Plant Physiol. (1992) 99, 1493-15010032-0889/92/99/1 493/09/$01 .00/0

Received for publication November 8, 1991Accepted March 16, 1992

Cloning and Characterization of an Arabidopsis thalianaTopoisomerase I Gene'

Joseph J. Kieber2, Alain F. Tissier, and Ethan R. Signer*

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

ABSTRACT

cDNA and genomic clones encoding DNA topoisomerase I wereisolated from Arabidopsis thaliana Xgtl1 and XFix libraries by lowstringency hybridization with a Saccharomyces cerevisiae TOPiprobe. The cDNA clones include a 2748-base pair open readingframe predicting an amino acid sequence that is highly homologousto sequences encoded by TOP1 from yeast and human sources.The sequence of the upstream genomic region reveals two putativeTATA-like elements and a purine-rich region, but no other obviouscontrolling elements. Southern blot analysis shows that the gene ispresent as a single copy in the Arabidopsis genome. When ex-pressed in a S. cerevisiae top1 mutant under the control of theGAL1 promoter, the gene complements the phenotype caused byloss of topoisomerase activity and directs the expression ofa protein that cross-reacts with a human anti-topoisomerase Iantibody.

DNA topoisomerases are a class of enzymes that share theability to alter the topological state of DNA. Type I topoisom-erases change the linking number of DNA in steps of one bythe transient breakage of a single strand of DNA, whereastype II enzymes alter the linking number in integrals of twoby introducing double-strand breaks (6, 11, 29, 30). Genesfor the type I enzyme have been cloned from bacteria (26),Saccharomyces cerevisiae (25), Schizosaccharomyces pombe (27),and human cells (8, 13). The bacterial and eukaryotic genesare not homologous. However, the yeast clone is able tocomplement a bacterial topoisomerase I-deficient mutant (4),and recently a novel topoisomerase I gene from yeast withhomology to the bacterial gene also was described (28).Topoisomerases have been implicated in a number of proc-

esses affecting DNA, including transcription, replication, andrecombination. The enzymes are thought to resolve the top-ological constraints imposed by the double-stranded, helicalnature of DNA. For example, topoisomerase II is required forresolution of recombined chromosomes in yeast (23). Topo-isomerase I is required for plasmid recombination in Esche-richia coli (10) and for the pairing of covalently closed circularplasmids in vitro (7).

1 Supported by grant No. GM40725 from the National Institute ofHealth (E.R.S.), training grant No. GM 07287 from the NationalInstitute of Health (J.J.K.), and a Lavoisier Fellowship from theMinistere des Affaires Etrangeres and the Soci&n Nationale ElfAquitaine (A.F.T.).

2 Present address: Plant Science Institute, Department of Biology,University of Pennsylvania, Philadelphia, PA 19104-6018.

How topoisomerases are involved in recombination is notyet clear. Yeast topl and top2 mutations show increasedrecombination specifically at rDNA3 repeats (5, 16) and notat other repeated sequences. A yeast mutation that giveshyperrecombination of repeated sequences, hprl, maps to agene with homology to the carboxyl terminus of TOP1 (1).Also, a yeast gene (TOP3) with homology to bacterial type Itopoisomerase was identified in a screen for mutants withelevated rates of recombination between co elements (28).These genes probably have overlapping functions becausedouble mutants of topl with either a top3 or an hprl mutationhave severely decreased growth rates, even though singletopl deletions grow normally. The HPR1 and TOPIII genesseem to affect only intrachromosomal repeats, and it is notclear whether analogous genes are involved in interchromo-somal homologous recombination.We are interested in genetic recombination in Arabidopsis

thaliana and have begun to analyze individual componentsthat may be involved. Elsewhere (14), we report the purifi-cation and characterization of a type I topoisomerase fromthe closely related crucifer Brassica oleracea var italica (broc-coli). Here, we describe the cloning and characterization of agene from A. thaliana that encodes a type I topoisomeraseand that we have named TOP1. The gene is present as asingle copy in the Arabidopsis genome. The cloned cDNA hasan open reading frame of 2748 nucleotides, predicting ahighly charged, basic protein with a mol wt of 104,000. Thepredicted protein sequence has 45% identity to that of the S.pombe gene. When expressed in a S. cerevisiae topl mutant,in which recombination at rDNA is specifically elevated (5),the clone depresses rDNA recombination, indicating that itcomplements the mutation, and directs the expression of aprotein that cross-reacts to an antibody made to humantopoisomerase I.

MATERIALS AND METHODS

Strains and Libraries

Strains and plasmids used and their sources are listed inTable I. The XFix (Stratagene) Arabidopsis genomic libraryand the Xgt1l (31) poly(A)-primed cDNA library were kindlyprovided by H. Goodman. The random hexamer-primedXgtll Arabidopsis cDNA library was kindly provided by G.Fink. The human antitopoisomerase I antibody (AFCDC9)was from the Centers for Disease Control (Atlanta, Georgia).

'Abbreviation: rDNA, ribosomal DNA.1493

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Table I. Strains and Plasmids UsedStrain or Relevant Source orPlasmid Characteristics Reference

E. coliXL1 recA l lac endA I gyrA96 thi

hsdR 17 supE44 relA 1; F'proAB+ /ac/Q lacZAM15 Tn Io

Y1090 A/acUl69 proA+ A/on araD139strA supF [trpC22::Tn 10] hsdR-hsdM+ mcrA- pMC9 (for thegrowth of the XgtlO and Xgtl 1libraries)

ER1458 Y1090, mcrB- (for the growth ofthe XFix library)

strainsXTop104 XFix containing a 11.0-kb TOP1

genomic fragment in the Xholsite

XTopl 15 XFix containing a 14.5-kb TOP1genomic fragment in the Xholsite

eastCY184 MATa ade2-4 ura3-1 his3-1 1, 15

trpl-l leu2-3, 112 canl-100rDNA::ADE2

CY185 CY184, top 1-7::LEU2YAB100 CY184, pTop17OYAB101 CY184, pCP125YAB102 YAB100 screened for URA- phe-

notypelasmidsPCB12 pYCp5O with a 3.8 kb HindlIl in-

sert containing the yeast TOPIgene

pKS- ColEl; Apr (for sequencing andsubcloning)

pTop 140 pKS- with 1.5-kb Arabidopsis ge-nomic TOPI fragment

pTop141 pKS- with 7.5-kb Arabidopsis ge-nomic TOPI fragment

pTop142 pKS- with 8.5-kb Arabidopsis ge-nomic TOPI fragment

pTop150 Subclone of cDNA insert fromArabidopsis TOP1 frompoly(A)-primed library in pKS-

pTop152 Subclone of cDNA insert fromArabidopsis TOPI frompoly(A)-primed library in pKS-

pTop160 pKS- with a complete copy ofthe Arabidopsis TOP1 cDNA inthe Sacl site

pTop170 pCP125 with a complete copy ofthe Arabidopsis TOP1 cDNA inthe BamHl site

pCP125 pYIP5 with CEN-ARS1 andGAL1-GAL1O

Stratagene

31

22

This work

This work

M. Christman

M. ChristmanThis workThis workThis work

25

Stratagene

This work

This work

This work

This work

This work

This work

This work

24

Xho l/Eco RI Eco RV Hind III Eco RI Hind III

MET----PPvu 11

pTop1 52

Eco RI/Spe

Bam HI

pTopl 500.5kb pTopl 54

Figure 2. Restriction maps of TOPI cDNA clones. The 5' end ofthe gene is indicated by MET, and the 3' end is indicated byAAAAA. The extent of the clones was determined by sequenceanalysis of the ends. pTop150 was derived from a poly(A)-primedcDNA library, whereas the others were from a random hexamer-primed library.

Cloning of the Arabidopsis TOPI Gene

A XFix genomic library was screened with radiolabeled 3.8-kb HindIII fragment containing the yeast TOPI cDNA asdescribed (2), with the following modifications. The plaqueswere fixed to the nitrocellulose filters and the DNA denaturedby autoclaving for 5 min. Hybridization and prehybridizationwere performed in 35% formamide, 6 x SSC, 0.25% nonfatdry milk, and 0.1% SDS. Denatured probe was added after1 h of prehybridization and hybridized overnight at 370C.The filters were washed with 2 x SSC, 0.1% SDS at roomtemperature three times for 10 min, followed by two washesin 0.2 x SSC, 0.1% SDS for 15 mi at 390C. The filters werethen dried and exposed for 2 d using Kodak X-Omat film.Fifteen positive plaques were identified, plated at low density,and rescreened. DNA was prepared from the clones andfurther analyzed. Two final TOPI genomic clones were usedto isolate cDNA clones from a random hexamer-primedand a poly(A)-primed cDNA library by screening at highstringency.

DNA Sequencing

DNA sequencing was performed using a Sequenase kitfrom United States Biochemical Corp. Sequencing deletionclones were obtained using nuclease Bal31 from New Eng-land Biolabs and were cloned into pKS- (Stratagene). DNAsequence analysis was performed using the University ofWisconsin Genetics Computer Group software as imple-mented locally at the Whitaker College Computer Facility.

Construction of pTopl 70

pTopl70 (see Fig. 7), which contains a complete copy ofthe Arabidopsis TOPI gene under the control of the yeastGALI promoter, was constructed as follows. The 1.9-kb SpeI-PvuII fragment from pTopl50, the 1.2-kb XhoI-PvuII frag-

Figure 1. Restriction maps of genomic TOPIclones. Only inserts are shown. The approxi-mate extent of the coding region, as deter-mined by sequencing and comparison of therestriction maps of the cDNA and genomicclones, is shown by the dotted line. Restrictionsites are abbreviated as follows: B, BamHl; C,C/al; H, HindlIl; L, Sall; S, Sacd; V, EcoRV; X,Xbal.

s SXTopll5 E

S LDEIs LJE

).ToplO 4

pTopl 40pTopl41,

pTopl42

p y aX S

1 kbC E

- - - - - - - Coding Region

x

yl

Pi

1494 KIEBER ET AL.

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ARABIDOPSIS TOPI GENE

ment from pTopl52, and pKS- cut with SpeI and XhoI were

ligated together and used to transform XL1, giving plasmidpTopl60. pTop160 was cleaved with SpeI and XhoI, the endsblunted with Klenow, BglII linkers added, and the 3.1-kbfragment gel-purified in low-melting point agarose. This frag-ment was ligated into pCP125 cleaved with BamHI. A clonewas chosen that had the open reading frame of the TOPIgene in the correct orientation to be transcribed by the GALlpromoter and was designated pTopl70.

,uL) was added, the suspension spun to remove debris, and20 uL loaded for the western blot or assayed for topoisom-erase activity.

Other Procedures

Southem blotting, westem blotting, subcloning, and otherDNA manipulations were as described (2). Yeast cells weretransformed by the lithium acetate method as described (12).Yeast cultures were monitored by measurement in a Klett-

Growth of Yeast for Protein Extraction Summerson photoelectric colorimeter using a green filter(Klett Mfg. Co., New York, NY). Topoisomerase assays were

Cells were grown to mid-log phase in 25 mL of SD medium as described (14, 21).

(bacto-yeast nitrogen base without amino acids, 0.67%, andwater [2]) containing 2% sucrose as the sole carbon source,then induced by the addition of galactose to 2% for 2 h. The RESULTS AND DISCUSSION

cells were harvested by filtration and washed once in buffer Cloning of the TOPI Gene from Arabidopsis(50 mm Hepes, pH 7.4, 1 mm PMSF, and 2 mm DTT). Glassbeads were added to the cell pellet, which was then vortexed An Arabidopsis (ecotype Landsberg erecta) genomic library3 times for 30 s each with 30-s intervals on ice. Buffer (500 in XFix was screened with a S. cerevisiae TOPI cDNA probe.

1 GGAGAGAGAT AGGCTTCGCTA CACAATTTCT TCAATCTCTGY GAAGAAGAAG T1GTCTIGAG61 CCTTCATCAA CG7TTGTCTTT CGGGA86 ATC GGC ACT GAA ACA GTT TCA AAA CCT GTG ATG GAT AAT GGG TCT GGA GAC AGT GAT GAT

M G T E T V S K P V M D N G S G D S D D

146 GAC AAG CCT TTA GCG TTC AAC AGG AAT AAT ACA GTG GCT TCT AAT TCG AAT CAA TCT AAA

D K P L A F K R N N T V A S N S N Q S K

206 TCC AAT TCC CAG AGA AGC AAG GCA GTT CCT ACC ACC AAG GTA TCA CCT ATG AGA TCA CCT

S N S Q R S K A V P T T K V S P M R S P

266 GTG ACT AGC CCA AAT GGA ACC ACT CCT TCG AAT AAA ACT TCT ATA GTC AAA TCC TCT ATGV T S P N G T T P S N K T S I V K S S M

326 CCA TCA TCT TCT TCT AAG GCT TCA CCA GCA AAG TCA CCA TTG CGG AAT GAT ATG CCC TCT

P S S S S K A S P A K S P L R N D M P S

386 ACT GTT AAG GAT AGG AGC CAG TTA CAG AAA GAT CAG TCT GAA TGT AAA ATT GAG CAT GAGT V K D R S Q L Q K D Q S E C K I E H E

446 GAT TCT GAG GAT GAT AGA CCT TTA AGT TCC ATA CTA TCT GGA AAT AAA GGG CCA ACC TCT

D S E D D R P L S S I L S G N K G P T S

506 TCG CGG CAG GTT TCT TCA CCG CAG CCA GAG AAA AAG AAT AAT GGT GAT CCA CCT CTT CAT

S R Q V S S P Q P E K K N N G D R P L D

566 AGA GCA AGC AGA ATT ATA AAA CAC GAG TCA GAT GAT GAA ACT CCA ATC TCG TCA ATG TTT

R A S R I I K D E S D D E T P I S S M F

726 CGG AAC AAG ATT GAT AGT GGG ATG TCA GGG GGC AAC CAG CTC TCC AAT GAT GAA AAG AAA

R K K I D S G M S G G N Q L S N D E K K

786 CCT TTG GTT CAA AAG TTG CAT CAG AAT GGT TCC ACA GTT AAG AAT GAA GTG CCG AAT GGTP L V Q K L H 0 N G S T V K N E V P N G

846 AAG GTTA TTA GGT AAC AGA CCT CTC GAG AAG AAT AGT TCT GCA GAT CAA TCT TCC TTG' AAGK V L G K R P L E K N S S A D Q S S L K

906 AAG GCC AAA ATA TCA GCT TCA CCT ACT TCA GTG AAA ATC AAA CAA GAT TCT GTC AAG AAAK A K I S A S P T S V K M K Q D S V K K

966 GAG ATA GAT CAT AAG GGA AGG GTTTCG GTT TCA CCA AA ATG AAA GCT AAA CAG TTA TCTE I D D K G R V L V S P K M K A K Q L S

1026 ACC AGA GAA GAT GGA ACT GAT GAT GAT GAT GAT GAT GAT CTT CCG ATA TCC AAG AGA TTCT R E D G T D D D D DD D D V P I S K R F

1086 AAA TCA CAT TCC TCC AAC AGT AAC ACA TCA TCT GCA AAG CCA AAA GCA GTA AAA CTA AATK S D S S N S N T S S A K P K A V K L N

1146 TCT ACT TCA TCA GCT CCA AAG CCA AAA GCG AGA AAT GTC GTT TCT CCT AGG TCC AGA GCAS T S S A A K P K A R N V V S P R S R A

1206 ATG ACC AAA AAC ACT AAG AAA GTA ACG AAG GAC TCA AAA TAT TCC ACG TCT TCA AAA TCAM T K N T K K V T K D S K Y S T S S K S

1266 TCA CCT TCC TCT GCA GAT GGG CAA AAG AAA TGG ACT ACT TTiG GTG CAC AAC GGT GTA ATAS P S S G D G 0 K K W T T L V H N G V I

1326 'TT CCA CCG CCA TAC AAG CCT CAT GGC ATT AAG ATT TT'G TAC AAG GGG AAA CCT GTT GACF P P P Y K P H G I K I L Y K G K P V D

1386 CTA ACT ATT GAA CAA GAA GAG GTT GCG ACT ATG TTT CCA GTG ATG AGA GAG ACA GAT TATL T I E E E V A T M F A V M R E T D Y

1466 TAT ACT AAA CCT CAA TTT CGG GAG AAT TTC TGG AAT GAC TGG CGA AGA CTG CTT GGC AAGY T K P Q F R E N F W N D W R R L L G K

1526 AAA CAT GTG ATA CAC AAC TT'G GAT GAT TGT CAC TTT ACT CCC ATA TAT GAG TOG CAT TTGK H V I Q K L D D C D F T P I Y E W H L

1586 GAA GAG AAA GAG AAG AAG AAA CAA ATG AGC ACA GAG GAG AAA AAA GCT TT AAA GAG GAGE E K E K K K Q M S T E E K K A L K E E

1646 AAA ATG AAG CAA GAG GAG AAA TAT ATG TGG GCT GTT GTT GAT GGT GTC AAA GAG AAG ATTK M K Q E E K Y M W A V V D G V K E K I

1706 GGT AAT TTC AGA GTG GAA CCG CCT GGC CTT TTC AGA GGC CGT GGA GAA CAT CCC AAG ATGG N F R V E P P G L F R G R G E H P K M

1766 GGA AAA CTA AAG AAG CGT ATC CAT CCA TGC GAG ATC ACC TTA AAT ATC GGG AAA GGT GCTG K L K K R I H P C E I T L N I G K G A

1826 CCT ATT CCA GAA TGT CCA ATT GCT GGT GAA AGA TGG AAA GAA GTA AAG CAT GAT AAC ACAP I P E C P I A G E R W K E V K H D N T

1886 GTC ACC TGG CTA GCT TTC TGG GCT GAT CCT ATT AAC CCA AAA GAA TTC AAG TAT GTA TTTV T W L A F W A D P I N P K E F K Y V F

1946 TTG GGA GCT GGC AGT TCT CTG AAA GGG CTG AGC GAC AAA GAG AAG TAC GAG AAA GCT AGGL G A G S S L K G L S D K E K Y E K A R

2006 AAT CTT ACG GAT CAT ATA GAT AAT ATA AGA ACA ACA TAC ACC AAG AAT TTT ACT GCC AAGN L T D H I D N I R T T Y T K N F T A K

2066 GAT GTT AAA ATC AGG CAA ATT GCT GTG GCG ACA TAT CTC ATT GAT AAA TTG GCG CTT AGGD V K M R Q I A V A T Y L I D K L A L R

2126 GCT GGA AAT GAG AAG GAT GAT GAT GAG GCC GAC ACT GTT GGT TMT TCT ACT TTA AAA GTTA G N E K D D D E A D T V G C C T L K V

2186 GGA AAT GTG GAG TGT ATT CCT CCA AAT AAA ATA AAG TTT GAT TTT CTT GGT AAG GAT TCAG N V E C I P P N K I K F D F L G K D S

2246 ATC CAG TAT CTG AAC ACA GTC GAG GTT GAG CCT CTT GTT TAC AAG GCC ATT GGA CAG TTCI Q Y V N T V E V E P L V Y K A I C Q F

2306 CAG GCG GGA AAA TCC AAA ACA GAT GAT CTT TTT GAT GAG TTA GAC ACA AGT AAA CTG AATQ A G K S K T D D L F D E L D T S K L N

2366 GCT CAT TTG AAG GAA CTT GTC CCT GGT CTC ACA GCT AAA GTA TTC CGT ACA TAT AAT GCTA H L K E L V P C L T A K V F R T Y N A

2426 TCC ATC ACT TT'G GAT GAA ATG CTG AGT CAA GAA ACC AAA GAT GGC GAT GTT ACT CAA AAAS I T L D E M L S Q E T K D C D V T Q K

2486 ATA GTA GTT TAT CAG AAA GCA AAT AAG GAG GTT GCC ATA ATC TGC AAT CAT CAG CGT ACTI V V Y Q K A N K E V A I I C N H Q R T

2546 GTA TCA AAG ACA CAT GGT GCG CAA ATT GAA AAA TTG ACT GCG AGA ATA GAA GAA CTC AAGV S K T H G A Q I E K L T A R I E E L K

2606 GAG GTT CTT AAA GAG CTC AAA ACT AAT CTT GAC CGG GCT AAA AAG GGA AAA CCA CCA TTGE V L K E L K T N L D R A K K G K P P L

2666 GAA GGT TCT GAT GGA AAG AAG ATT AGA AGC TTA GAA CCA AAC GCG TGG GAG AAG AAG ATTE G S D G K K I R S L E P N A W E K K I

2726 GCT CAA CAG AGT GCT AAG ATC GAG AAA ATG GAA CGA GAC ATG CAT ACA AAA GAG GAC TTGA Q Q S A K I E K M E R D M H T K E D L

2786 AAA ACA GTG GCA CTC GGC ACG TCT AAG ATC AAC TAC CTG CAT CCT AGA ATC ACA GTT GCAK T V A L G T S K I N Y L D P R I T V A

2846 TGG TGC AAA CGT CAT GAA CTA CCA ATC GAA AAG ATA TTC ACC AAG TCT CTT TFCC GAG AAGW C K R H E V P I E K I F T K S L L E K

2906 TTT GCG TGG GCA ATC GAT GTA GAA CCC GAG TAC AGA TTC TCG CGC CGT TAGGTATAGAGAAGCF A W A M D V E P E Y R F S R R .

2969 TCTTCTCGGATGGAGCAAGTATTIGGAGGGCAAAAGAGCTAATAGATAAGAGCAAAGAGAAAACCGGCCTTTCTTCTAT3049 TCATCTTCCTCCCTTIGTTTAGAATTTTATTACCTTAGCTACTAAAAAACAT'GAACATGTCAAGCTGTTTCATTAGTGTCT3129 TTTTTAACATTACTATGACTCAAATATGTA'TTAFTTTTAACATTACTAGATTAAAGAAATAGAAAATCCGAAGAAGAA3201 CAAAAACAAAAAASA

Figure 3. Sequence of the Arabidopsis TOPI gene. The sequence shown is derived from the pTop150 and pTopl52 cDNA clones. Thederived amino acid sequence is shown in one-letter code below the DNA sequence. Nucleotides are numbered on the left, with the firstcDNA nucleotide designated as +1. The first in-frame methionine is used as the start of the translation.

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Eight positive plaques were identified, which fell into twoclasses by restriction pattem. The larger of the classes (fivemembers) encodes a protein with homology to yeast pyro-phosphatase and is described elsewhere (15). The remainingtwo clones, XTopl04 and ATop115 (see Fig. 1), overlap attheir ends as shown by Southern analysis and DNA sequenc-ing (data not shown), and represent a 25-kb stretch of Ara-bidopsis genomic DNA. The inserts from subclones pTop140and pTopl42 (Table I, Fig. 1) were used to isolate fourrandom hexamer-primed cDNA clones and a single poly(A)-primed cDNA clone, maps of which are shown in Figure 2.The clones span a stretch of approximately 3 kb. This is thesize anticipated for a TOPI cDNA, assuming a protein witha molecular mass of approximately 100 kD, which is the molwt of topoisomerase I from most other eukaryotes (29, 30),although topoisomerase I purified from broccoli has a molec-ular mass of 80 kD (14). The inserts from these X clones weresubcloned into the vector pKS- and sequenced.

A0 200

1 I400

I I I

Sequence of TOPI cDNA

The nucleotide sequence and predicted protein sequenceof the 2748-bp open reading frame is shown in Figure 3.There are four methionine codons in frame within the first150 amino acids at the start of the open reading frame. Thefirst two, at nucleotides 85 and 115, match the consensussequence for plant translational start sites fairly well (PuA/C A/C AUG GC, with GC at +4, 5 the most critical; 17).Because it is not clear which one is the authentic start codon,we have assumed the first in further analysis. Upstream are90 bp that have no extended open reading frame in any ofthe six frames and presumably represent an untranslatedleader. Downstream of the stop codon is a 180-bp purine-rich stretch with a poly(A) tail at the 3' end. The predictedprotein is highly basic and has an estimated molecular massof 104 kD. The type I topoisomerase purified from broccoliis also very basic, although the purified protein is only 80

600 800I I I I

- B00

- 600

'-4

0

.0

0

U]

- 400

- 200

I/ I I I- I I I -0

Arabidopsis TOP1

Figure 4. A dot matrix analysis comparing the predicted Arabidopsis TOPI amino acid sequence with that of the S. pombe TOPM gene. A,The dotplot comparing the S. pombe (vertical axis) with the Arabidopsis TOP1 (horizontal sequence) predicted amino acid sequences usingthe dot matrix analysis program from the University of Wisconsin Genetics Computer Group set at a stringency of 18 and a window of 30 isshown. The region of homology extends from approximately amino acid 200 to the end of the gene. (Fig. 4B on facing page.)

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ARABIDOPSIS TOP1 GENE 1497

BArab. 1 MGTETVSKPVMDNGSGDSDDDKPLAFKRNNTVASNSNQSKSNSQRSKAVPTTKVSPMRSPVTSPNGTTPSNKTSIVKSSMPSSSSKASPAKSPLRNDMPHuman 1 MSGDHLHN ------------------DSQIEADFRLNDSHKHKDKH- --------------------------------------------------

pombe 1 MS-----------------------------SSDSVSLSIRRRQR- -----------------------------------------------------

cer. 1 MT-----------------------------IADASKVN------------------------------------------------------------

Arab. 103 KDRSQLQKDQSECKIEHEDSEDDRPLSSILSGNKGPTSSRQVSSPQPEKKNNGDRPLDRASRIIKDESDDETPISSMFRKKIDSGMSGGNQLSNDEKKHuman 29 KDREHRHKEHKKEKDREKSKHSNSEHKDS-EKKHKEKEKTKHKDGSSEKHKDKHKDRDKE- -------------------------------------

pombe 17 --RGSSKRISMKESDEESDSSENHPLSESLNKKSKSESDEDDIPIRKRRASSKKNMSNSS- -------------------------------------

cer. 11 ----------HELSSDDDDDVPLSQTLKKRKVASMNSASLQDEAEPYDSDEAISKIS- -------------------------------------

Arab. 205 KLHQNGSTVKNEVPNGKVLGKRPLEKNSSADQSSLKKAKISASPTSVKMKQDSVKKEIDDKGRVLVSPKMKAKQLSTREDGTDDDDDDDVPISKRFKSHuman 87 -------------------- KRKEEKVRASGDAKIKKEKENGFSSPPQIKDEP------EDDGYFVPPKEDIKPLKRPRDEDDVDYK-----PKKIKTpombe 75 -------------------- SKKRAKVMGNGGLK---- NGKKTAVVKEEE------- DFNEIAKPSPKHKRVSKANGSKNGA-----KSAVKKcer. 58 -------------------- KKKTKKIKTEPVQS------SSLPSPPAKKS------------------ ATSKPKKIKKEDG--DVK-----VKTTKK

Arab. 307 SNTSSAKPKAVKLNSTSSAAKPKARNVVSPRSRAMTKNTKKVTKDSKYSTSSKSSPSSGDGQKKWTTLVHNGVIFPPPYKP--HGIKILYKGKPVDLTHuman 159 KE------KKRKLEEEEDGKLKKPKNKDKDKKVPEPDNKKKKPKKEEEQKWKWWEEERYPEGIKWKFLEHKGPVFAPPYEPLPENVKFYYDGKVMKLSpombe 137 TD------DSVPLRAVSTVSLT-PYKSELPSGASTTQNRSPNDEEDEDEDYKWWTSENIDDTQKWTTLEHNGVIFAPPYEPLPKNVKLIYDGNPVNLPcer. 110 NE------KK---------------KRE-------EEEEEDKKAKEEEEEYKWWEKENEDDTIKWVTLKHNGVIFPPPYQPLPSHIKLYYDGKPVDLP

Arab. 407 EVATMFAVMRETDYYTKPQFRENFWNDWRRLLGK------KHVIQKLDDCDFTPIYEWHLEEKEKKKQMSTEEKKALKEEKMKQEEKYMWAVVDGVKEHuman 255 EVATFFAKMLDHEYTTKEIFRKNFFKDWRKEMTNEEKNI ---- ITNLSKCDFTQMSQYFKAQTEARKQMSKEEKLKIKEENEKLLKEYGFCIMDNHKEpombe 233 EVAGFYAAMLETDHAKNPVFQDNFFRDFLKVCDECN---FNHNIKEFSKCDFTQMFHHFEQKREEKKSMPKEQKKAIKEKKDEEEEKYKWCILDGRKEcer. 185 EVAGFFAALLESDHAKNPVFQKNFFNDFLQVLKESGGPLNGIEIKEFSRCDFTKMFDYFQLQKEQKKQLTSQEKKQIRLEREKFEEDYKFCELDGRRE

*** * * ** * * **** * * * * * *

Arab. 503 FRVEPPGLFRGRGEHPKMGKLKKRIHPCEITLNIGKGAPIPECPIAGERWKEVKHDNTVTWLAFWADPINPKEFKYVFLGAGSSLKGLSDKEKYEKARHuman 353 FKIEPPGLFRGRGNHPKMGMLKRRIMPEDIIINCSKDAKVPS-PPPGHKWKEVRHDNKVTWLVSWTENIQ-GSIKYIMLNPSSRIKGEKDWQKYETARpombe 332 FRIEPPGLFRGRGEHPKTGSLKRRVYPEQITINIGEGVPVPE-PLPGHQWAEVKHDNTVTWLATWHENIN-NNVKYVFLAAGSSLKGQSDLKKYEKSRcer. 287 FKVEPPDLFxGRGAHPKTGKLKRRVNPEDIVLNLSKDAPVPP-APEGHKWGEIRHDNTVQWLAMWRENIF-NSFKYVRLAANSSLKGQSDYKKFEKAR

* *** ** *** *** * ** * * * * * * * * *** * ** * * ** * * ** * * * *

Arab. 605 HIDNIRTTYTKNFTAKDVKMRQASVATYLIDKLALRAGNEKDDDE-ADTVGCCTLKVGNVECIPPNK-----IKFDFLGKDSIQYVNTVEVEPLVYKAHuman 453 CVDKIRNQYREDWKSKEMKVRQRAVALYFIDKLALRAGNEKEEGETADTVGCCSLRVEHINLHPELDGQEYVVEFDFLGKDSIRYYNKVPVEKRVFKNpombe 432 YIDDIRKGYRKDLKNELTVERQRGTAMYLIDVFALRAGNEKGEDE-ADTVGCCSLRYEHVTLKPPRT-----VVFDFLGKDSIRYYNEVEVDPQVFKNcer. 387 YIDAIRRDYTRNLKSMVMLERQKAVAIYLIDVFALRAGGEKSEDE-ADTVGCCSLRYEHVTLKPPNT-----VIFDFLGKDSIRFYQEVEVDKQVFKN

* ** * ** * * ** ***** ** * ******* * * ********* * * * *

Arab. 702 -QAGKSKTDDLFDELDTSKLNAHLKELVPGLTAKVFRTYNASITLDEMLSQET-KDGDVTQKIVVYQKANKEVAIICNHQRTVSKTHGAQIEKLTARIHuman 555 MEN-KQPEDDLFDRLNTGILNKHLQDLMEGLTAKVFRTYNASITLQQQLKELTAPDENIPAKILSYNRANRAVAILCNHQRAPPKTFE --------

pombe 529 KRPPKKEGDLIFDRLSTNSLNKYLTSLMDGLSAKVFRTYNASYTMAEELK-KMPKNLTLADKILFYNRANRTVAILCNHQRSVTKNHDVQMERFAERIcer. 484 KRPPKQPGHQLFDRLDPSILNKYLQNYMPGLTAKVFRTYNASKTMQDQLD-LIPNKGSVAEKILKYNAANRTVAILCNHQRTVTKGHAQTVEKANNRI

* ** * ** * ** ********** * * ** * ** *** ***** *

Arab. 801 ------KEVLKELKTNLDRAKK- -----------------------------------------G GKPP------- LEGSDGKKIRSLEPNAWEKHuman 642 -------KSMMNLQTKIDAKKEQLADARRDL- ---------------------KSAKADAKVM-----------------------------KDAKTKpombe 630 YQRMRLRKMMLNLEPKLAKSKPELLAKEEGITDSWIVKHHETLUELEKEKIKKKFDRENEKLAAEDPKSMLPESELEVRLKAADELK-KALDAELKSKcer. 585 WQKIRCKRAILQLDKDLLKKEPKYFEEIDDLTKEDEATIHKRIIDREIEKYQRKFVRENDKRKFEK-EELLPESQLKEWLEKVDEKK-QEFEKELKTG

Arab. 843 QSA---------KIEKMERDMHT----- KEDLKTVALGTSKINYLDPRITVAWCKRHEVPIEKIFTKSLLEKFAWAMDVEPE-YRFSRRHuman 683 ------VESKKKAVQRLEEQLMKLEVQATDREENKQIALGTSKLNYLDPRITVAWCKKWGVPIEKIYNKTQREKFAWAIDMADEDYEF ---pombe 730 -PGRSSMEQLEKRLNKLNERINVMRTQMIDKDENKTTALGTSKINYIDPRLTYSFSKREDVPIEKLFSKTIRDKFNWAADTPPD-WKE---cer. 686 KSSWNSVEKIKAQVEKLEQRIQTSSIQLKDKEENSQVSLGTSKINYIDPRLSVVFCKKYDVPIEKIFTKTLREKFKWAIESVDENWRF ---

***** ** *** * ***** * ** **

Figure 4. B, Comparison of various TOP1 amino acid sequences. The predicted amino acid sequences of the Arabidopsis (Arab.), human, S.pombe (pombe), and S. cerevisiae (cer.) were aligned with the University of Wisconsin Genetics Computer Group clustal program. An asterisk(*) indicates that the amino acids are identical across all species; a dot (-) indicates that the sequences are conserved.

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Plant Physiol. Vol. 99, 1992

Figure 5. Nucleotide sequence of the up-stream region of the Arabidopsis TOPI gene.Two possible TATA regions are indicated inbold, an upstream intron is underlined, and apurine-rich stretch is italicized. The start of thecDNA sequence is shown. The nucleotides arenumbered on the left, with the start of thecDNA corresponding to +1.

-814-754-594-534-474-414-354-294-234-174-114-84-2427

kD, a discrepancy that may be due to proteolysis of thebroccoli enzyme.The protein sequence has striking homology to other se-

quenced TOPM genes (Fig. 4). The predicted amino acidsequence is 45% homologous to the S. pombe gene. Homologyis extended throughout the carboxyl two-thirds of the genes,whereas the amino terminus is variable, with the Arabidopsisgene showing approximately 100 extra amino acids (depend-ing on which AUG is the correct start codon). The Arabidopsisgene also has a deletion of approximately 60 amino acids justbefore the conserved carboxyl region. The tyrosine residueshown in yeast to be covalently linked to DNA as an inter-mediate in the topoisomerase I reaction (18) is presumed tobe at position 840 in the Arabidopsis enzyme, as judged fromconservation of the residues surrounding it.

i Sj H t; i

AATAATAATT CAGGTTAATA ATATTAAGAA TTATACATAA AATGGTAGTA CCT[TCTTTAATCTATATAC TATGGATGAT TAATAAGTTT ATGCATTAAG TAATGAAAAT TTTTAAGTTTTITrG=TGAC AAAATTCATT TTAACCTTTT TTAGTACTTG AAATAACTTT AATAATTTACAAATTAACTT TCAAATGGTT AAAAGTTTTG ATGACACATT GTTTCGTATA TCTAAATAGTATCTAAATGC TAAATTATCC GTTGATACGT AATTTTATCC CATAAGAATG TGGATATCCAAATGCTAAAT TATCTATCGA CACGTAAATT TTTCTGATAA AAAATGTTAA GAGAATCCAAACGTTAAATT ATTTGATGAT ACATCGGATA AAAAATTGGA TTTCTTTTTA TTTCTGATTTTATCCCCTAT TTTATTTACT TAAATATGCT TCACAATTTA GTAAATTAAC CGATGAGTCAAT[GTTTAATC AAlTTTTTCA TCTTAACCCA AAAATTAAGG GGGATTTGGC AATTAAAAAGTTTACGGTAA TAAGAGTGAA AAAAATTGTC TATGGCGGTA AGATTAAACG ATTTGGCGCGACTCAATACG ACGTCGAAAC GAAAACAAAA CCCTCAAGGG GTGAACTCTC TAATCTAAAGAGAGTCTCCT TATGCCTTAG TCTCCTCGTC CTTAAAACGA TAGA"CCAT CGAAGCAGAAGAGAAGAGAG AACAGAGAGA CGGA <start cDNA> GGAGAG AGATAGGCTT CGCTAGACAATTTCTTCAAT CTCTGGAAGA AGGTAGCTTC TTCAATCCAA CGTCAAATCT CTGAAATTTGA

TTGTATTAAG CTT... (remainder of intron not sequenced) AAGTTGGTCTGAGCCTTC ATCAACGTTG TTCTTTCGGGA ATG

Upstream Sequence of the TOP1 Genomic Region

Upstream of the portion of the TOPI genomic sequence(Fig. 5) that corresponds to the start of the cDNA clone (Fig.3) are two TATA-like sequences (19, 20). No homology toother plant upstream activating sequences is obvious. Justbefore the likely RNA start site and overlapping the cDNAis a stretch of 45 nucleotides containing 42 purines. Thesignificance of this sequence, if any, is not clear, although wehave found a similar purine-rich stretch at the start of acDNA encoding an Arabidopsis pyrophosphatase enzyme(15). The 5' untranslated leader sequence appears to containan intron, part of which has been sequenced. The introndonor splice site is consistent with the consensus plant se-quence (3).

TOP1 Is Present as a Single Copy in the ArabidopsisGenome

We digested Arabidopsis DNA (ecotype Columbia) withseveral restriction enzymes and analyzed it by Southern

j.-

Figure 6. Southern blot of Arabidopsis genomic DNA probed withTOPI. Total DNA from Arabidopsis ecotype Columbia was cut withvarious restriction enzymes, blotted to nitrocellulose, and probedwith the 1.4-kb carboxyl-terminal EcoRI fragment from pTopl 50. S,Sspl; H, Xhol; K, KpnI; N, Narl; X, Xbal; P, Spel; B, BamHI; and C,Clal. The sizes of marker DNA fragments are indicated in kb on theleft.

Figure 7. Map of pTopl 70, constructed as described in "Materialsand Methods." The direction of transcription of the genes is shown.

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ARABIDOPSIS TOPI GENE

Figure 8. Growth of various yeast strains inglucose and galactose medium. Cultures wereinoculated in SD medium containing glucoseor galactose as the sole carbon source to astarting Klett reading (see "Materials and Meth-ods") of 20 from a fresh overnight culture grownin SD plus glucose. The cultures were grown at30'C with moderate shaking and the Klettmonitored at various intervals. YAB100 carriespTop170 and YAB101 carries the parent plas-mid (no insert) pCp125; see Table I for a moredetailed description of the strains.

blotting using the 3' 1.5-kb EcoRI fragment from pTopl50 as

a probe. In most cases, a single band was detected undermoderately stringent conditions (Fig. 6), indicating that thegene is present as a single copy in the Arabidopsis genome.

Some enzymes (for example XbaI) gave a different pattern ofbands than expected from the restriction map of the TOP1genomic region (see Fig. 2). This is likely due to the fact thatthe restriction enzyme-digested DNA was from ecotype Co-lumbia, whereas the TOPI clones (from which the probe wasderived) were isolated from a library made with ecotypeLandsberg erecta DNA.

Expression of the Clone in Yeast

A complete copy of the cDNA sequence of TOPI was

constructed in vitro (see 'Materials and Methods') from a

poly(A)-primed cDNA clone (pTopl50) and a random hex-amer-primed cDNA clone (pTopl52) and placed under thecontrol of the yeast GAL1 promoter (pTopl 70, Fig. 7). A topl-strain of S. cerevisiae was transformed with pTopl70. Thetransformant, YAB100, proved unable to grow in the pres-ence of galactose (Fig. 8). This may be due to overexpressionof the Arabidopsis TOP1 gene, although the vector (CENorigin) should be present in only a few copies per cell. Evenwith glucose as the sole carbon source, YAB100 has a slight

Figure 9. Growth of YAB101 (left) and YAB100 (right) on SD agarplus adenine. Colonies were grown for 2 d at 30'C, then shifted to4°C for 1 week to help visualize the red sectoring. Dark sectorsindicate Ade- cells due to recombination at rDNA.

growth defect, the colonies being approximately 80% the sizeof YAB101 on minimal (SD) plates (J.J. Kieber, E.R. Signer,unpublished observations). This is evidently an instance of a

more general phenomenon. Both yeast TOP1 (R.K. Johnson,W.K. Eng, personal communication) and human TOP1 (L.F.Liu, P. D'Arpa, M.-C. Fann, personal communication), ex-

pressed from plasmids behind the GAL4 promotor, are alsolethal to yeast growing with galactose, but not glucose (orraffinose), and because that is true as well of mutant yeastTOP1 in which the active site tyrosine codon has been re-

placed by a phenylalanine codon (9), topoisomerization ac-

tivity appears not to be the cause.

The yeast strain CY185 carries an ADE2 gene inserted intoits rDNA. Recombination between tandem rDNA repeatsloops out ADE2 and makes the cells Ade-. In a wild-typestrain, recombination in this region is repressed and the cellsbecome Ade- very infrequently (5). However, CY185 carriesan insertion in the TOP1 gene eliminating topoisomerase Iand consequently has increased recombination in this region.On adenine agar, where wild-type colonies are white andAde- colonies are red, this can be visualized as red sectoringof the white colonies. When pTopl70, but not pCP125 (the

Table II. Arabidopsis TOPI Complements the Phenotype of a YeasttopI MutantTen independent colonies were scraped from minimal plates

(SD) containing glucose, adenine, and the appropriate supplementsfor growth of the strains and suspended in 1 mL of sterile water.The suspension was diluted appropriately and plated on SD mediacontaining glucose, adenine, and the appropriate supplements. Theplates were grown for 3 d at 30'C and then shifted to 4'C for 1week to allow the red color to develop. Approximately 500 colonieswere counted for each original colony, and the mean frequency ofred colonies (adel) is shown.

Strain Relevant Phenotype Frequency of Ade-

CY184 rDNA::ADE2 1.11 x 10-2 ± 0.01CY185 CY184, topl-7::LEU2 1.19 x 10-1 ± 0.04YAB100 CY185, pTopl7O 7.3 X 10-3 ± 0.003aYAB101 CY185, pCP125 1.29 X 10-' ± 0.05YAB102 YABQ00 cured of pTop170 1.24 X 10' ± 0.04a These colonies were paler red than the other strains.

200

100 .oof~ ~~~~~- -l~~~~~~ili am

00

-0-

-a--a

-di

YABIOI, gicCY185, gic

YAB100, gic

YABIOI, gal

CY185, gal

YAB100, gal

10

Time (Hrs)

20

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30-

'4

Figure 10. Western blot of extracts from yeast carrying the parentplasmid pCpl25 (101) or pTop170 (100). Cells were grown insucrose-containing SD to mid-log phase, one half of the cultureinduced by the addition of 2% galactose (gal) and the other was

not (suc). The cells were extracted as described in "Materials andMethods," and the protein blot probed with the human antitopo-isomerase antibody AFCDC9. The sizes of the marker proteins are

shown on the left.

parent plasmid lacking the Arabidopsis TOPI gene), is intro-duced into CY185, the frequency of Ade- colonies is reducedapproximately 10-fold relative to TOPI+ strains on platescontaining glucose as the sole carbon source (Fig. 9, Table II),as expected if the Arabidopsis TOPI gene complements theyeast deficiency and suppresses rDNA recombination. Al-though glucose represses the GALI promoter, enough Arabi-dopsis topoisomerase I activity is still presumably expressed.The failure of the strain to grow in galactose precludes testingthe derepressed condition. The Ade- colonies of YAB100were much paler in color than the other strains used in thisstudy. This may be related to the growth defect of this strain.YAB100 was plated on rich medium and then replica plated

to medium lacking uracil to screen for URA' colonies (whichpresumably have lost pTopl70). One such colony was se-

lected for further study (YAB102) and this strain shows a

frequency of Ade- colonies comparable to the topl parentstrain (Table II). This indicates that the suppression of thesectoring phenotype is linked to pTopl70.

Despite the phenotypic complementation, no topoisomer-ase I activity was detected in extracts of YAB100 grown withglucose and then shifted to galactose (not shown). This mightbe due to low activity of the Arabidopsis enzyme made inyeast, or to instability of the protein, either after cessation ofgrowth in galactose or in the extract. We probed extracts ofYAB101 (no insert) and YAB100 with human antitopoisomer-ase I antibody (AFCDC9). This antibody recognizes an ap-proximately 100-kD protein in extracts made from YAB100cells grown in galactose, but not control extracts made fromYAB101 (Fig. 10). Two other proteins of higher molecularmass are also detected by the antibody, but these bands are

also present in extracts made from strains carrying the parentvector plasmid alone. Subsequent blots also showed a 30-kDband from YAB101 cells grown in galactose (not shown),indicating that significant proteolysis of the Arabidopsis TOPIprotein takes place under certain conditions, which mayexplain the lack of detectable activity in extracts made fromYAB100.

In conclusion, we have cloned and characterized a TOPigene from Arabidopsis that is homologous to other eukaryoticTOP genes, particularly that of S. pombe. When the gene isexpressed in S. cerevisiae, although topoisomerase activity isnot detected in extracts, a phenotypic assay indicated thatthe gene product functions to complement a topl mutationand western analysis indicates that a protein is expressedfrom the clone that cross-reacts with an antibody directedagainst human topoisomerase I.

LITERATURE CITED

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