THE JOURNAL OF BIOLOGICAL CHEMISTRY (0 1989 by The American Society for Biochemistm ’ and Moleculaz ’ Biology, Inc

Vol. 264, No. 30, Issue of October 25. pp. 17924-17930, 1989 Printed in U.S.A.

Molecular Cloning and DNA Sequence Analysis of Escherichia coli topB, the Gene Encoding Topoisomerase 111”

(Received for publication, May 4, 1989)

Russell J. DiGateS and Kenneth J. Marianss From the Program in Molecular Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center,

”

New York, New York 10021

Escherichia coli contains two type 1 topoisomerases, topoisomerase I and 111. Although topoisomerase I11 can be purified as a potent decatenase, its role in DNA metabolism is unclear. In order to address this issue, the gene encoding topoisomerase I11 from E. coli has been molecularly cloned and its DNA sequence deter- mined. The cloned fragment of DNA contains an open reading frame that can encode a polypeptide of 73.2 kDa. The first 20 amino acids of this open reading frame are identical to those of topoisomerase I11 as determined by amino-terminal gas-phase microse- quencing. Expression of the polypeptide encoded by this open reading frame, using a bacteriophage T, tran- sient expression system, results in the accumulation of a 74-kDa polypeptide. Soluble extracts prepared from cells overexpressing this gene product show a dramatic increase in topoisomerase activity when compared with control extracts. We propose that this gene be designated topB.

DNA topoisomerases are enzymes that alter the topological state of DNA by the transient breakage and resealing of either one strand (type 1 topoisomerase) or both strands (type 2 topoisomerase) of the DNA duplex (1). Three topoisomerases have been purified from Escherichia coli, DNA gyrase (2), topoisomerase I (Topo I) (3),’ and topoisomerase I11 (Topo 111) (4-6). DNA gyrase has been shown to be an essential enzyme in E. coli (7-11). Although deletions of the gene ( topA ) encoding Top0 I exist, these strains rapidly acquire compen- satory mutations in either the genes encoding the DNA gyrase subunits (12-14) or in a locus known as toc (14). It has been shown that these two topoisomerases are involved in the maintenance of the topological state of the E. coli chromosome

* This work was supported in part by National Institutes of Health Grant GM34558 and American Cancer Society Grant NP-648. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “eduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 505076.

$Recipient of National Institutes of Health National Research Service Award GM11381.

Recipient of an Irma T. Hirschl, Monique-Weil Caulier Career Scientist Award.

The abbreviations used are: Topo, topoisomerase; form I, super- coiled circular duplex DNA; form 11, nicked circular duplex DNA; form 111, linear duplex DNA ss(c), single-stranded covalently closed circular DNA ORF, open reading frame; SDS, sodium sodium dodecyl sulfate; SST, 0.015 M Tris-HC1, pH 7.5, 0.15 M NaC1, 0.0025 M EDTA; SSC, 0.015 M sodium citrate, pH 7.0, 0.15 M NaCI.

(l), as well as in transcription (15-17), and possibly recom- bination (18, 19).

Topo I11 was originally identified as a superhelical DNA relaxing activity in soluble extracts prepared from strains in which topA had been deleted (4). However, the purification of Topo I11 based on this activity proved unreliable. Subse- quently, this laboratory identified and purified Top0 I11 as a factor required to segregate replicating pBR322 daughter DNA molecules in uitro (6). Highly purified Top0 I11 was shown to be a potent decatenase, the Kc,, for decatenation was 9-fold higher at 30 “C than the Kcat for the relaxation of negatively supercoiled DNA, thus explaining the difficulty encountered purifying the enzyme based on the latter assay.

In order to facilitate both the biochemical analysis of the properties of this enzyme and the determination of its role in DNA metabolism, the gene encoding Top0 I11 of E. coli has been molecularly cloned. In keeping with the designation of the genes encoding the A and B subunits of DNA gyrase as gyrA and gyrB, and that encoding Top0 I as topA, the gene encoding Top0 I11 has been designated topB.

MATERIALS AND METHODS

E. coli Strains and Bacteriophage-Competent DH5a and DH5aF’ cells for transformation were obtained from Bethesda Research Lab- oratories. E. coli strain BL21 and bacteriophage X-CE6 were provided by Dr. F. William Studier of Brookhaven National Laboratory. Bac- teriophages X-328 and X-329 (20) were provided by Dr. C. Squires (Columbia University).

DNA-pBS-M13 (Bluescribe) was obtained from Stratagene. M13 Mpl8 and Mp19 form 1 (supercoiled) DNAs were obtained from Bethesda Research Laboratories. Genomic DNA was prepared from E. coli strain HMS-83 ( r h lys thy polAl polB100 lacZam st$). Plasmid pet3C was provided by Dr. F. William Studier.

Enzymes-Restriction enzymes were from Bethesda Research Lab- oratories and New England Biolabs. Sequenase was from U. S. Biochemicals.

DNA Sequencing-DNA fragments were molecularly cloned into M13 mp18 and M13 mp19 replicative form DNAs. Bacteriophage DNA was isolated and sequenced using Sequenase as suggested by the manufacturer. The products of sequencing reactions were sepa- rated through 6% polyacrylamide gels containing 50% urea using 100 mM Tris-borate, 1 mM EDTA as the running buffer. Gels were dried before autoradiography.

Reagents-Acrylamide, Zeta Probe membrane, and protein deter- mination kit was from Bio-Rad. Agarose ME was from Seakem (Rockland, MD). Immobilon was from Millipore.

Relaxation of Superhelical DNA-Superhelical DNA relaxation assays were performed as described previously (6) except that bacte- riophage 6x174 form I DNA was used as the substrate.

Transformation of Competent E. coli-Transformation of E. coli DH5a and DH5aF’ was performed as described by the manufacturers recommendations.

Southern Analysis-Southern analysis was performed using a Vacugene Apparatus (Pharmacia LKB Biotechnology Inc.). Transfer to Zeta probe was carried out in 0.4 N NaOH. All other procedures were as previously described (21,22) except 2 X SST (2 X SST is 0.3

17924

E. coli topB 17925

M NaCI, 0.03 M Tris-HCI, pH 7.5 at 22 "C, 0.005 M EDTA) was used in the place of 2 X S s c .

Amino Acid Microsequencing of Top0 III-E. coli Topo 111 was purified as previously described (6). Aliquots (totaling 40 wg) were electrophoresed through a 10% polyacrylamide gel containing 0.1% SDS and transferred to a polyvinylidene difluoride (Immobilon) membrane as described hy Matsudaira (23). The 74-kDa Top0 III polypeptide was located by staining with Coomassie Blue, excised, and used for amino acid microsequence analysis (courtesy of Dr. W. Lane, Harvard University).

RESULTS

Molecular Cloning of the Gene Encoding Top0 111-An amino-terminal sequence was obtained for the major (6040% purity) 74-kDa polypeptide present in preparations of E. coli Top0 I11 (4-6). Forty pg of Top0 I11 (fraction 5 as in Ref. 6) were electrophoresed through a 10% polyacrylamide gel (con- taining 0.1% SDS). Protein was electroblotted to Immobilon membrane (23) and the 74-kDa polypeptide identified by staining with Coomassie Blue. Twenty pmol of protein bound to the membrane (331% of the transferred sample) was sub- jected to automated Edman degradation in an Applied Bio- systems 470A gas-phase microsequencing apparatus. A 20 amino acid amino-terminal sequence was determined (Fig. 1A). Based on the degeneracy of the potential mRNA se- quence encoding this peptide sequence (Fig. 18), the codon usage frequency in E. coli (25), and allowing G-T base pairing in the wobble position, an oligonucleotide probe (Fig. 1 C) was designed as a "best guess'' to locate the Top0 I11 gene. This best guess sequence was shown, after sequence analysis of the isolated gene (as described below), to be 83% correct (Fig. 10).

The best guess oligonucleotide was used to identify, from

an enriched plasmid library of E. coli genomic sequences, a plasmid DNA carrying the gene for Top0 111. Southern blots of genomic DNA digested with restriction endonucleases pos- sessing 4-base pair recognition sequences were probed with the best guess oligonucleotide. A 2.5-kbp DNA fragment was identified in HaeIII-digested genomic DNA that hybridized to the oligonucleotide under conditions of 85% stringency (22). This DNA fragment was not digested by either the EcoRI, BurnHI, or HindIII restriction endonucleases. There- fore, a plasmid library enriched for the 2.5-kbp Hue111 DNA fragment was prepared by first digesting E. coli genomic DNA (20 pg) simultaneously with the HueIII, EcoRI, BarnHI, and HindIII restriction endonucleases. This digest was fraction- ated by electrophoresis through an agarose gel. The region of the gel surrounding the 2.5-kbp DNA fragment was excised and the DNA electroeluted and recovered by ethanol precip- itation. This DNA preparation was ligated into SmaI-digested pBS-Ml3 RFIII DNA (Bluescribe). This mixture of ligated DNA was used to transform competent E. coli DH5a cells.

Recombinant plasmid DNAs were isolated from cells grown from 65 white colonies. The plasmid DNA was electropho- resed through agarose gels, blotted, and probed with the radiolabeled Top0 I11 oligonucleotide. One positive signal was obtained. A restriction map of the genomic DNA insert in this plasmid (pRD15) is shown in Fig. 2. The map was constructed using Southern blot analysis with the Top0 111 probe.

The 800-bp AccI, 400-bpAccI/EcoRV, 685-bp HincII/BglII, and 670-bp BglII/HincII DNA fragments (Fig. 2) were sub- cloned into M13 mp18 and mp19 RF DNA, and the insert regions in the recombinant M13 single-stranded DNA were sequenced in both directions. The DNA sequence of the cloned

A) Met Arg Leu Phe I l e Ala Glu Lys Pro Ser Leu A l a Arg Ala Ile Ala Asp Val Leu PTO

FIG. 1. The amino-terminal se- quence of Top0 I11 and probe design. A, the first 20 amino acids of Top0 111 as determined by microsequence analysis. 8, mRNA degeneracy for the Top0 111 amino-terminal sequence C, sequence o f the best guess oligonucleotide probe. An examination of the cloned Top0 111 gene DNA sequence (D) reveals that this probe was 83% homologous to the actual Top0 111 DNA sequence, and 89% ho- mologous if one assumes G-T base pair- ing neither strengthens nor weakens the DNA duplex.

G G G G G G G G G G G G G G G T T T T T T T T T T T T T T T c c c c c c c c c c c c c c c c c

B) ATG CGA CTA TTT ATA GCA GAA AAA CCA TCA CTA GCA CGA GCA ATA GCA GAT GTA CTA CCA AGA TTA AGC TTA AGA TTA

G G T G G G

C) ATG CGT CTG TTT ATT GCT GAA AAA CCG TCT CTG GCT CGT GCT ATT GCT GAT GTT CTG CCG

D) TAC GCC AAC AAA TAA CGG CTT TTT GGC TCA GAC CGC GCG CGG TAA CGA CTA CAG GAC GGG 5' 5' ATG CGG TTG TTT ATT GCC GAA AAA CCG AGT CTG GCG CGC GCC ATT GCT GAT GTC CTG CCC

811 RV H A A H l l l

2.5 kbp Hae 111

I , ; I I I I I I ! ! I

I

0.7 kbp

nmc 111 801 II

801 IU Hlnc I1 (MCS) 0.7 kbp

FIG. 2. Restriction map of the cloned 2.5-kbp HaeIII DNA frag- ment containing the putative Top0 I11 gene. The indicated DNA fragments derived from the fragment were sub- cloned into M13 mp18 and mp19 RF DNA as described in the text. HIIZ, HaeIII; A, AccI; RV, EcoRV; BZI, BglII; H, HincII. MCS indicates that the HincII restriction endonuclease site was located in the (m)ulti-(clloning (s)ite of the vector DNA.

E. coli topB m , m 2

1

10 20 30 4 0 *Ar 50 60 70 80 CCACTTGAGC W T G T G I C AGGGGCICTGG TGTGCWCA CGCGTCWCT ATGAAGCGAT CCCGAAACTC CCCGGIGITG AAGAGTACAT TAAGTTGGGC GCAGTACCTG GCGGCACTGA ACGTAACiTT GCCAGCTACG GTCAICIGA:

90 100 110 12c 13; 1 4 5 ,x

160 170 180 I90 200 210 220 230 240 250 260 210 2eO 290 3CZ W T G A M T G CCGCGTCAAG TGCGCGATCT GCTGTGTGAT CCGCAMCTT CTGGCGGTTT GCTGCTGGCG GTCATGCCGG RAGCAGAAM TGAGGICAAA GCTACAGCCG CCGAGTTTGG CATTGMCTG ACGGCMTTG GCGMCTGGT

1200 1210 1220 1230 1240 1250 1260 1270 1280 1 1290 CGG GM TCA GM TCC K G CCG CTG C C I T T T TCG CTT TCA GCG TTG CAG ATT GM GCG GCA AM CGT T T T GGT CTG ACT GCG CAG M C GTG CTT GAT ATC TGC CAG AM CTG T I C G M ACG CAC MG Arm d l u Ser Glu SCI Ala Pro Leu Pro Phe Ser Leu Ser A 1 1 Ley G l n Ile G I u 1\11 A l a L y l Arq Phe G l y Leu Ser A l a G l n Aln V a l Leu Asp Ile Cy$ G l n Lys Leu Tyl Glu T h l His L y d

1300 1310

T R T M T G G C T G a M A T A T RMTffiIM TTTTGMCGG A T G A I I A M G M M T A A C G G 2490 2500 2510 I' 2520 2510 2540

FIG. 3. The DNA sequence of the 2.5-kbp HaeIII DNA fragment. The 653-amino acid ORF (nucleotide 345-2306) has been translated. Two other ORFs are present on this fragment. One, extending from the beginning of the fragment to 4 bp before the Top0 111 initiation codon (nucleotides 1-340), and another extending from

E. coli topB 17927

TABLE I Codon usage of the topB gene

was obtained from Konigsberg and Godson (25). The frequency of codon usage from 25 nonregulatory E. coli genes

Amino acid Codon -___ % codon use

topB E. coli

Phe

Leu

Ile

Met

Val

Ser

Pro

Thr

Ala

UUU 64 UUC 36

UUA 5 UUG 16 CUU 18 CUC 3 CUA 5 CUG 53

AUU 42 AUC 50 AUA 8

AUG

GUU 13 GUC 20 GUA 11 GUG 56

ucu 10 UCC 16 UCA 6 UCG 13 AGU 32 AGC 22

ccu 8 ccc 10 CCA 17 CCG 65

ACU 4 ACC 50 ACA 11 ACG 37

GCU 12 GCC 24 GCA 15 GCG 49

44 56

6 8 9 7 2

69

37 62 1

38 13 23 27

27 26 8

11 6

22

9 6

20 65

24 51 6

20

28 19 23 30

Lmino acid Codon 9% codon use

tODB E. coli

UAU UAC

UAA UAG UGA

CAU CAC

CAA CAG

AAU AAC

AAA AAG

GAU GAC

GAA GAG

UGU UGC

UGG

CGU CGC CGA CGG AGA AGG

GGU GGC GGA GGG

75 41 25 59

100 88 0 4 0 8

47 39 53 61

30 27 70 73

28 24 72 76

71 77 29 23

65 51 35 49

70 73 30 27

58 42 42 58

33 58 43 35 6 2

18 3 0 1 0 0.25

30 48 32 41 11 5 27 7

HueIII DNA fragment is shown in Fig. 3. The DNA sequence of nonoverlapping clones was confirmed by using synthetic primers complementary to DNA sequences upstream of the AccI and BgZII restriction sites with Mp19 SS(c)DNA con- taining the entire 2.5-kbp DNA insert as a template.

The 2.5-kbp HueIII DNA fragment contained an open reading frame (ORF), extending from nucleotide 345 to 2303, with a coding capacity of 653 amino acids. The first 20 amino acids of this open reading frame were identical to those of Topo I11 (Figs. 1 and 3). The predicted molecular mass of this polypeptide, 73.2 kDa, was in excellent agreement with the observed molecular mass of 74 kDa for Top0 I11 (4-6).

Table I shows the codon usage of topB and 25 other E. coli genes that encode abundant, nonregulatory proteins (25). Although the topB gene does not contain the extremely rare AGA and AGG arginine codons, there is a bias toward less frequently used codons. This is particularly evident for the

AGU serine, AUA isoleucine, and CGG arginine codons. An analysis of the DNA sequence upstream of the large

ORF revealed a potential ribosome-binding site 13 bp before the translational initiation codon (Fig. 3). Although a consen- sus -35 promoter sequence was present from nucleotide 263- 272, no -10 box was observed. Interestingly, DNA sequences resembling the consensus u54 nitrogen starvation promoter (24) could be observed upstream of the topB gene (Fig. 4).

Immediately downstream of the termination codon is a potential p-independent transcription terminator, a 16-bp inverted repeat that includes a stretch of 8 consecutive thy- midine residues (Fig. 3, nucleotide 2378-2385).

Immediately upstream and downstream of the gene are open reading frames. The upstream ORF extends from the beginning of the DNA fragment to nucleotide 340. The down- stream ORF begins at nucleotide 2434 and extends to the end of the HueIII DNA fragment (Fig. 3). No match was found when the predicted amino acid sequences for these two ORFs were compared with the GenBank protein sequence data base.

Top0 ZII Shows Homology to Top0 I-The predicted amino acid sequence for Top0 I11 was used in a homology search of the translated GenBank, Doolittle, and Dayhoff protein se- quence data banks. There was only one significant homology detected. This homology was to E. coli Top0 I. Top0 I11 and I share a 24% identity and 46% similarity over a stretch of 308 amino acids (Fig. 5) resulting in an optimized score of 271 using the Fast P homology program.' Two tyrosine resi- dues at amino acid positions 320 and 328 of topB are located in a highly conserved region between the two topoisomerases; therefore, this region is a good candidate for the active site of the enzyme. Although the two enzymes show a high degree of homology in the middle of the proteins, they diverge rapidly in the NHz-terminal and COOH-terminal regions.

Expression of the Putative Top0 IIZ Gene Results in In- creased Topoisomerase Activity in Crude Extracts-The pu- tative Top0 I11 gene was introduced into a transient expres- sion DNA vector such that it was under the control of a bacteriophage T7 RNA polymerase promoter. The gene prod- uct was expressed after infection of the plasmid-containing cell with bacteriophage X (CE6) carrying the bacteriophage T7 RNA polymerase gene under the control of PL (26, 27).3 Extracts prepared from cells bearing the putative Top0 I11 ORF showed a dramatic increase in the amount of a 74-kDa polypeptide (Fig. 6A); in addition, virtually all of the polypep- tide was present in the soluble fraction (compare lune 2, Fig. 6A with the insoluble cellular debris (lane 1 , Fig. 6A)). The overproduction of this polypeptide was correlated with a greater than 15,000-fold induction of topoisomerase activity (Fig. 6B) .

The demonstration of a translated amino-terminal amino acid sequence in the putative Top0 I11 ORF that matched exactly the sequence determined experimentally from genuine Top0 I11 (Figs. 1 and 3) and the correlation of overexpression of the 74-kDa polypeptide with the pronounced induction of topoisomerase activity allows one to conclude that the ORF does, in fact, encode E. coli Top0 111.

Mapping of the Top0 III Gene on the E. coli Chromosome-

' The next highest homology was to the Barley B1 hordein gene which resulted in an optimized score of 60. Top0 I11 had a self- homology score of 3225.

R. J. DiGate and K. J. Marians, manuscript in preparation.

nucleotide 2434 to the end of the Hue111 DNA fragment. S.D. indicates a potential ribosome-binding site. Z.R. indicates the inverted repeat that is a potential p-independent terminator. Pertinent restriction endonuclease sites are shown above their recognition sequence.

17928

FIG. 4. The topB gene possesses DNA sequences resembling a RNA polymerase u64 promoter. The region immediately upstream of the topB gene initiation codon is compared with the nifM and nifF promoter sequences (24). The sequences similar to the promoter consensus are boxed, and another ho- mologous sequence between the three genes is underlined. The arrow indicates that the insertion of a cytosine residue in the topB DNA sequence would result in a match to the consensus sequence.

E. coli topB

CTGGC TTGCA c o n s e n s u s

TCAGCCAGCCGTGGCTGGCCGGAAATTTGCAATACAGGGAT ni fM n n

T GTATGCAAAGCAACCTGGCACAGCCTTCGCAATACCCCTGC

I I I" 260 1 2 7 0 I 280 290 -

TACAGCCGCCGAGTTTGGCATTGAACTGACGGCAATTGGCG U 4

ni fF

t opB

190 200 210 220 t O p B TILGRNAGYQGVLSVGRVQTPVLGLVVRRDEEIENFVAKDFFEVKAHIVTPADERFTAIW

230 240

tOpA VSPLLWKKIARGLSAGRVQSVAVRLAVEREREIKAFVPEEFWEVDASTTTPSGEAL-ALQ . . . . . . . . . . . . . . . . . . . . . . . . ::.,::...:.::.: ..::..: . :.

180 190 200 210 220 230

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESPNRYASKENS-QEAHEAIRPSDVMTMAESLKD~ADAQKLYQLIWRQWACQMTPMY

350 360 370 380 390 400

4 30 440 450 460 470 RKCVIELDIRKGKFVRKRRFLREAGWRTLLGSKERDEENDGTPLPWRKGDELLCEKGEV

480

. . . . . . . . . . . . . . DSTTLTVGAGDFRLKARGRILRFDGWTXV"PAW(GDED-RILPAVNKGDALTLVELTP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

410 420 430 440 450 4 60

490 500 510 520 530 540 VERQTQPPRHFTDATLLSAMTGI-QD~LKKILRATDGLGTEATRAGIIELLFKRGF

AQHFTKPPARFSEASLVKELEKRGIGRPSTYASIISTIQDRGYVRVENRRFYAEKMGEIV . . . . . . . . . . . . . . . . . . . . . . . .

410 480 4 90 500 510 520

FIG. 5. E. coli Top0 I11 shows protein sequence homology to E. coli Top0 I. (:) indicates identity and (.) indicates a conservative change.

Kohara and co-workers (20) have established a restriction endonuclease map for the E. coli chromosome and have also constructed a bacteriophage X library of the E. coli genome. It is therefore possible to map a gene by performing Southern analysis of genomic DNA digested with the eight restriction endonucleases used by Kohara et al. (20), and placing the pattern obtained on the existing map. In this manner, a genomic restriction map was constructed for the Top0 I11 gene. A search of the restriction map of E. coli revealed only one probable map position for the Top0 I11 gene, at 38.7 min, between pheT and pabB.

To confirm that the Top0 I11 gene was located at this point, bacteriophage X containing this region of the genome was obtained and the DNA was isolated and analyzed by Southern analysis using a Top0 I11 gene-specific probe (Fig. 7A). The Top0 111-specific probe hybridized to identically sized DNA fragments in both the bacteriophage X and plasmid DNA restriction endonuclease digests, confirming that the Top0 I11 gene is located at 38.7 min on the E. coli map near (within 15 kbp) the gene encoding exonuclease I11 ( x t k 4 ) (Fig. 7B) .

C

DISCUSSION

The gene encoding E. coli Top0 I11 has been molecularly cloned, sequenced, and mapped in the E. coli genome. DNA sequence analysis of the DNA fragment containing the Top0 I11 gene revealed a large ORF capable of encoding a polypep- tide of 73.2 kDa. When this ORF was expressed using a T7 transient expression system, a 74-kDa polypeptide was ob- served. In addition, soluble extracts prepared from cells con- taining the overproduced polypeptide had dramatically in- creased topoisomerase activity.

Since the genes encoding the subunits of DNA gyrase are designated gyrA and gyrB and that encoding Top0 I is desig- nated topA, we propose that the gene encoding Top0 I11 be designated topB.

Analysis of codon usage in topB indicates the presence of codons that are usually ascribed to rare proteins in E. coli (25). In fact, Top0 I11 is present at only 1-10 copies/cell (6). However, codon usage as a means to control the concentration of Top0 I11 in the cell is unlikely, since the protein can be overproduced in large quantities using the T7 transient expres- sion system.

The topB gene has a potential ribosome-binding site and -35 box, however, a -10 box was not observed. In addition, a p-independent terminator is located immediately down- stream of the gene. The lack of a good promoter sequence upstream of the Top0 I11 gene, the presence of an ORF that terminates within four nucleotides of the Top0 I11 initiator ATG, and the presence of the RNA polymerase transcription terminator sequence after the Top0 I11 gene suggest that topB may be the terminal gene of a multicistronic message.

The presence of a potential nitrogen starvation promoter upstream of the topB gene indicates that the expression of the gene may be alternately controlled in the absence of a nitrogen source. Experiments to determine the mRNA initi- ation sites are in progress.

The topB gene shows considerable homology to the topA gene; however, this homology is present in the middle of both polypeptides, with the two diverging rapidly at their amino- and carboxyl-terminal ends. Analysis of the structural do- mains of Top0 I by mutagenesis (28) indicated that the carboxyl-terminal 11% of Top0 I was insensitive to premature termination by inserted nonsense codons; however, nonsense mutations that removed more than 11% of the carboxyl- terminal domain of Top0 I completely eliminated activity. Interestingly, these mutations are well outside the region of homology between Top0 I and 111.

The deletion analysis of Top0 I correlates with the loss of two of three putative zinc fingers (29) in Topo I. A search of topB for a zinc finger motif revealed no matches to any of the

E. coli topB 17929

A . 1

B. 1

2 3 4 5 6 7 kDa

- 200

- 97.4

- 68

- 43

FIG. 6. Expression of the Top0 I11 ORF resul ts in the accu- mulation of a 74-kDa polypeptide and superhelical DNA re- laxation activity. The putative Top0 I11 ORF, or the putative Top0 I11 ORF containing a 1-bp deletion within the first 20 nucleotides of the gene: was cloned into the T, transient expression vector pet-3c resulting in plasmids pDEl and pDE2, respectively. A , cells contain- ing the pDEl and pDE2 plasmids were infected with bacteriophage X CE6, incubated at 37 "C for 3 h, and collected by centrifugation. The cells were lysed by incubation with lysozyme in the presence of 0.1% Brij-58 for 30 min a t 0 "C. The cellular debris was removed by centrifugation, and the supernatant fluid was retained. Solid ammo- nium sulfate (0.39 g/ml) was added to the supernatant fluid and the solution was stirred slowly at 0 "C for 30 min. The precipitate was collected by centrifugation and resuspended in the same amount of buffer as the soluble extract. The pellet remaining after centrifugation of the lysis mixture was also resuspended in the same amount of buffer as the soluble extract. One volume of 2 X SDS gel dye was added, and each sample was heated to 90 "C for 5 min. Equal volumes (2 pl) of the samples were then electrophoresed through a 10% polyacrylamide gel containing 0.1% SDS and the protein visualized by staining with Coomassie Blue. Lane I , the resuspended insoluble pellet from cells harboring plasmid pDE1. Lane 2, the soluble extract prepared from cells harboring plasmid pDE1. Lane 3, the resuspended ammonium sulfate pellet from cells harboring plasmid pDE1. Lane 4 , high molecular weight protein markers. Lane 5, the resuspended ammonium sulfate pellet from cells harboring the control plasmid, pDE2. Lane 6, the soluble extract prepared from cells harboring pDE2. Lane 7, the resuspended pellet from cells harboring plasmid pDE2. A densitometer scan of lane 3 revealed that the 74 kDa polypeptide represented >80% of the protein present in the sample. B, the resuspended ammonium sulfate pellet prepared from both cultures was assayed for superhelical DNA relaxation activity as described under "Materials and Methods." Reaction mixtures con- tained no protein (lane I ), 0.4 ng (lanes 2 and 6 ) , 2 ng (lanes 3 and 7 ) , 10 ng (lanes 4 and 8), or 50 ng (lanes 5.and 9) of the resuspended ammonium sulfate pellet prepared from cell-harboring plasmid pDEl (lanes 2-5) or plasmid pDE2 (lanes 6-9).

A.

2915 bp-

1175 bp-

- 29

2 3 4 5 6 7 8 9

- Form I1 - Form I11

B. 28

1 2 3 4 5 6 7

- w,

.

28. 1 1 35.6 38. 4 3 ,8.7 rnin

++ + + direction of transcription

FIG. 7. T h e topB gene maps to 38.7 min on the E. coli chromosome. A , Southern analysis of the topB clone and bacterio- phage X-329 DNA. Lane I , size markers. Lane 2, the original topB- containing plasmid digested with the EcoRI and BamHI restriction endonucleases to excise the 2.5-kbp HaeIII DNA fragment (the cloning procedure eliminates the original HaeIII sites). Lane 3, bac- teriophage X-329 DNA, containing the region about 38.7 min on the E. coli chromosome, digested with the HaeIII restriction endonucle- ase. Lane 4 , the topB-containing plasmid digested with the HincII restriction endonuclease. Lane 5, the bacteriophage X-DNA digested with HincII restriction endonuclease. Lane 6, the bacteriophage X- 329 DNA digested with the PuuII restriction endonuclease. Lane 7, genomic DNA digested with the PuuII restriction endonuclease. The blot was probed with nick-translated plasmid DNA carrying the HincII amino-terminal fragment of the topB gene. The topB contain- ing plasmid and the bacteriophage X-DNA contain identically sized HaeIII and HincII DNA fragments (lanes 2-5) that are complemen- tary to the Top0 I11 oligonucleotide (the 3.2-kbp band observed in the lanes containing plasmid DNA are vector sequences that hybridize because the probe DNA was in the same vector as the to@-containing plasmid). The observed hybridization of the probe is not because of contamination of the bacteriophage X-DNA with E. coli DNA, since the PuuII restriction endonuclease digest of the bacteriophage X- DNA and genomic DNA are distinctly different (lanes 6 and 7; the bacteriophage X-329 DNA encompasses only one of the PuuII restric- tion enzyme sites present in the genomic DNA PuuII fragment). B, the map position of the topB gene places it just outside of the T2 replication fork terminator. A map of this region is shown. This drawing is not to scale. The direction of transcription of top& topA (34), cysB (34), and xthA (35) are also indicated.

consensus sequences (30,31). These data suggest that Top0 I and I11 are functionally distinct in the cell.

In the absence of Top0 I, E. coli rapidly acquires compen- satory mutations in gyrA, gyrB (12-14) and a locus known as toc (14). The toc locus maps to 66 min, near tolC (14). Genomic mapping of the topB gene to 38.7 min excludes the possibility that topB is toc. The lack of A topA compensatory mutations in topB may not be surprising, since preliminary data suggest that overproduction of Top0 I11 is toxic to the cell:

R. J. DiGate and K. J. Marians, unpublished data.

17930 E. coli topB

The role of Top0 111 in the cell is unclear. There is a strain of E. coli, BW9109, that contains a chromosomal deletion from xthA (38.4 min) to pncA (39 min). This strain, which should lack topB, is viable, suggesting that topB is not an essential gene. However, it is not known whether this strain, similar to A topA strains, has acquired compensatory muta- tions. The construction of a A topA topB strain of E. coli is currently under investigation.

The map position of the topB gene is particularly interesting in view of the location of topA near cysB and trp A-E, just outside of the TI replication fork terminator (32, 33). Simi- larly, topB is located just outside of the T2 replication fork terminator (Fig. 7 B ) .

The T, and T2 DNA sequences act as polar replication fork terminators that essentially act as a replication fork trap, confining the replication forks to the region between the two sites (32, 33). If Top0 111 is involved in the segregation of nascent daughter DNA molecules, the location of the topB gene just outside of the T2 terminator may serve to regulate its expression during the cell cycle. The topB gene could be inactive during the majority of the cell cycle either from topological constraints, or as a result of the action of a specific repressor. However, transcription of the topB gene could be activated by the passage of the replication fork through the region just prior to its entrance to the replication fork termi- nator region. This means of control may serve two purposes: 1) to time the expression of Top0 111 production, and 2) since transcription and translation are coupled in E. coli, the loca- tion of the topB gene near the region where termination of E. coli DNA replication occurs may serve to concentrate the enzyme near its site of action. This model remains to be tested.

The genes encoding the two type 1 topoisomerases of E. coli are located symmetrically about the terminator region. When combined with the evidence that Top0 I11 shows sig- nificant protein homology to Top0 I and the polar nature of the replication fork terminators, it is possible that this region arose by the duplication and inversion of a single terminator and topoisomerase locus.

Acknowledgments-We thank Drs. J . Hunvitz, F. Dean, S. Rabkin, and S. Shuman for their critical reading of the manuscript and D. Valentin for the artwork.

Note Added in Proof-E. coli Top0 111 exhibits an almost identical

the product of the TOP3 gene of yeast (Wallis, J . W., Chrebet, G., molecular mass and shows extensive protein sequence homology to

Brodsky, G., Rolfe, M., and Rothstein, R. (1989) Cell 5 8 , 409-419).

REFERENCES 1. Wang, J. C. (1985) Annu. Reu. Biochem. 54,665-688 2. Gellert, M., Mizuuchi, K., ODea, M., and Nash, H. (1976) Proc.

3. 4.

5.

6.

7. 8.

9.

10. 11.

12. 13.

14.

15.

16.

17.

18.

19.

20. 21.

22. 23. 24. 25.

26.

27.

28. 29.

30. 31. 32.

33.

34.

Natl. Acad. Sci. U. S. A . 73 , 3872-3876 Wang, J. C. (1971) J. Mol. Biol. 5 5 , 523-533 Pastorcic, M. (1982) Purification and Characterization of a New

Type I Topoisomerase in E. coli. Ph.D. thesis, University of Chicago

Srivenugopal, K. S., Lockshon, D., and Morris, D. R. (1984) Biochemistry 2 3 , 1899-1906

DiGate, R. J., and Marians, K. J. (1988) J. Biol. Chem. 263 ,

Drlica, K., and Snyder, M. (1978) J. Mol. B i d . 120, 145-154 Kreuzer, K. N., and Cozzarelli, N. R. (1979) J. Bacteriol. 140 ,

Orr, E., Fairweather, N. F., Holland, I. B., and Pritchard, R. H.

Steck, T. R., and Drlica, K. (1984) Cell 36 , 1081-1088 Bliska, J. B., and Cozzarelli, N. R. (1987) J. Mol. Biol. 194 , 205-

Pruss, G. J., Manes, S. H., and Drlica, K. (1982) Cell 31,35-42 DiNardo, S., Voelkel, K. A., Sternglanz, R., Reynolds, A. E., and

Raji, A., Zabel, D. J., Laufer, C. S., and Depew, R. E. (1985) J .

Jovanovich, S., and Lebowitz, J . (1987) J . Bacteriol. 169, 4431-

Menzel, R., and Gellert, M. (1987) Proc. Natl. Acad. Sci. U. S. A .

Sternglanz, R., DiNardo, S., Voelkel, K. A., Nishimura, Y., Hir- ota, Y., Becherer, K., Zumstein, L., and Wang, J. C. (1981) Proc. Natl. Acad. Sci. U. S. A . 78 , 2747-2751

Ikeda, H., Aoki, K., and Naito, A. (1982) Proc. Natl. Acad. Sci. U. S. A. 79,3724-3728

Fishel, R. A., and Kolodner, R. (1984) J. Bacteriol. 160 , 1168- 1170

Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50,495-508 Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular

Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

13366-13373

424-435

(1979) Mol. Gen. Genet. 177 , 103-112

218

Wright, A. (1982) Cell 31,43-51

Bacteriol. 162, 1173-1179

4435

84,4185-4189

Lathe, R. (1985) J. Mol. Biol. 183 , 1-12 Matsudaira, P. (1987) J. Biol. C k m . 262 , 10035-10038 Ausubel, F. M. (1984) Cell 37,5-6 Konigsberg, W., and Godson, G. N. (1983) Proc. Natl. Acad. Sci.

Rosenberg, A. H., Lade, B. N., Chui, D.-S., Lin, S.-W., Dunn, J.

Studier, F. W., and Moffatt, B. A. (1986) J . Mol. Biol. 189 , 113-

Zumstein, L., and Wang, J. C. (1986) J . Mol. Bid. 191,333-340 Tse-Dinh, Y.-C., and Beran-Steed, R. K. (1988) J. Biol. Chem.

Berg, J. (1986) Nature 319,264-265 Evans, R. M., and Hollenberg, S. M. (1988) Cell 5 2 , 1-3 Hill, T. M., Henson, J . M., and Kuempel, P. L. (1987) Proc. Natl.

DeMassy, B., Bejar, S., Louarn, J., Louarn, J.-M., and Bouche,

Wang, J . C., and Becherer, K. (1983) Nucleic Acids Res. 11,1773-

U. S. A . 8 0 , 687-691

J., and Studier, F. W. (1987) Gene (Amst . ) 56 , 125-135

130

263 , 15857-15859

Acad. Sci. U. S. A. 8 4 , 1754-1758

J.-P. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,1759-1763

1790

(1988) J . Bacteriol. 170 , 4542-4547 35. Saporito, S. M., Smith-White, B. J., and Cunningham, R. P.