Proc. Nati. Acad. Sci. USAVol. 86, pp. 9431-9435, December 1989Genetics

Characterization and immunological identification of cDNA clonesencoding two human DNA topoisomerase II isozymes

(two genes/oligonucleotide probes/anti-peptide antibodies/comigrating mRNAs)

THOMAS D. Y. CHUNG, FRED H. DRAKE, K. B. TAN*, STEVEN R. PERt, STANLEY T. CROOKEt,AND CHRISTOPHER K. MIRABELLI-IResearch & Development, Smith Kline & French Laboratories, P.O. Box 1539, King of Prussia, PA 19406

Communicated by Peter B. Dervan, August 29, 1989 (received for review June 20, 1989)

ABSTRACT Several DNA topoisomerase II (Topo II; EC5.99.1.3) partial cDNA clones obtained from a human Raji-HN2cDNA library were sequenced and two classes of nucleotidesequences were found. One member of the first class, SP1, wasidentical to an internal fragment of human HeLa cell Topo [1cDNA described earlier. A member of the second class, SPli,shared extensive nucleotide (75%) and predicted peptide (92%)sequence similarities with the first two-thirds of HeLa Topo II.Each class of cDNAs hybridized to unique, nonoverlappingrestriction enzyme fragments of genomic DNA from severalhuman cell lines. Synthetic 24-mer oligonucleotide probes spe-cific for each cDNA class hybridized to 6.5-kilobase mRNAs;furthermore, hybridization of probe specific for one class wasnot blocked by probe specific for the other. Antibodies raisedagainst a synthetic SP1-encoded dodecapeptide specifically rec-ognized the 170-kDa form of Topo II, while antibodies raisedagainst the corresponding SPli-encoded dodecapeptide, or asecond unique SPil-encoded tridecapeptide, selectively recog-nized the 180-kDa form of Topo II. These data provide geneticand immunochemical evidence for two Topo II isozymes.

DNA topoisomerase II (Topo II; also called type II topo-isomerase; EC 5.99.1.3), a target of several anticancer drugs,is involved in chromosome segregation and is implicated inthe regulation of gene structure and function (1-4). Theenzyme functions as a dimer that passes a DNA segmentthrough a transient double-stranded break (4). Topo II genesequences from Saccharomyces cerevisiae (5), Schizosac-charomyces pombe (6), and Drosophila melanogaster (7)have been published. Recently, a Topo II cDNA sequencefrom a human cervical carcinoma cell line (HeLa) wasreported and the gene was mapped to chromosome 17 (8). Ineach case, Topo II was reported to be encoded by a single-copy gene.Topo II has been purified from various sources and is most

commonly isolated as a protein of about 170 kDa (7, 9-11).Reports of smaller polypeptides that reacted with Topo II-specific antibodies were attributed to proteolytic degradationof the 170-kDa protein (9, 11, 12). However, we recentlypurified two forms of functional Topo II from P388 murineleukemia (13) and U937 human monocyte cell lines (14). Oneform migrates on SDS/PAGE as the previously reported170-kDa polypeptide, while the other migrates as a polypep-tide of 180 kDa (13, 14). Polyclonal antisera specific for eitherthe 170- or the 180-kDa polypeptide demonstrate that both arealso present in the human Burkitt lymphoma cell line Raji (15),in the human colon carcinoma cell line COLO 201 (16), and inthe mouse fibroblast cell line 3T3 (17). The two enzymes differin a number of biochemical and pharmacological properties(14). They appear to be differentially regulated, and the ratio

of their levels changes in response to cellular proliferation rate(14), oncogenic transformation (17), and selection for resis-tance to Topo II inhibitors (13) or alkylating agents (15).These results led us to seek genetic evidence for Topo II

isozymes. We constructed a human cDNA library in Agtllfrom a mechlorethamine-resistant Burkitt lymphoma cell line(Raji-HN2), which contains increased amounts of both the170- and the 180-kDa form of Topo II (15, 18). Our resultsdemonstrate that two cDNA§ classes representing unique butsimilarly sized mRNAs encode either the 170- or the 180-kDaTopo II enzyme.

MATERIALS AND METHODSMethods. The construction, screening, and subcloning of

the Raji-HN2 cDNAs have been described (15). Oligonucle-otides were synthesized on a model 380DNA synthesizer andpurified over OPC columns (Applied Biosystems). Syntheticpeptides were obtained from Multiple Peptide Systems (SanDiego, CA).

Nucleotide Sequencing and DNA Analysis. cDNAs weresequenced by the Sanger dideoxynucleotide method (19) withSequenase (United States Biochemical) on alkali-denatureddouble- or single-stranded templates (20), using syntheticsequencing primers. Sequence information was analyzedwith the University of Wisconsin Genetics Computer Group(21) and DBSYSTEM programs (22). Southern analyses ofgenomic and cDNAs were as described previously (15).RNA Analysis. Poly(A)+ RNA from exponentially growing

U937 (14) was isolated by oligo(dT) chromatography, elec-trophoresed in a 1.5% agarose vertical gel containing 2 Mformaldehyde, and transferred to Optiblot (International Bio-technologies) membranes (23). A nonstringent hybridizationofeach replicate strip to 32P-labeled oligonucleotides (23) wasdone separately, according to Wood et al. (24), withoutdextran sulfate and including Escherichia coli tRNA at 50kkg/ml, then washed in 3 M trimethylammonium chloridecontaining 0.1% SDS at 42-500C (24).

Specific Anti-Peptide Antibodies and Western Blot Analysis.Synthetic peptides were glutaraldehyde-linked to hemocya-nin, mixed with Freund's complete adjuvant, and injectedintradermally into rabbits (23). High-titer antisera were pu-rified after IgG fractionation by affinity chromatography onmedia prepared by covalently linking each peptide to Affi-Prep 10 (Bio-Rad) according to the manufacturer's instruc-tions. Following SDS/PAGE (25), proteins were transferred

Abbreviation: Topo II, DNA topoisomerase II.*To whom correspondence and reprint requests should be ad-dressed.

tPresent address: Quality Biologics, Copewood Street, Camden, NJ08103.tPresent address: ISIS Pharmaceuticals, 2280-B Faraday Avenue,Carlsbad Research Center, Carlsbad, CA 93008.§The sequence reported in this paper has been deposited in theGenBank data base (accession no. M27504 for the 180-kDa form).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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to Immobilon membranes (Millipore) in 10 mM 3-(cyclohex-ylamino)-1-propanesulfonate (Caps), pH 11 (26), for 1 hr at400 mA in a Hoefer Transfor unit. Immunoblotting was asdescribed previously (13).

RESULTSAnalysis and Identification of Novel cDNA Clones. After the

Raji-HN2 library had been screened with a cDNA probeencoding Drosophila Topo II (7), 26 positive cDNA cloneswere obtained. Restriction enzyme analysis of 12 clonesdemonstrated the presence oftwo classes ofcDNAs (8 clonesin class I and 4 in class II). Representatives of class I (SP1)and class II (SP11 and SP12) cDNAs were completely se-quenced from both strands. Confirmatory sequencing wasdone on the remaining clones. The longest class I cDNA,SPi, was identical (Fig. 1) to the internal 3031-base-pairEcoRI-EcoRI fragment of the published HeLa Topo II se-quence (8). The longest class II cDNA, SP11, an EcoRI-SstI insert rescued from a Agtll recombinant, contained about1.1 kb of phage arm at its 5' end. The restriction map of SP1lhad notable differences from the HeLa map: no Kpn I andXba I sites, additional Bgl II sites, one different Nco I site,and different BamHI, EcoRI, and Pst I sites.SP11 Encodes a Protein with Features Conserved Among

Type II Eukaryotic Topoisomerases. Alignments betweenSP11 and the HeLa (SP1) nucleotide (Fig. 2) and encodedprotein (Fig. 3) sequences were done according to Smith andWaterman (31) and Gribskov and Burgess (32), respectively.The nucleotide sequences were colinear with no gaps andshared a 75% overall identity. Replacements occurredthroughout the alignment with over 80% of the replacementsbeing in the codon third base position. The 2685-base-paircoding sequence of SP11 ended at an internal EcoRI site(AG I AATTC), since no additional linker-derived G (GGA-ATTCC) preceded this restriction site.The HeLa and SP11 peptide sequences were also colinear

(Fig. 3) and shared a 92% protein similarity, consistent withcodon third base redundancy. All other reading frames werepunctuated with numerous stop codons. The SP11-encodedprotein spanned (Fig. 1) the first two-thirds (59%o) of the HeLaprotein and comprised 895 amino acid residues (102 kDa).On the basis of alignments with Escherichia coli (33, 34)

and Bacillus subtilis (35) gyrases, eukaryotic Topo II isproposed to consist of three regions (see ref. 7): an N-terminal third homologous to the B or ATPase gyrase sub-unit, a central portion homologous to the N-terminal regionof the A or breaking-rejoining subunit, and a highly chargedlysine-rich C-terminal third homologous to histones H1 andH5, which may be involved in DNA binding (36). The

N Hc B Hc E H Hc Bg K N X B PIr ,~ ... .

E H HcBgKN X B P

HeLa it

Class I

\SP 1

Bg Bg P H Hc Bg N B Ne ESP1-- - S IfI

Class I I P H Hc Bg N B E

\SP12-- I

B E

B E

1 kb

FIG. 1. Restriction maps of class I (HeLa and SP1) and class II

(SPli and SP12) cDNAs. HeLa map generated from sequence in ref.8. Alignment of inserts and selected restriction sites are indicated: B,BamHI; Bg, Bgl II; E, EcoRI; Hc, HincII; H, HindI1; K, Kpn I; N,Nco I; P, Pst I; X, Xba I. Solid regions are noncoding sequences andbroken lines are Agtll phage sequences. kb, Kilobase.

SP11-encoded protein spanned some of the first and all of thesecond region (indicated in Fig. 3).The SP11-encoded protein showed greater homology (32)

with the HeLa Topo II protein (91%) than with the Drosoph-ila (68%) or Saccharomyces (60%) Topo II proteins. TheSP11-encoded, HeLa, and Drosophila protein sequencesremained colinear (refer to alignments in ref. 7) until aminoacid 1140 (HeLa) and contained several highly conservedpeptide stretches (indicated in Fig. 3). The strictly conservedtwo amino acid motif Arg-Tyr, at position 677 of SP11 (803 inHeLa), corresponds to the critical tyrosine (Tyr-122) in E.coli gyrase A (27) to which the DNA is covalently linkedwhen the enzyme transiently breaks DNA during strandpassage. Three of the conserved potential eukaryotic glyco-sylation (Asn-Xaa-Thr) sites (28) were also present in SP11(indicated on Fig. 3): asparagines 243 (Asn-Pro-Thr), 253(Asn-Met-Thr), and 818 (Asn-Gly-Thr). Two of the con-served potential sites (Arg-Xaa-Yaa-Ser/Thr) for phospho-rylation by protein kinase C (29) and calmodulin-dependentprotein kinase of eukaryotic Topo II enzymes (7) werepresent in the SP11-encoded protein: serine 370 (Arg-Glu-Ala-Ser) and threonine 805 (Arg-Thr-Trp-Thr). TheHeLa protein possesses a putative "leucine zipper" (30)motif (Leu-Xaa6-Leu-Xaa6-Leu-Xaa6-Leu) near its C-terminal third (37), but the SP11 protein lacked the fourthleucine. This may be important in protein dimerization.

Class II cDNA Detects Different Genomic Sequences. TheTopo II gene appeared to be similarly organized in normal(MRC5) and neoplastic (Raji-HN2) cells. Unique and non-overlapping fragments were detected when BamHI-, HindIII-,or Pst I-digested genomic DNAs from Raji-HN2 and MRC5cell lines were probed with either class I (SP1) or class II(SP12) cDNAs (Fig. 4). Genomic DNA from these and othercell lines digested with EcoRI, Pvu II, Msp I, or Sst I alsoyielded unique hybridization patterns (data not shown). Theseresults indicate that the two classes of cDNA were derivedfrom different genomic sequences and suggest that a differentgene may exist for each Topo II isozyme.The Two cDNA Classes Represent Comigrating mRNAs.

32P-labeled cDNAs of either class detected an apparently6.5-kb mRNA (15, 16). To rule out cross-hybridization oftheselong probes to the same mRNA, oligonucleotides complemen-tary to a 24-base region where the aligned sequences of SP11and HeLa greatly differed (indicated on Fig. 2) were synthe-sized for use as probes. Probe I (5'-pAATGGTTGTAGAAT-TAAGAATAGC-3') was specific for SP1, while probe II(5'-pTACTGTGTTTCTGTCCACTACAAA-3') was specificfor SP11 sequences. Both oligonucleotide probes detected a6.5-kb mRNA. However, when 32P-labeled probe I was hy-bridized to identically prepared strips containing electropho-resed poly(A)+ RNA from U937 cells in the presence ofunlabeled probe I or II, only unlabeled probe I reduced theintensity of the radioactive signal in a concentration-depen-dent manner (Fig. 5). Even a 100-fold molar excess of unla-beled probe II did not reduce the hybridization signal of32P-labeled probe I. The converse experiment with 32P-labeledprobe II yielded complementary results. These results dem-onstrate that SP1 and SP11 represent two different mRNAswhich comigrate under the electrophoretic conditions used.

Class II cDNAs Encode the 180-kDa Topo II. Specificantibodies were raised against synthetic dodecapeptides rep-resenting corresponding but dissimilar protein stretches inthe alignment (indicated on Fig. 3) of the proteins encoded bySP1 or HeLa (beginning at residue 874, N I R R L M D G EE P L) and by SP11 (beginning at residue 748, N V R R M LD G L D P H). Affinity-purified antibodies directed againstthe SP1-dodecapeptide (class I) selectively recognized the170-kDa protein on Western blots containing equal amountsof purified 170- and 180-kDa U937 enzymes (17) or freshwhole cell lysates (Fig. 6). Antibodies directed against the

H H =L:lm

9432 Genetics: Chung et al.

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Genetics: Chung et al. Proc. Natl. Acad. Sci. USA 86 (1989) 9433

1 OTAGAACACAAGGTGGAGAAAGMATGTTCCTGCTTAATTTTTOGACAOCT MAACATCCAGTAACTATGATGATGATGAGAAAAAAGTTACAGGTGGTCGTAATGGTTATGGTGCAII 11111111 II II II 11111 111111llllll ll~llllll llll111111lllllllIIIIIIIIIII 111111 Illl~llllll 11111 1111111l

379 GTTGAACACAAAGTTGAAAAGATGTATGTCCCAGCTCTCATATTGGACAGCTCCTAACTTCTAGTAACTATGATGATGATGMAAAGAAAGTGACAGGTGGTCGAAATGGCTATGGAGCC121 AAACCTTT^ATATTTTCACTACAAArriACAGTAGuACAGCTTGCAAAGAATACACACACGTTTTAGCAGACATGGATG^TAATATGATGAAGACTTCTGAAGCCAAAATTAAi

111 1 11111 11111111111 11 11111 1111111111 1I 1 111111111 1I1ii lllll~lllllll 11111111 II 111 11 111499 AAATTGTGTAACATATTCAGTACCAAATTACTGTGGAAACAGCCAGTAGAGMATACAAGAAAATGTTCAAACAGACATGGATGGATAATATGGGAAGAGCTGGTGAGATGGAACTCAAG241 CATMTGATCGTGAAGATTiCACATGCATAACATTCCAACCAGATCTGTCCAAATTTIACA&TCAAAAACTTGACAACCATATTGTGCCCCTCATGACTGACAGCCGCATTCATGTmGGCi

1 11111111 11111 11 11 1 11IIIIII p111 1111111 II III 11 11111 1111 1111111 111111H I1111 1III

819 GCTATACATATACAAGATAAACACAGGTGAAGTGTG~CACTCAACTATGTAA~mAAAGTTTCAA TGCACAAAACATATTCAACACATCTACATTCACAAGGTGCATAGACATGTGAT3C1 CTATCCTCTAGACAGTGTGGTCAACCCATCI ATTGAAGACTAGAWGTTACCTAAATCCiGCGTOTATTCAiTGACCATGTCAAGTACAMTCCTATAAACTCGGTTTTACCAATGCC~

1111 I 11 1111 1iii IIII 1111 111 1111 111 11111111 11111 11111 IIIi III11 11111 11 11IIIIIIIII I 11111

9739 TCATACTGATCAMGATTGTGCTAAAT~CTTGTTGCATGTTTGAAGAGAAGACAGGGTGGTGTTGATGTCAACACATCTmGAGGACGTGAATCCATGAMTGGATGTAATGCCTTCAA481 CTATTGAATiCAACTCiTTCAATCAGCATCCAAGGAACiTGACATCTGCAGCCCAAAMMGGAT GGCCCAGCAMCA iGTCAAGTAAiTTTTTATACAAMGTCACGCCTACGTGG.ATi

liii 11111IIIi 111111111111I11IIII Illllll 11111111111111IHM 11 11111111 II 11111 I 11l III8699 ATTGAATA'1CCCAA M'1CCMGA T11MTTTTCTCAAAAGAA ATGACTTCAACCCACTTTGC1 CACMATG1CCATTGAGC1CTGAAAAA ATCAAAGCTGCCATGAC1TGTGTATT

841 GTATCGAAAGATCMCTGCTTGTCCT-rAAGCTCATTG CTCAGCTGMMACAAGAACTGGTCATCAGTCiAATCAmCGTAMATCAAGATCCCAATATGGATC.TTTTAATTATTGCi111111111liii 11111 lii liii1111 1111111 11111 I1HI1I1I11 IIII 11111 11111III 11111I liiiIIII

9719 GTAGAAACCATACTAACATGGTGAC MMCAAGTCCAGlTTCCATTMAAlACAAGAAGTlTCATTG~lTAMAAACA ATAlGATCAAGAATTCCAAClTCGATCATCAATGTGCCTA721 GTTGGMTAAACTTCCC;TGGTGTACACTMGATATACAGAGGACTCTGC;CCCAAAGTCTGGCGTGCTATGGCATTAGTGTCCATGACA;T|GAC AGATCGAGCTCTTTGTCCCCAG

1111111 IllI llIII 11111II11 11 111111111111 1 11HI I11111 11111 11 I I11I1111 HI I11IIIll 11111l ll 1111339 ATGGGGCGACTCCMCACTGCTGTACGCTTATCCATGACTGGGAATCACCAAAAG~CTTGCATGMCTT CMTAG¢TCAGGCGCCATTGGCTGTGGTGAAACAAGGTTCCTCTTAG

1841 GGACAAATATCCTTCAA ACTGGGTAA mGCTT~CAACACiTCAGTGGAMTMGCTGAACATCGTMAATATCTAAATAMACMAGiTCATGGTACTTAQ AGTGACATGCTAACAATGCi11 1111111 11111 11 1111111 1111111IIII ll 111 111111 11111 II 1111 1111 111 11 11 11 111 II11

1439 CGGAAACAATCTCAATTCTGAGTCAAGCTTATCTCATAA AGCAGATTCATGGAAAATCGCTGAGTACAACTCAGACTTGGTGGGTCTTCAGTCAGACAAAATATGAGA~TGAAGATTCAG

10167 TTGAGCM TTCGTTATCT AAATAAMCTGATTATGACAGATCACGACCATGATGGMTMTCCC ATCAAAGTTGCMTGATTAAT TACCATMCACACTGGCCCTCATCTCTCACAsT

1111 11111 111111111111 111111111111111'11111 11 11111 III 11111 11111111 11111 11 11111 III

1699 CTTGMCTGACGGAACG ATCGMACTCCATTGTGACAAG ATCTAAAACACAAGAATGGTCCCATMACAMGCCTTCCTGAATMTTTGAAGAGTGGAAGGCTAGCCTCTCAATCATGCACAAHIM1111 11 11 11111 IIIIIIIIIIIII III IIIIIIIIIIIlii11111111 111111111111111 liii11111111IIIII1III

13219 GGAAATCTTCMAAATTACATAAGTCTTTGGCACCAGCACACAAAAAGCTAAGAATACCTTCTGCAGATATTGAAAAA ATCGTATCCGTTAAAMCTATTCTGTCCTACAGATAGCT11111 II1 111 1111 11111 11111111II1 111111111 111 11111 111111 I 11111111 IIIIIIIIIII 11III 11 111 I

1939 cGCTATTCATGcAGCC ATcccATTAGACGATMGAGTATCGAAGAM~ATGcGTTACTAAT ATGGGGTAGAAGACAACcuG^AAAGTTTGGzGCGTTCCTGGATTcACTTATGA^

4168 ACGMTOCACTAAcATTTAiTTATATcGTAiTTCAGTCAcA~CTA AGGAATTG-ACCTCT~ATTCAGATATGcAAAc.AiATC~CATCCTTImGATTGAT6GCTcTAA^AiTGCCATGCT111111 I111111 11111 II IIIIIIIIIIII 1111I111111111 11 11 1111 IIIIIIIII 1III I 1 Hl11111 11 III11111111111 1 11 1111 11111 11111 11 11 111 111 11111 11 11111 11111 111111 liii 11111 III 11 1111111111 111111

2819 TCAAACTcACC TATCGATTAAT~ccGcACTCATCAcA~CAAGAA~CTMATCTTCCAATT~GCCATATAMMACG ATCGTATCCCGTTCATATGTGGTGGTTCTGAAACCAGTCGATGA

5217 AGGcTT~TTGTTTmATGCTTCAACGAGATATGACAACAGAAGAAGA^GGTTGsCCAATTAGTGATCGT~~cGGCTGAcAATGTCTCTATCAATGGcTG~AGATGTCACTATATGATGACC

2939 AlTTTCAA M GGlCTCGATTTGTGGGTAGCAAAATlClTAAlGACCTCTTGCAGCCCATTlliGGlTAGTTTGTACCAGCAlCAlTGTGGCAGGTGCT~lTAGTAGATACAcTCTlTT

2198 ACATGCTCMAGCmCTC GTCATATGATTA CATCMCAAGCA^TTCACACGTTGGTTTTMATCATACTGATCATCAACCAGTTCTGACTGAAGTCTTCCTATCCC,.ACTGCATGC

l 111 1 l1111111 11111li ii1111111ii11II1II11111111 1111 1111 11111111 11 111 111 11111 11 111111111 1

2069 GTCTATAcAcATGTGCTGAcAGGATCGTGACTGGTGGTC~CTGCAAATCTCCCTTcTGAMTGTGCTc GAcAATTGTAATACcTcATCGCG GTGGATGGGmcAcAGACTcAGCAG

2801 MATGTTCCAiATACAAWCTTTAA¢AG¢AATCATMCTCAAWCTGT^iAAceCCAGTTiGCGcTCAGT6GcTGAAATA'T'TccTTAGTGGAicAr.ACAACA'GTA ATTCACATGACTTC

11 11111111111111IIIIIIIIIIIIIII III 11111 11 11 11111 1I1111 1111111111111 11 111111111

2779 A^GTCA TGAAAcTGGCCCGCTcA~CAAACAccG^AGTTCAAACGAGTTGAAcTGGACGATGAcAGACCTGCCTCTATAACAACTTATcAGcGGATGACATACGAcTACATGATGAAA9621 TTTGTGATTiTGACGciAGAsGACTiG~CACGTC~TAWGCTGTGACTGCA~cTAiGTTTGTcAA~TCAACTc TCTTcACTTGicAATCCATGcTAC IIIIC.cTCGATATGTT.c

1111111 1111111111 1111111 III 111111 1 11111 1111111111111 1111 11111 1111I11 I 1111 111 111 11 111

2899 TTGTATC~GTGAA CTGAAcTTGAACTGGAGCAGAcT~TAGAGAGTTGGACTAAccATGTCTCAACGTCAcAACcTAGCTACAGTG~CAACGTCTATGGTAGCTTTTGACCTAGCA2041 TGCTGA"TGGATMGTGCAmACcTAGcTATT~CTGcWTcTGAGC SPcTAGTemJAcAAJicTTA~cTATGTTTcTA~Tc

III IIIIIIIIIII III 1111 11111 III 11 1111IIIIIII IliIIIIIIIIli 11111 1

3419 AcTGTTTAAGAATTGCACGGcTGTTGGTTT~lICTAACGATGCCTT AACT~H*AGTGCCACTGTACTATGTCTCTATTCCT

SP11-dodecapeptid (lass111 111111eetveyrcgnzdte180 hav11111 p111II1 activity1 as111assaye by111the 111111 eden

2419 . . * . . *a p2161 As ta phsp ate xcange39.Sc class I sequnceswrec

secondSP11-encode peptide1(beginnin at reidu 85 subsetof1the111aisequence11hese1immunological result

2659 also providedadditil evidencat the 170Tkla

11111 11111 11111111111 11111 111 111111 1 111111 11 11111 11 11111 1111111 111 11 1111 11111111 111111111 1I11

2641 TGTCTG^AAGAATATGAAACTGTGCAAGACATTCTGMAAAAATTC SP113819 TGTTTAAAGAATATGACACGGTGTTGGATATTCTAAGAGACCTT HeLa

FIG. 2. Nucleotide sequence alignment of SP11 and HeLa (numbered as in ref. 6) over coding regions. Region from which complementaryoligonucleotide probes were synthesized is bracketed.

SPil-dodecapeptide (classII)selectivelyrecognizedthe 1d e80- have Topo II activity as assayed by the ATP-dependentkDa enzyme, but they also cross-reacted with a protein of unknotting of phage P4 DNA (38) and amsacrine-stimulatedabout 145 kDa. Another affinity-purified antibody, against a phosphate exchange (39). Since class I sequences were asecond SP11-encoded peptide (beginning at residue 785, E I subset of the HeLa sequence, these immunological resultsF V V D R N T V E I T), a tridecapeptide corresponding to also provided additional evidence that the 170-kDa enzymethe region to which complementary oligonucleotide probes isolated by us corresponds to the protein encoded by thewere directed, also selectively recognized the 180-kDa en- sequence of Tsai-Pflugfelder et al. (8).zyme, but it did not detect the 145-kDa protein, indicating DSUSOthat the dodecapeptide beginning at residue 748 probably DSUSOcontained a cross-reacting determinant. The corresponding A second class (class II) of partial cDNAs has been obtainedHeLa tridecapeptide elicited no immune response in the two from a Raji-HN2 cDNA library whose nucleotide sequence israbbits injected. These results immunologically identified distinct from but 75% identical to that of a human Topo IIclass I cDNAs as encoding the 170-kDa form of Topo II and derived from a HeLa cell line (8). The predicted peptideclass II as encoding the previously isolated 180-kDa form (13, sequence of the longest representative of this class, SPil, has14). We have previously shown that both the 170- and the extensive similarity (92%) with the predicted HeLa sequence180-kDa enzymes isolated from P388 (13) or U937 (14) cells and spans two of the functional domains proposed for type II

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I VEHKVEKVYVALIFGQLLTSSNYDDDEKKVTCCRNCYCAXLCNIFSTKFTVETACKEYKHSFKQTWM ITEAKIKHFDCEDYTCITFQPDLSKFKMEKUDKDIVALMTRRAYDLA

11111111111111iiiIIIIIIIIIIIIIIIIII 11111 I IIIIIIIIIIIIIIIII111 It 111111111III IlIllII1111 III IlllllIllll 1* .*247 GSTKDVKVFLNCNWLPVKGFRSYVDMYDLKDETGNSLKVIHEQVWRqCLTMSEKGFQQFVNSI AISKGGRVDYVADQIVTKLVDVM GCVAVKAHQVKNHMWFVNAL

241 IENP>TFDSQTKENVTLQPKSFCSKCQLSEKFKANGIVESILNWVKFKAQTQLNKKCSSVKYSKIKGIPKLDDANDAGGKHSLECTLILTEGDSAKSLAVSGLGVIGRDRYGVFPLR367 IEWPTFDSQ-TKBVTLQFKS~FGSTCQLSEKFIKAAIGCGIVESILNWVKFKAQVQL NKKCSAVKHNRIKGIPKDDADADGNSTECTLILTEGDSAKVSGLGVGRDKYGVFPLR

3,81 IGKILNVREASHWQIMENANiIKIVeGLQYKKSYDDAESUKTLRYeGKIlMTDDQDGSeHI[KeLLIFiHHNWPSLLKHGFLEEFITPiVKASKWKQELSFYSIPEFDWKKHIENQK487 GKILNVREASH~gIMEIKIVKGLRY YED EEDSLKTaRYKIIMIMDQDQDGSHIKGLLIWFIHWtSLLRHRFLEEFITPIVKVSKWKQEMAFYSLPEFEEWKSSTPNHKK481 WKIKYYKGLGTSTAKEAKEYiFADMERHR3ILFRYAGPEDDMTTn-AFsKKKIDDRKEWLTNi~rEDRRQRRLHeGLPEQFLYC.TATKHLTYNDFIWKELILFSNSDNERSIPSLVDGFKPGQi607 GLKYCT GSTSKEAKYADIRQFRKY KYSPEDDAAISLAFSKQIDDRKEILTWMSEDRRQRKLLGLPE1YLYGQTTTYLTYNDFIWKELILFSNSDNERSIPSMVDGLKPR

* ~~~~GYRASE 6 (ATPose ACTIVITY) aGYRASE A (BREAK-REJOIN ACTVITY)

601 KV`LFTCFKRsDKREK QLAGSVAEMSAYHHGEQALMMTIVNLAQWFVCSNNINLLQPIGQFGTRLHGGKDAASPRYIFTMLSTLARLLFPAVDDNLLKFLYDDNQRVEPEWYIPI IPM727 KVLFTCF RKEVQLGSVAEMSSYHHGEMSLMMTIINLAQNFGSNNLNL;LQPIGQFTRLHGGKDSASPRY FTMLSSLARLLFPPKDOHTLKFLYDDNQRVEPEWYIPIIPM

As! . r .,~~~~I . t . *721 VLIWCAElITGW~ACKLPNYDAREIWCRLDOLPC WNKKTIQELCQNY^YAIV~VSEIFVDRTiELPVRTWT4VYKEQVLEPMLNGTDKTPALISDYKEYHTDrrVK847 VLIN¢AEGI¢TG"CKIPNFDRIVNNIRRLMDGEEPLPLPSYK¢KTIEAPNQY IVISEVAILNSTTIEISELPVRTt 1TY_1EVLEPMLGTEKTPPLITDY1EYHT TTV

+__841 FVVKMTEEKLAQAEAAGLHK'VFKLQTTLTCNSMVLFDHMGCCKKYETVQDILKEF SP11

487 F____TEEKEAGLW__QTSLTCNS__L__HVGCLKKIDLH _L

FIG. 3. Alignment of the predicted amino acid sequences of SP11 and HeLa TopoII. The conserved critical tyrosine ( I), three glycosylationsites (*), two phosphorylation sites (t), and "Ieucine zipper" (t) leucines are indicated (refs. 27-30, respectively). Amino acid residues that areidentical in the alignment of S. cerevisiae (5), D. melanogaster (7), and the two human Topo, II protein sequences are underlined. Antibodieswere directed against the bracketed peptides. The two gyrase-like domains (7) are indicated.

topoisomerases (7). The peptide sequence also retains severalof the peptide stretches, the critical tyrosine residue, three ofthe possible glycosylation sites, and two of the potentialphosphorylation sites that are conserved among the type IIeukaryotic topoisomerases (7). These results provide strongevidence that this partial cDNA, SPli, encodes more than halfof an authentic but previously undescribed human Topo II.Evidence relating the two classes of Topo II cDNAs with

the previously isolated 170- and 180-kDa Topo II enzymes(13) was provided by specific anti-peptide antibodies whichselectively recognized either the 170- or the 180-kDa isozymeand immunologically validated that class I (SP1 or HeLa)sequences encoded the 170-kDa enzyme, while SP11 (classII) encoded most of the 180-kDa Topo II.

spi

R MB- BHBH P BH P

23.1 -

0

6.6 - *

SP12

R MBHP BHPBH P BHP

6.5 -

'p

* V4.4 - * * * W,

Gw *..

2.2 -

2.0 -

FIG. 4. Southern blots of genomic DNA. Raji-HN2 (R) andMRC5 (M) DNAs were probed with 32P-labeled (23) SP1 or SP12cDNA. They show nonoverlapping restriction fragments after diges-tion with BamHI (B), HindIII (H), and Pst I (P). Marker fragmentsizes of A phage DNA digested with HindI1I are indicated in kilobasepairs.

We previously noted that the 170- and 180-kDa isozymeshave differences in cleavage site preferences, processivity,and inhibition by "A+T-rich" oligonucleotides (14). Theputative DNA-binding region (36) of the 170-kDa protein (8)corresponds to the more variable C-terminal third of Topo IIprotein sequences (7). We speculated that differences in thisregion account for the biochemical differences observed (14).Unfortunately, our 180-kDa isozyme partial sequence lackedthis region.The existence of two cDNA classes implies the existence

of two mRNAs. However, only one apparent 6.5-kb mRNAwas detected in Northern blots of U937, Raji-HN2 (15), andCOLO 201 cells (16) when probed with either cDNA class.

I* :11*I Itl[____ :I

SPIrO 0.5 2.5 515Fw5 2.5 5ibH

-9.5-7.5

-4.4

-2.4

-1.42 3 4 5 6 7 8 9 10 11 12 13

FIG. 5. Autoradiogram of competition hybridization in Northernblots. Replicate strips containing 0.5 ,ug of U937 poly(A)+ RNA werehybridized separately. Lane 1 was hybridized to 32P-labeled (23) SP1as described (15). Lanes 2-7 were hybridized (24) with 32P-labeledprobe I (1*) (1 pmol/ml, 1.6 x 107 cpm/ml), specific for SP1, in thepresence of indicated molar excesses of unlabeled probe I (lanes 3-6)or H (lane 7). Lanes 8-13 were hybridized to 32P-labeled probe 11 (11*)(1 pmol/ml, 9.1 x 106 cpm/ml), specific for SPil, in the presence ofunlabeled probe II (lanes 9-12) or I (lane 13). RNA marker positionsare indicated in kb.

9434 Genetics: Chung et al.

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Proc. Natl. Acad. Sci. USA 86 (1989) 9435

PurifiedTopoisomerase 11

-20!180 kDa _

170 kDa "

Total CellLysate

-103 kDa

1 23

-205 kDa

-103 kDa

4 5 6

FIG. 6. Western blots of strips containing equal amounts ofpurified U937 170- and 180-kDa Topo II (lanes 1-3) and U937 wholecell lysates (lanes 4-6). Lane 1 was immunoblotted with antiserumraised against murine P388 Topo II; lanes 2 and 4, with anti-SP1-dodecapeptide; and lanes 3 and 5, with anti-SP11-dodecapeptide(see text and Fig. 3). Lane 6 was immunoblotted with antiserum tothe SP11-specific tridecapeptide.

The Northern analysis and competition experiment withspecific oligonucleotide probes demonstrated that there mustbe at least two comigrating mRNAs. The apparent 10-kDadifference between the 170- and 180-kDa enzymes (13, 14)corresponds to about 93 amino acid residues, or a 0.27-kbdifference (23) in mRNA size, which may not resolve in ouragarose gels. However, there is no requirement for messagesof different sizes because the HeLa coding sequences com-prise only 4590 base pairs, leaving about 1.9 kb of untrans-lated sequences. The additional coding sequences requiredfor the 180-kDa enzyme could be compensated for by shorteruntranslated sequences in its mRNA.The hybridization of each class of cDNA to unique non-

overlapping DNA restriction fragments in Southern blots(Fig. 5) suggests the presence of two Topo II genes. Thealignment of the SP11 and HeLa sequences argues againstalternative splicing (40), unless the exons are very short andmultiply shuffled. However, this does not rule out the pos-sibility of alternative splicing in the regions 5' or 3' to thosespanned by the cDNA sequences presented here.Taken together, these results provide strong immunologi-

cal and genetic evidence that the 170- and 180-kDa formsrepresent two different Topo II enzymes. These results arenot unique to the Raji-HN2 cells, because the 170- and180-kDa enzymes have been detected in human U937 (14,17), Raji (15), and COLO 201(16) cell lines and in murine P388(13) and NIH 3T3 (17) cell lines.To avoid confusion with other proteins designed by their

molecular weights, such as the glycoprotein P170 encoded bythe multidrug-resistance gene, and because the isozymesappear to arise from different genes, are differentially regu-lated, and may have different physiological functions, wesuggest that the 170-kDa enzyme be referred to as Topo II-aand the 180-kDa enzyme as Topo II-,B.

Further understanding of the physiological functions ofTopo TI-a and Topo II-f3, especially with regard to theneoplastic state, may be relevant to consideration of Topo IIas a target for drug therapy. If only one enzyme is implicatedin neoplasia, then it may be possible to develop a drug thatis selectively more toxic to neoplastic cells. The availabilityof antibodies and nucleic acid probes that are specific foreither Topo Il-a or Topo II-,B will allow us to investigate theinvolvement of these enzymes in differentiation, regulation ofgene expression, and gene -structure.

We are most grateful to Drs. Tao-shih Hsieh and Maxwell Lee forproviding us with the Drosophila cDNA probe. We thank Ms.Rebecca Boyce and Lois Funk for technical assistance in mainte-nance of cell lines and screening of cDNA clones, Mr. JonathanMarsh and Joseph P. O'Connor for synthesizing oligonucleotideprimers and probes, Dr. Klaus Esser for technical advice on anti-peptide antibodies development, and Mr. Francis L. McCabe forskillful injection and bleeding of the rabbits. We thank Drs. David J.Ecker, Michael Mattern, and Randall K. Johnson for useful discus-sions. We also thank Dr. Russell Greig for support of this work. Thiswork was supported in part by Grant CA-40884 from the NationalCancer Institute.

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