomerase I11 belongs to the type I-group I DNA topoisomerases and is the second enzyme of this group from D. amylolyticus.

The specific properties of Dam topoisomerase I11 add to the list of differences among type I-group I topoisomerases. Dam topoisomerase I11 has a lower molecular weight than Dam reverse gyrase and has less affinity to phosphocellulose and heparin. The enzymes differ also in their affinity for single-

stranded DNA; Dam topoisomerase I11 needs melted regions in DNA for relaxation and unwinding, whereas reverse gyrase can work on DNA with low duplex stability. The ATP- independent activity of Dam topoisomerase I11 was detected in freshly prepared lysates in the presence of multiple protease inhibitors (Fig. 1). In addition, the ratio of relaxing to positive supercoiling activites depends on the growth phase of D. amylolyticus; it is higher in the early exponential than in the stationary phase.' I t is also important to emphasize that both activities are not limited to D. amylolyticus cells; they were also detected in other extremely thermophilic archaebacteria (72, 73). Thus, our current knowledge is in the favor of the presence of Dam topoisomerase I11 with its DNA relaxing activity in the cell.

D N A Topoisomerization at High Temperatures-We have shown that the direction and products of DNA topoisomerization by Dam topoisomerase I11 crucially depend on temperature (Figs. 9-12). To explain this effect, we should take into consideration the DNA helix-coil transition. In the melting range of linear DNA, the completely relaxed topoisomer should be partly melted. If its degree of denaturation is 1 - Oo, then its linking number, L h , is less than that of the relaxed completely duplex DNA, L b d , and is equal to Oo. Lkod. The higher the temperature, the less is 29" and the smaller is L h , compared with L b d . Thus, whatever the initial Lk of a negatively supercoiled topoisomer, at some temperature it becomes equal to L b = Oo. Lkod. Below this temperature, Dam topoisomerase I11 relaxes DNA by increasing Lk, whereas above it, the enzyme changes the direction of relaxation and decreases Lk. Above the melting range of linear DNA, complete relaxation means complete separation of DNA strands, i.e. Lko = 0. At these conditions, the enzyme always reduces Lk. Thus the novel DNA topoisomerase can be used for generation of topoisomers with required Lk up to Lk = 0 (including forms V, I**, I*, U, or X (48-57, 61)).

Our data can be used also to compare two alternative mechanisms of positive supercoiling activity by thermophilic reverse gyrase (27, 62). I t was suggested recently that reverse gyrase could be a chimeric protein that consists of a DNA helix-tracking ATPase and relaxing topoisomerase that is specific to DNA conformation (62). This model differs sub- stantially from another mechanism that is based on the recognition of mutual orientation of two single strands of DNA by the enzyme (27). The "tracking plus relaxation" mechanism works very well at moderate temperatures where the topoisomerase increases Lk due to the relaxation of negative superhelicity. However, at the high temperatures of the reverse gyrase activity (up to at least 100 "C (24)), negatively supercoiled duplex DNA is unstable and undergoes helix-to- coil transition. So, any relaxing topoisomerase (like Dam topoisomerase 111) would decrease Lk instead of increasing it.

Possible Functional Role of Dam Topoisomerase III-Ironi- cally, although ATP-independent Dam topoisomerase I11 is topochemically closer to eubacterial relaxing DNA topoisomerases I than reverse gyrase is, its physiological role appears to be different. At ambient temperatures of D. amylolyticus up to 97 "C) it denatures DNA rather than stabilizes the double helix. I t is unlikely that Dam topoisomerase I11 takes part in regulation of cellular DNA supercoiling and relaxes positive superhelicity because of the greater affinity of reverse gyrase to single-stranded DNA and its ability to positively supercoil DNA to an extent unaccessible to Dam topoisomerase I11 activity.

Another functional role of bacterial DNA topoisomerases consists in the decatenation of parental strands at the end of chromosome replication (63-68). All known Eco topoisomer-


ases (I, 11, 111, and IV) are able to do this work, although Eco topoisomerase I11 was suggested to be the best candidate for kinetic reasons (19). Dam topoisomerase I (reverse gyrase) should be excluded from this list because of its inability to reduce Lk and hence to unlink parental strands. The final products of the extensive unwinding of DNA by Dam topoisomerase I11 at high temperatures should be comletely separated complementary strands. We suggest that Dam topoisomerase I11 is the archaebacterial counterpart of Eco topoisomerase I11 and that their function is possibly the segregation of a pair of newly replicated molecules. However, determination of the role of Dam topoisomerase 111, as well as establish- ing the phylogenetic relationships with known type I topoisomerases, awaits its more detailed characterization.

Acknowledgments-We thank Lisa Bonch-Osmolovskaya, Jutta Seger, and Konrad Eichinger for their great help with growth of D. amylolyticus, Michel Duguet for suggestions and discussions, and Diana Golden for her great help with the manuscript. We deeply appreciate Fraser McBlane and Marty Gellert for their critical read- ing of the manuscript, fruitful discussions, and a lot of valuable suggestions.

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