dna topoisomerase i domain interactions impact enzyme activity

DNA Topoisomerase I Domain Interactions Impact EnzymeActivity and Sensitivity to Camptothecin*

Received for publication, December 23, 2014, and in revised form, March 17, 2015 Published, JBC Papers in Press, March 20, 2015, DOI 10.1074/jbc.M114.635078

Christine M. Wright‡1, Marié van der Merwe§1,2, Amanda H. DeBrot‡, and Mary-Ann Bjornsti‡3

From the ‡Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294and §Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105

Background: Despite similarities in mechanism and architecture, human DNA topoisomerase I (Top1) is �100-fold moresensitive to camptothecin (CPT) than yeast Top1.Results: Reciprocal swaps of conserved and divergent protein domains alter chimeric Top1 activity.Conclusion: Conserved core and C-terminal domains dictate Top1 biochemical behavior and intrinsic CPT sensitivity.Significance: Interactions between nonconserved structural domains of Top1 impair cell viability, independent of enzymecatalysis.

During processes such as DNA replication and transcription,DNA topoisomerase I (Top1) catalyzes the relaxation of DNAsupercoils. The nuclear enzyme is also the cellular target ofcamptothecin (CPT) chemotherapeutics. Top1 contains fourdomains: the highly conserved core and C-terminal domainsinvolved in catalysis, a coiled-coil linker domain of variablelength, and a poorly conserved N-terminal domain. Yeast andhuman Top1 share a common reaction mechanism and domainstructure. However, the human Top1 is �100-fold more sensi-tive to CPT. Moreover, substitutions of a conserved Gly717 resi-due, which alter intrinsic enzyme sensitivity to CPT, induce dis-tinct phenotypes in yeast. To address the structural basis forthese differences, reciprocal swaps of yeast and human Top1domains were engineered in chimeric enzymes. Here we reportthat intrinsic Top1 sensitivity to CPT is dictated by the compo-sition of the conserved core and C-terminal domains. However,independent of CPT, biochemically similar chimeric enzymesproduced strikingly distinct phenotypes in yeast. Expression of ahuman Top1 chimera containing the yeast linker domainproved toxic, even in the context of a catalytically inactive Y723Fenzyme. Lethality was suppressed either by splicing the yeastN-terminal domain into the chimera, deleting the human N-ter-minal residues, or in enzymes reconstituted by polypeptidecomplementation. These data demonstrate a functional interac-tion between the N-terminal and linker domains, which, whenmispaired between yeast and human enzymes, induces celllethality. Because toxicity was independent of enzyme catalysis,the inappropriate coordination of N-terminal and linker do-mains may induce aberrant Top1-protein interactions to impaircell growth.

Eukaryal DNA topoisomerase I (Top1)4 catalyzes the relax-ation of local domains of positive and negative DNA supercoils,which are generated by processes such as DNA replication,transcription, and recombination (1– 4). Top1 is a monomericenzyme that forms an asymmetric protein clamp, which encir-cles double-stranded DNA. During the reaction cycle, theactive site tyrosine (Tyr723 in human Top1) attacks a singlestrand of duplex DNA to form a 3�-phosphotyrosyl intermedi-ate. Within this covalent Top1-DNA complex, DNA strandrotation allows for the relaxation of DNA supercoils. The 5� OHof the cleaved DNA strand then acts in a second transesterifi-cation to resolve the phosphotyrosyl bond and religate theDNA.

Top1 is the cellular target of the camptothecin (CPT) class ofchemotherapeutics (5–7). These drugs reversibly stabilize thecovalent Top1-DNA intermediate by intercalating into the pro-tein-linked DNA nick. During S phase, the interaction of thereplication machinery with the ternary CPT-Top1-DNA com-plex itself or with the positive supercoils that accumulate aheadof the fork creates irreversible DNA lesions that induce celldeath (5– 8). Increased concentrations of the covalent interme-diate can also be induced with Top1 mutants, where alteredrates of DNA cleavage/religation effectively convert the en-zyme into a cellular poison (9 –12).

Biochemical and x-ray crystallographic data reveal anunusual architecture of monomeric Top1. The human nuclearenzyme consists of four distinct domains: a charged N termi-nus, a conserved core domain, a linker domain formed by anextended pair of �-helices (residues 636 –712), and a conservedC-terminal domain that contains the active site Tyr (1, 7,13–17). Fig. 1A depicts the co-crystal structure of humanTopo70, which lacks the N-terminal 174 amino acid residues,noncovalently bound to DNA (18). As summarized in Fig. 1B,the N-terminal and linker domains of yeast and human Top1share only 17 and 12% amino acid identity, respectively,whereas the conserved core and C-terminal domains are 58 and

* This work was supported, in whole or in part, by National Institutes of HealthGrant CA58755 (to M.-A. B.). This work was also supported by University ofAlabama at Birmingham Cancer Center Core Grant P30CA013148.

1 These authors contributed equally to this work.2 Present address: Dept. of Health and Sport Sciences, University of Memphis,

Memphis, TN 38152.3 To whom correspondence should be addressed: Dept. of Pharmacology and

Toxicology, University of Alabama at Birmingham, 1670 University Blvd.,Birmingham, AL 35294. Tel.: 205-934-4579; Fax: 205-934-8240; E-mail:[email protected].

4 The abbreviations used are: Top1, DNA topoisomerase I; Topo70, N-ter-minally truncated 70-kDa version of DNA topoisomerase I; CPT, camp-tothecin.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 19, pp. 12068 –12078, May 8, 2015© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

12068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 19 • MAY 8, 2015

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62% identical, respectively (43).The conserved core domain ofTop1 forms a protein clamp around duplex DNA. We previ-ously demonstrated that locking the human Top1 clamparound the DNA prevents strand rotation within the Top1-DNA intermediate (19). Similar impediments to DNA rotationwere observed with the analogous studies of yeast Top1 (20),suggesting a conservation of protein clamp enzyme architec-ture and catalytic mechanism.

The core domain of Top1 provides most of the catalytic res-idues. However, Tyr723 is in the conserved C-terminal domainand is positioned within the catalytic pocket of the core domainby the extended coiled-coil linker domain, yet the linkerdomain does not appear to be required for activity. Top1enzymes found in some bacteria and the poxviruses lack boththe N terminus and the linker domain (21, 22). In addition,fragment complementation experiments demonstrate thatpolypeptides comprising only the human Top1 core and C-ter-minal domains can reconstitute an active enzyme in vitro (23),albeit with reduced specific activity.

In the co-crystal structure of the covalent Topo70-DNAintermediate, the linker is not resolved (7). However, when theTopo70-DNA intermediate is bound by CPT or the analogtopotecan, the linker becomes extended at an oblique angle tothe DNA helix, as in the noncovalent structure in Fig. 1 (7,

15–17). Although the role of the linker domain in mediatingenzyme sensitivity to CPT is not clear, the “linker-less” poxvirusTop1 enzymes are resistant to CPT (24). Moreover, the in vitroCPT resistance of the two subunit Leishmania Top1, andreconstituted human Top1, demonstrates that the physicallinkage of the linker to the core domain is necessary for CPTsensitivity (23, 25). Studies of mutant yeast and human enzymesfurther suggest a correlation of linker flexibility with CPT resis-tance (7, 26 –30).

The function of the N-terminal domain also remains poorlyunderstood, in part because of a lack of structural information.In vitro, residues 1–190 of the nuclear enzyme are dispensablefor enzyme activity and CPT sensitivity. However, amino acids191–206 are required for DNA binding and enzyme pro-cessivity (31–34). The N terminus has also been implicated innumerous interactions with other proteins, including TATA-binding protein, nucleolin, and SV40 large T antigen (35–38).Additional studies suggest a role for the N-terminal domain inthe opening and closing of the Top1 clamp around DNA (20)and in CPT-induced alterations in the intracellular localizationof Top1 (39).

Despite the considerable conservation of yeast (yTop1) andhuman (hTop1) enzyme mechanism, structure, and function,there are notable differences. Importantly, wild-type yTop1 andhTop1 exhibit an �100-fold difference in intrinsic sensitivity toCPT in vitro. Moreover, mutant-induced alterations in enzymesensitivity to CPT are not always manifested in cells. Forinstance, substitution of Asp for a conserved Gly residue, whichis posited to form a flexible hinge with the linker domain, justN-terminal to the active site Tyr in yeast and human Top1,renders these mutant enzymes intrinsically more sensitive toCPT in vitro. However, expression of yTop1G721D in yeastenhanced cell sensitivity to CPT, whereas cells expressing thecorresponding hTop1G717D mutant were no more sensitive toCPT than cells expressing wild-type hTop1 (28).5

Such results prompted us to ask whether differences inintrinsic CPT sensitivity might be attributed to the domaincomposition of Top1 and whether the different phenotypesinduced by mutations of conserved residues within the catalyticpocket of yTop1 and hTop1 were due to distinct architecturalfeatures of these enzymes. To address these questions, crystal-lographic and biochemical data were used to design a series ofchimeric enzymes that comprise reciprocal swaps of the yeastand human N-terminal and linker domains. Here, we reportthat the composition of the conserved core and C-terminaldomains dictate intrinsic Top1 enzyme sensitivity to CPT,whereas the functional interaction of the N terminus with thelinker domain impacts cell viability, independent of CPT andenzyme catalysis. Thus, intramolecular interactions betweenpoorly conserved protein domains dictate the critical con-served function of Top1.

EXPERIMENTAL PROCEDURES

Yeast Strains and Plasmids—Saccharomyces cerevisiae strainEKY3 (MAT�, ura3–52, his3�200, leu2�1, trp1�63, top1::

5 M. van der Merwe, C. M. Wright, A. H. DeBrot, and M.-A. Bjornsti, unpublisheddata.

FIGURE 1. The structure of a C-terminal 70-kDa fragment of human DNAtopoisomerase I (Topo70) in a noncovalent complex with a 22-base pairDNA duplex. A, ribbon diagram of PDB file 1A36 (18), viewed perpendicularto helical axis of the DNA duplex (backbone is in orange), the core domain ofTopo70 forms a protein clamp (shades of blue). The linker domain (purple)extends from the core at an oblique angle to the DNA, and the C-terminaldomain is in green. The active site Tyr is mutated to Phe (indicated inmagenta). B, based on the structure in A and the alignment of yeast Top1(yTop1) and human Top1 (hTop1) sequences (43), the percentages of identi-cal and (similar) amino acid residues in the four domains are indicated. Thesame domain designations are depicted in Figs. 3A, 4A, and 9A.

Activity of Human/Yeast DNA Topoisomerase I Chimeras

MAY 8, 2015 • VOLUME 290 • NUMBER 19 JOURNAL OF BIOLOGICAL CHEMISTRY 12069


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TRP1) was previously described (11). Herein, “h” and “y” desig-nations distinguish human and yeast TOP1, respectively.Plasmids YCpGAL1-yTOP1 and YCpGAL1-hTOP1 expresswild-type yeast TOP1 and human TOP1 cDNA from the galac-tose-inducible GAL1 promoter in an ARS/CEN, URA3 vector(11, 19), whereas YCpGAL1-hTopo70 expresses an N-terminaltruncated human Top1 (residues 175–765), referred to asTopo70. All of the constructs used in these studies contained anN-terminal Flag epitope (DYKDDDDK) recognized by the M2monoclonal antibody (Sigma). Yeast-human chimeras weregenerated using homologous recombination of PCR-generatedchimeric junctions as described in Ref. 40 and diagrammed inFig. 2A. See Table 1 and Fig. 2B for primer design and chimerajunctions. Plasmids were recovered from individual yeast colo-nies as in Ref. 40, amplified in Esherichia coli, and sequenced toconfirm yTop and hTop sequences.

Vector pESC-ura (Stratagene) contains the bicistronic, gala-catose-inducible GAL1-10 promoter cassette with FLAG andmyc tags, and terminator sequences in a 2-�m, URA3 vector.The entire GAL1-10 cassette flanking polylinker sequenceswere PCR-amplified and used to replace the polylinker se-quences of pRS416, by homologous recombination, as in Ref.

40, to create YCpGAL1-10. Reconstituted Top1 protein expres-sion vectors were generated as described in Table 2. As anexample, the linker and C terminus of hTop1 were PCR-ampli-fied from YCpGAL1-hTOP1 and inserted 3� to the myc epitope(EQKLISEEDL) sequences under the GAL1 promoter in YCp-GAL1-10 to create YCpGAL1-10 hTopo14. The hTop1 Nterminus and core domain sequences were then PCR-ampli-fied from YCpGAL1-hTOP1 and inserted 3� to the FLAGepitope sequences of the GAL10 promoter to create YCp-GAL1-10 hTopo76/14. Bicistronic constructs were also createdwithout the myc tag. In all cases, mutant sequences were con-firmed by DNA sequencing. Primer sequences are availableupon request.

Yeast Cell Sensitivity to CPT—To assay yeast cell sensitivityto CPT, cultures of EKY3 (top1�) yeast cells, transformed withthe indicated YCpGAL1-TOP1 vector, were serially 10-folddiluted. Aliquots (4 �l) were spotted onto synthetic completeagar lacking uracil (SC-ura) medium containing 25 mM Hepes(pH 7.2), 2% dextrose or galactose, and the indicated concen-tration of CPT in a final 0.125% DMSO. Cell growth wasassessed after incubation at 30 °C.

DNA Topoisomerase I Expression and Purification—Galac-tose-induced cultures of yeast top1� cells expressing wild-type,mutant, or chimeric Top1 enzymes were lysed with prechilled(�20 °C) glass beads in TEEG buffer (50 mM Tris, pH 7.4, 1 mM

EDTA, 1 mM EGTA, 200 mM KCl, 10% glycerol) and completeprotease inhibitors (Roche). Lysates were normalized for totalprotein, and Top1 protein levels were verified by immunoblot-ting and chemiluminescence using the M2-FLAG antibody(Sigma). Wild-type and chimeric Top1 were partially purifiedby phosphocellulose chromatography or purified to homoge-neity by phosphocellulose and FLAG affinity chromatography,as described (12, 28). Top1 protein fractions were adjusted to afinal concentration of 30% glycerol and stored at �20 °C. Pro-tein integrity was assayed by immunoblot analysis (28, 41).

Plasmid Relaxation Assays—The specific activity of the Top1preparations was measured in plasmid DNA relaxation reac-tions as described (9, 41). Briefly, serial 10-fold dilutions of puri-fied Top1 protein or cell lysates (corrected for protein concen-tration) were incubated in 20-�l reaction volumes with 0.3 �gof negatively supercoiled pHC624 plasmid DNA in 20 mM Tris(pH 7.5), 10 mM MgCl2, 0.1 mM EDTA, 50 �g/ml gelatin, and50 –200 mM KCl (as indicated), at the temperatures indicated,for 30 min. DNA products were extracted with phenol/chloro-form, resolved by agarose gel electrophoresis, and visualizedfollowing staining with ethidium bromide or GelRed (Phenix).

DNA Cleavage Assays—Intrinsic enzyme sensitivity to CPTwas assessed in DNA cleavage assays, as previously described(12), using a 480-base pair DNA fragment that contained a highaffinity Top1 cleavage site and was 32P-end-labeled at a single 3�end. The cleavage reaction products were treated with SDS at75 °C; digested with proteinase K; ethanol precipitated;resolved in denaturing 8% polyacrylamide, 7 M urea gels; andvisualized by PhosphorImage analysis.

Co-immunoprecipitations—Lysates of galactose-inducedcultures of EKY3 cells expressing the indicated Top1 reconsti-tution vectors, prepared as above, were split into two 400-�laliquots, and Triton X-100 was added to a final concentration of

FIGURE 2. Generation of yeast/human Top1 chimeras in yeast. A, strategyfor generating chimeric human/yeast Top1 proteins using homologousrecombination in yeast. In this example, sequences encoding the N-terminaldomain of human Top1 were PCR-amplified with primers containing an addi-tional 40 nucleotides at the 5� end, which were complementary to sequencesin the GAL1 promoter (black) or sequences flanking the N-terminal domain ofyeast Top1 (pink). The chimera junction is defined by the 3� primer sequence.YCpGAL1-yTOP1 was cut at a unique restriction site within the region to beexchanged. The PCR-amplified DNA and linearized plasmid were co-trans-formed into yeast cells. Homologous recombination of the DNA ends regen-erated a circular plasmid, and the resulting transformants were selected foruracil prototrophy. The plasmid DNA was extracted, and the identities of thechimeric sequences were confirmed by DNA sequencing. B, amino acidsequence alignment of the portion of human and yeast Top1 N-terminaldomains used in the chimeras. Purple represents the first six residues of theTopo70 constructs. Red and green indicate the junctions of (yN)-hTop1 and(yN137)-hTop1, respectively. The corresponding residues surrounding thejunction lines designate (hN)-yTop1 and (hN209)-yTop1. The gray box denotesthe beginning of the Top1 core domain.




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1%. 20 �l of protein A/G Plus-agarose (Santa Cruz) was added,and the samples were rotated end over end for several hours at4 °C. Following centrifugation, the supernatant was added to30 �l of protein A/G Plus-agarose and 5 �g of either anti-FLAG or anti-myc antibody and rotated end over end over-night at 4 °C. The samples were washed five times with 1�TEEG containing 200 mM KCl, 1% Triton, and protease inhib-itors. The proteins were resolved by SDS-PAGE and visualizedby immunoblotting.

RESULTS

N-terminal Human/Yeast Top1 Chimeras—To address thefunction of Top1 protein domains in regulating enzyme activityand sensitivity to CPT in cells, homologous recombination (asdescribed in Ref. 40 and diagrammed in Fig. 2A) was used togenerate reciprocal swaps of the N-terminal and linker domainsof yTop1 and hTop1. The poorly conserved N-terminaldomains have been shown to contain nuclear localization sig-nals (42) and to mediate Top1 interactions with other proteins(35–37). Hence, N-terminal domain chimeras were first engi-neered to ask whether yeast protein interactions with the N-ter-minal domains of yTop1 or hTop1 could account for theobserved alterations in enzyme activity in cells. hTopo70 (resi-dues 175–765) has been extensively used for biochemical and

structural studies of hTop1. However, available structuralinformation is restricted to the core, linker, and C-terminaldomains (residues 215–765) (Fig. 1, A and B). Within the N-ter-minal domain, residues 1–190 are dispensable for hTop1 catal-ysis, whereas residues 191–206 are required for efficient DNAbinding (32). Based on sequence homology and codon usage, weengineered two sets of reciprocal swaps that spanned these crit-ical residues (summarized in Fig. 2 and Table 1).

In h120yTop1, human N-terminal residues 1–191 were fusedto yeast residues 120 –769, whereas y192hTop1 contains yeastN-terminal residues 1–119 fused to human residues 192–765(Figs. 2B and 3A). For ease of discussion, hereafter these chime-ras are referred to as (hN)-yTop1 and (yN)-hTop1, respectively.As shown in Fig. 3B and Table 3, exchanging these portions ofthe N-terminal domains had no effect on the pattern of in vivoCPT sensitivity in yeast. CPT-induced toxicity of cells express-ing (hN)-yTop1 mirrored that of wild-type yTop1 and was onlyobserved at high drug concentrations. Conversely, cellsexpressing either wild-type hTop1 or (yN)-hTop1 were sensi-tive to 100-fold lower concentrations of drug (compare cell via-bility at 5 versus 0.05 �g/ml in Fig. 3B and in Table 3). The levelsof chimera protein expressed and the specific activity of thepurified N-terminal chimeric proteins were similar to that ofthe wild-type enzymes (Fig. 3, C and D). Similar results wereobtained with additional constructs: 1) a second set of N-termi-nal chimeras (y210hTOP1 and h138yTOP1) that containedjunctions at human residue 210 or yeast residue 138, respec-tively, just N-terminal to the core domain, and 2) a yeast/hu-man chimera y�201hTOP1, where deletion of 8 residues wasengineered in the yeast sequences just upstream of the human201 chimera junction (Figs. 2B and 3D, Table 1, and data notshown). In all cases, the N-terminal composition of the chime-ras had no effect on enzyme catalysis in vitro or on yeast cellsensitivity to CPT (Fig. 3 and data not shown). Thus, it isunlikely that yeast protein interactions with N-terminal Top1domain residues alone dictate enzyme sensitivity to CPT inyeast cells.

Introducing the Yeast Linker Domain into hTop1 InducesYeast Cell Lethality—The other nonconserved domain of Top1is the coiled-coil linker, whose flexibility has been implicated inaltering enzyme sensitivity to CPT (7, 26 –28). We thereforeengineered reciprocal exchanges of the linker domains. To

TABLE 1Composition of yeast/human Top1 chimerasChimeric Top1 enzymes were generated by homologous recombination of PCR products as diagrammed in Fig. 2. Chimera junctions were engineered in the PCR primers.Primer sequences are available upon request.

Chimeraa Compositionb Junction sequencec

(hN)-yTop1 hTop1 (1–191)-yTop1 (120–769) DKKVPEPDNK/EDKKAKEEh138yTop1 hTop1 (1–209)-yTop1 (138–769) EQKWKWWEEE/NEDDTIKW(yN)-hTop1 yTop1 (1–119)-hTop1 (192–765) EKKKREEEEEE/KKKPKKEEEQKWy210-hTop1 yTop1 (1–137)-hTop1 (210–765) EEEYKWWEKE/RYPEGIKWK(hL,hC)-yTop1 yTop1 (1–560)-hTop1 (635–765) VAILCNHQR/APPKTFEKSMM(hL)-yTop1 yTop1 (1–560)-hTop1 (635–713)-yTop1 (717–769) QATDREENK/QVSLGTSKINY727

(yL,yC)-hTop1 hTop1 (1–634)-yTop1 (561–769) VAILCNHQR/TVTKGHAQTVEK(yL)-hTop1 hTop1 (1–634)-yTop1 (561–716)-hTop1 (713–765) QLKDKEENS/QIALGTSKLNY723

a The heterologous Top1 protein domains are in parentheses and are designated h for human or y for yeast. N, L, and C refer to N-terminal, linker, and C-terminal domains,respectively. For example, (hN)-yTop1 contains the human N terminus and the yeast core, linker, and C-terminal domains. (yL,yC)-hTop1 contains the human N terminusand core domains, with the yeast linker and the C terminus.

b The numbers indicate the yeast or human amino acid residues contained in the chimeras, with 1 referring to the first Met residue. The (hL,hC)-yTop1 chimera was used togenerate (hL)-yTop1, whereas the (yL,yC)-hTop1 chimera was used to generate (yL)-hTop1.

c Residues spanning the chimera junctions, indicated by a slash (/). Y727 and Y723 indicate the active site tyrosine residues of yTop1 and hTop1, respectively.

TABLE 2Composition of reconstituted Top1 enzymesReconstituted enzymes were created by PCR amplification or restriction digest ofDNA sequences encoding the indicated y or hTop1 linker� hTop1 C-terminaldomains and ligation behind the GAL1 promoter in a bicistronic vector. Subse-quently, DNA encoding hTop1 N-terminal � core domains or core domain alonewas PCR-amplified or excised by restriction digest and inserted behind the GAL10promoter.

Reconstitutedenzymea

GAL10 expressedconstructb GAL1 expressed constructc

hTopo76/14 hTop1 (1–634) hTop1 (635–765)hTopo78/12 hTop1 (1–659) hTop1 (658–765)(yL)-hTopo76/14 hTop1 (1–634) yTop1 (561–716)-hTop1 (713–765)hTopo56/14 hTop1 (175–634) hTop1 (635–765)hTopo58/12 hTop1 (175–659) hTop1 (658–765)

a The numbers refer to the mass of the polypeptides comprising the reconstitutedenzyme.

b All constructs contained an N-terminal FLAG tag. hTop1(175– 634) andhTop1(175– 659) polypeptides, which comprise the large subunits ofhTopo56/14 and hTopo58/12, respectively, were also engineered with and with-out an N-terminal NLS (PKKIKTED). Parenthetical numbers refer to hTop1amino acid residues comprising the expressed polypeptide.

c All constructs were created with an N-terminal myc tag, followed by the indi-cated y/hTop1 residues.




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accomplish this, the linker and C terminus of each enzyme wasfirst replaced with the corresponding residues of the other.These chimeras were then used in reciprocal swaps to reintro-duce the corresponding C-terminal domains, such that the(yL)-hTop1 chimera consisted of the human N-terminal, core,and C-terminal domains, yet it contained the yeast linker (Fig.4A). The precise chimera junctions correspond to the con-served residues found at the N- and C-terminal ends of thecoiled-coils of the linker domain, evident in the co-crystal

structure of the drug-bound covalent Topo70-DNA complex(7). The linker domain spans hTop1 residues 635–712 andyTop1 residues 561–716 (Table 1).

Introducing the shorter hTop1 linker into yeast Top1, in(hL)-yTop1, induced a �10-fold reduction in specific enzymeactivity in vitro and induced a CPT-resistant phenotype in yeast

FIGURE 3. Reciprocal swaps of the N-terminal domains of yeast andhuman Top1 do not alter enzyme activity or yeast cell sensitivity to CPT.A, scheme of N-terminal residues exchanged between yeast and human Top1via homologous recombination. The structurally and biochemically defineddomain junctions are labeled. (hN)-yTOP1 encodes the yeast enzyme with ahuman N-terminal domain; (yN)-hTOP1 encodes human Top1 with the yeastN terminus. B, exponential cultures of top1� cells, transformed with the indi-cated YCpGAL1-TOP1 constructs, were serially diluted and were spotted ontoSC-uracil agar containing dextrose (Dex) or galactose (Gal) and the indicatedconcentration of CPT in 0.125% DMSO. The viability of two independenttransformants was assessed following incubation at 30 °C. C, equal concen-trations of purified Top1 proteins were serially 10-fold diluted and incubatedin a plasmid DNA relaxation assay. The reaction products were resolved byagarose gel electrophoresis and visualized with ethidium bromide. The rela-tive positions of relaxed (R) and supercoiled (�) DNA topoisomers are indi-cated. Lane C indicates DNA alone. D, extracts of galactose-induced top1�cells, expressing the indicated FLAG-tagged Top1 proteins, were correctedfor protein concentration, resolved in SDS acrylamide gels, and immuno-blotted with an anti-FLAG antibody. The asterisk indicates the relative posi-tion of an unrelated yeast protein also recognized by the FLAG antibody.Immunostaining of GAPDH served as a loading control. The data are repre-sentative of n 3 experiments.

TABLE 3Viability and CPT sensitivity of top1� cells expressing yeast/humanchimeras

TOP1 allelea

Number of viable cells forming colonies(relative to vector control)b

No drug 0.05 �g/ml CPT 5.0 �g/ml CPT

Vector 1.0 1.0 0.0024 1.0 0.0098yTOP1 1.1 0.2 1.0 0.087 �0.00004hTOP1 0.83 0.031 0.0001 0.000046 NDc

(yL)-hTOP1 �0.00004 �0.00001 ND(yN,yL)-hTOP1 0.38 0.06 �0.00001 NDhTopo70 1.2 0.26 0.0016 0.0006 ND(yL)-hTopo70 1.1 0.14 0.0011 0.0003 ND

a top1� cells were transformed with galactose-inducible YCpGAL1 constructsthat express the indicated TOP1 allele and selected on SC �uracil medium sup-plemented with dextrose. Under these conditions, the Top1 proteins were notexpressed.

b Yeast cell transformants, selected as in footnote a, were grown in SC �uracilmedium and serially diluted, and aliquots were plated in duplicate on SC �ura-cil medium supplemented with 2% galactose, 0.125% DMSO, and the indicatedconcentrations of CPT. Following incubation at 30 °C, the number of viable cellsforming colonies were counted and normalized relative to the number obtainedwith the no drug, vector control. Average values of three independent experi-ments are presented, with standard deviations from the sample mean indicated.

c ND indicates not determined.

FIGURE 4. The introduction of the yeast N-terminal domain suppressesthe lethal phenotype of the (yL)-hTop1chimera. A, a scheme of the N-ter-minal and linker domains used to generate the indicated chimeras. (yL)-hTOP1 encodes human Top1 with a yeast linker domain; (yN, yL)-hTOP1encodes human Top1 with a yeast N-terminal and yeast linker domain. B,serial dilutions of exponential cultures of top1� cells, transformed with theindicated YCpGAL1-TOP1 constructs, were spotted onto SC-uracil agar con-taining dextrose (Dex) or galactose (Gal) and CPT (as indicated). C, equal con-centrations of yeast lysates expressing hTop1 and chimeric proteins wereserially 10-fold diluted and incubated in a plasmid DNA relaxation assay con-taining the indicated concentration of KCl. The relative positions of relaxed (R)and supercoiled (�) DNA topoisomers are indicated. For B and C, the data arerepresentative of at least n 3 experiments.




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(data not shown). In contrast, replacing the shorter hTop1linker with the longer yeast linker, in (yL)-hTop1, induced asurprising phenotype in yeast. As shown in Fig. 4B and Table 3,galactose-induced expression of (yL)-hTop1 inhibited cellgrowth, even in the absence of CPT. Thus, the presence of thelonger yeast linker in the human enzyme is toxic to yeast. How-ever, when the corresponding yeast N-terminal domain wasintroduced into this chimera (to generate (yN,yL)-hTop1), suchthat the conserved core and C-terminal domains were hTop1,whereas the divergent N-terminal and linker domains were ofyeast Top1 origin, this toxic phenotype was largely suppressed(in Fig. 4B and Table 3). Moreover, CPT-induced lethality ofcells expressing the (yN,yL)-hTop1 chimera was evident at con-centrations sufficient to sensitize cells expressing wild-typehTop1, but not yTop1 (Fig. 4B and Table 3). Similar resultswere obtained with other N-terminal chimera junctions (Table1 and data not shown). These finding suggest that the CPTsensitivity of Top1 is dictated by the composition of the con-served core and C-terminal domains, whereas alterations inN-terminal domain-linker domain interactions adverselyimpact enzyme function in cells.

This model was further supported by assays of chimericenzyme catalysis. First, in cell lysates corrected for total protein,the specific activity of the (yN,yL)-hTop1 chimera was similarto that of wild-type hTop1 over a range of salt concentrations.The (yL)-hTop1 chimera exhibited a similar pattern of optimalactivity at 150 –200 mM KCl, yet was slightly more active (�5-fold) than comparable levels of wild-type hTop1 (Fig. 4C).Thus, (yL)-hTop1-induced toxicity could not be attributed togross alterations in enzyme catalysis. Second, comparable levelsof hTop1, (yL)-hTop1, and (yN,yL)-hTop1 proteins wereexpressed in cells (see Fig. 7B and data not shown). Moreover,in DNA cleavage assays performed with equal concentrationsof purified yTop, hTop1, and the (yL)-hTop1 and (yN,yL)-hTop1 chimeras, the intrinsic CPT sensitivity of the chimeraswere indistinguishable from that of intact hTop1. As seen inFig. 5, at least 100-fold higher concentrations of CPT wererequired to induce similar levels of covalent yTop1-DNA inter-mediates as that observed for wild-type hTop1. Thus, indepen-dent of N-terminal/linker domain composition, the intrinsicCPT sensitivity of the (yL)-hTop1 and (yN,yL)-hTop1 chimerasmirrored that of wild-type hTop1. Moreover, subtle differencesin sequence specificity between the yeast and human enzymes(as indicated by the arrowheads in Fig. 5) were also retained bythe hTop1 chimeras. Thus, the N-terminal and linker domaincomposition of hTop1 does not impact the intrinsic pattern ofenzyme sensitivity to CPT.

Because the toxic phenotype of (yL)-hTop1 expressing cellscould not be attributed to any obvious alterations in enzymebinding to DNA, sequence specificity, or shift in cleavage/reli-gation in the absence of CPT, we next investigated the role ofDNA cleavage in (yL)-hTop1 toxicity. The active site Tyr723 isessential for enzyme catalyzed DNA cleavage and formation ofthe covalent phosphotyrosyl intermediate, but not DNA bind-ing by the Top1 protein (1–3, 16). In (yL)-hTop1Y723F, theactive site Tyr723 was mutated to Phe to produce a catalyticallyinactive mutant protein. Nevertheless, expression of the (yL)-hTop1Y723F mutant also proved cytotoxic, inducing a similar

�3-log drop in cell viability as with (yL)-hTop1 (Fig. 6). Thesefindings demonstrate that the lethal phenotype induced by(yL)-hTop1 was not a consequence of alterations in enzymecatalysis.

Because the appropriate pairing of the yeast N-terminaldomain with the yeast linker partially suppressed the (yL)-hTop1 lethal phenotype (Fig. 4B), we then asked whetherN-terminal/linker domain interactions were required for (yL)-hTop1-induced toxicity. Here, we simply deleted the first 174amino acid residues (upstream of the chimera junction athuman residue 192) to generate (yL)-hTopo70. The hTop1nuclear localization signal (KPKKIKTED) (44) was also insertedafter the N-terminal FLAG epitope to ensure nuclear localiza-tion of all hTopo70 proteins. As shown in Fig. 7 and Table 3,removal of the N-terminal 1–174 residues completely sup-pressed the toxicity of (yL)-hTop1. Indeed, yeast cells express-ing (yL)-hTopo70 grew equally well as cells expressinghTopo70 in the absence of CPT and exhibited the same sensi-tivity to low concentrations of CPT as wild-type hTop1- andhTopo70 expressing cells (Fig. 7A and Table 3). In cell lysates,the levels of (yL)-hTop1 protein were comparable with hTop1,whereas hTopo70 and (yL)-hTopo70 levels were �2-foldhigher (Fig. 7B). As in Fig. 4B, (yL)-hTop1 was slightly moreactive than hTop1. However, the specific activities of hTop1

FIGURE 5. The core and C-terminal domains of Top1 dictate CPT sensitiv-ity and sequence specificity. Equal concentrations of the purified Top1 pro-teins (described in Fig. 4A) were incubated with a single 3� 32P-end-labeledDNA substrate in a DNA cleavage reaction containing 0, 0.5, 2, 10, or 50 �M

CPT in a final 0.125% DMSO. After incubation for 10 min at 30 °C, the covalentTop1-DNA complexes were trapped with SDS at 75 °C and treated with pro-teinase K. The reaction products were resolved in 8% polyacrylamide, 7 M ureagels and visualized using a PhosphorImager. Lane C is DNA alone, and theasterisk indicates a high affinity Top1 cleavage site. Arrowheads indicatecleavage products that differ between yTop1 and hTop1. (n 3 experiments).




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and (yL)-hTop1 were slightly lower than that observed withhTopo70 and to a lesser extent with (yL)-hTopo70 (Fig. 7C).

As with the full-length enzymes shown in Fig. 5, the intrinsicCPT sensitivity of hTopo70 and (yL)-hTopo70 also mirroredthat of full-length hTop1, in DNA cleavage assays performedwith equal concentrations of the purified proteins (Fig. 8). CPT-induced stabilization of covalent Top1-DNA intermediates wasobserved at low drug concentrations, with the same alterationsin sequence specificity as hTop1 (as indicated by the arrow-heads in Fig. 8). Taken together, these data indicate that the

toxic phenotype of (yL)-hTop1 is not solely a consequenceof the presence of the yeast linker domain but requires a func-tional interaction with the first 174 residues of the hTop1 Nterminus.

Reconstituted Human/Yeast Chimeras—Champoux and co-workers (23) demonstrated that an active enzyme could bereconstituted from separate polypeptides comprising the coreand linker-C-terminal domains of hTop1. However, the intrin-sic CPT resistance of such reconstituted enzymes argued that aphysical tethering of the core and linker domains was requiredfor drug sensitivity. Consequently, we wondered whether aphysical connection between the core and linker domain wasalso required for the (yL)-hTop1 chimera lethal phenotype. Alibrary of plasmids was engineered to coordinately expressTop1 fragments from a galactose-inducible, bicistronicGAL1–10 promoter (Fig. 9A and Table 2). The inclusion ofdistinct Flag and myc tags eased detection and immunoprecipi-tation of the expressed polypeptides. The constructs used toreconstitute hTopo76/14 or (yL)-hTopo76/14 were based onthe same linker domain junction used to generate the chimericenzymes (see Tables 1 and 2), although the hTopo78/12 con-struct recapitulated the polypeptide sequences reported by theChampoux laboratory. The corresponding hTopo56/14 andhTopo58/12 reconstituted enzymes each lacked the N-termi-nal 174 residues of Topo70.

As shown in Fig. 9B, cells co-expressing hTopo76/14 (thehTop1 N terminus � core and linker � C terminus) or

FIGURE 6. Catalytically inactive hTop1 containing a yeast linker is toxic toyeast cells. Exponentially growing cultures of top1� cells, transformed withthe indicated YCpGAL1-TOP1 constructs, were adjusted to an optical den-sity 0.3, serially 10-fold diluted, and aliquots were spotted onto SC-ura agarcontaining 25 mM Hepes (pH 7.2), 0.125% DMSO, and either dextrose (Dex),galactose (Gal), or galactose plus 0.5 �M CPT. Viability was assessed followingincubation at 30 °C (n 3 experiments).

FIGURE 7. The presence of the hTop1 N-terminal domain is essential forthe lethal phenotype seen with the (yL)-hTop1chimera. A, the hTop1 andhTopo70 constructs were transformed into exponentially growing top1�cells. Liquid cultures of the transformed cells were diluted to an optical den-sity of 0.3 and serially 10-fold diluted onto the indicated plates, as in Fig. 6.Viability was assessed after a 4-day incubation at 30 °C. B, as in Fig. 3D, extractsof galactose-induced top1� cells, expressing the indicated FLAG-taggedTop1 proteins, were subjected to immunoblot analysis with an anti-FLAGantibody, with GAPDH as a loading control. Images are of different lanes fromthe same gel. C, equal concentrations of yeast cell lysates (prepared as in B)were serially 10-fold diluted and incubated in a plasmid DNA relaxation assay.The reaction products were resolved by agarose gel electrophoresis and visu-alized with ethidium bromide. The relative positions of relaxed (R) and super-coiled (�) DNA topoisomers are indicated. Lane C indicates DNA alone (n � 3experiments).

FIGURE 8. The N-terminal domain of hTop1 does not impact enzyme sen-sitivity to CPT in vitro. As in Fig. 5, equal concentrations of partially purifiedhTop1, hTopo70, and (yL)-hTopo70 proteins were assessed for sensitivity to0.5 and 5 �M CPT. The reaction products were resolved in 8% polyacrylamide,7 M urea gels and visualized using a PhosphorImager. Lane C is DNA alone, andthe asterisk indicates a high affinity Top1 cleavage site. As in Fig. 5, arrow-heads indicate cleavage products specific for hTop1, not yTop1.




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hTopo56/14 (hTop1 core and linker � C terminus) were resis-tant to CPT. However, the catalytic activity of the reconsti-tuted enzymes was detectable in cell extracts, albeit onlyunder optimized reaction conditions of low salt and tempera-ture (Fig. 9C). Similar results were obtained with co-expressionof hTopo78/12 and hTopo58/12 (data not shown). The associ-ation of the hTopo76 and 14 polypeptides to form a reconsti-tuted enzyme was also evident in yeast. As shown in Fig. 9D,

myc-tagged hTopo14 was detectable in immunoprecipitates ofFLAG-tagged hTopo76 and vice versa. Thus, consistent withthe in vitro results of Champoux and co-workers (23), the phys-ical linkage of the core and linker domains also appears to berequired for CPT sensitivity in yeast and may reflect the lowenzyme activity of the reconstituted enzyme in cells.

Similar results were obtained with reconstituted (yL)-hTopo76/14. As shown in Fig. 9B, the lethal phenotype of cellsexpressing the intact (yL)-hTop1 chimera was not induced byco-expression of (yL)-hTopo76/14. However, in contrast to theCPT resistant phenotype of hTopo76/14, co-expression of(yL)-hTopo76/14 did confer modest sensitivity to CPT (Fig.9B). These results appear to be due to the presence of the longeryeast linker rather than any detectable alterations in enzymecatalysis. The specific activity of (yL)-hTopo76/14 was compa-rable with that of hTopo76/14 (Fig. 9C), as was the associationof the (yL)-hTopo76/14 fragments in co-immunoprecipitationexperiments (Fig. 9D). These data suggest that despite the phys-ical disconnect between the core and linker domain, the yeastlinker (in the context of full-length hTop1) has a modest impacton Top1 sensitivity to CPT.

Taken together, these findings support a model whereby theintrinsic CPT sensitivity of Top1 is dictated by the compositionof the core and C terminus, whereas the functional interactionof hTop1 with other proteins/protein complexes in yeast isaffected by linker composition. In the context of hTop1, theability of the yeast N-terminal domain to suppress the growthdefect induced by the yeast linker further implies the coordi-nated function of these poorly conserved domains in regulatinghTop1 interactions with other as yet unidentified proteins orprotein complexes.

DISCUSSION

The formation of clamp structures about a DNA strand orduplex is a common feature of proteins that function in DNAreplication, repair, and transcription. Examples include theDNA polymerase processivity factor proliferating cell nuclearantigen, the replicative Mcm2–7 helicase, DNA polymerases,the 9-1-1 checkpoint complex, and DNA topoisomerases (45–50). Eukaryal nuclear Top1 possesses some unique architec-tural features. First, the protein clamp, formed by the highlyconserved core domain of this monomeric enzyme, tightlyencircles duplex DNA (7, 13, 18). Second, Top1 comprises ahighly asymmetric structure. An extended coiled-coil of thelinker domain connects the core with the conserved C-terminaldomain, which contains the active site Tyr723. The positioningof residue Tyr723 within the protein clamp completes the cata-lytic pocket of the enzyme. This nuclear enzyme also contains ahighly charged N-terminal domain, for which little structuralinformation is available but has been implicated in regulatingthe intracellular localization of Top1, Top1-protein interac-tions, and the catalytic activity of Top1 (32, 42, 51).

The movement and functional interactions of Top1 proteindomains during enzyme catalysis and how the structural fea-tures of this enzyme impact drug sensitivity have been of con-siderable interest. One strategy used to address these questionsis the use of molecular modeling to design reversible disulfidebonds that lock the hTop1 clamp around duplex DNA (7, 13,

FIGURE 9. The physical connection between the core and linker domain isrequired for chimera-induced toxicity. A, the bicistronic YCpGAL1–10 vec-tor was used to express the Top1 N-terminal and core domains from theGAL10 promoter and the linker and C terminus from the GAL1 promoter(Table 2). B, exponential cultures of top1� cells transformed with the indi-cated constructs were serially 10-fold diluted, spotted onto the indicatedplates, and incubated at 30 °C. C, equal concentrations of lysates of galactose-induced yeast cells expressing the indicated Top1 constructs were 10-foldserially diluted and incubated in a plasmid DNA relaxation assay with a final50 mM KCl for 30 min at 30 °C. The reaction products were resolved by agarosegel electrophoresis and visualized with GelRed. The relative positions ofrelaxed (R) and supercoiled (�) DNA topoisomers are indicated. Lane C indi-cates DNA alone. D, lysates from C were incubated with precleared proteinA/G beads and either anti-FLAG or anti-myc antibody overnight. Input lysatesand the bead-bound immunoprecipitates (� FLAG IP, � myc IP) were resolvedby SDS-PAGE and visualized by immunoblotting with the indicated antibod-ies. Immunostaining of tubulin served as a loading control for lysate samples.The data are representative of n 4 experiments.




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18 –20, 52). Placing the disulfide “lock” in close proximity to theactive site Tyr723 prevented DNA strand rotation within thecovalent enzyme-DNA intermediate but did not affect CPT sta-bilization of the Top1-DNA complex (19). Parallel studies withyTop1 demonstrated a conservation of clamp architecture anda role for the N-terminal domain in Top1 clamp binding ofduplex DNA (20).

Structural and biochemical studies also implicate the flexi-bility of the linker domain as a determinant of enzyme sensitiv-ity to CPT. Increasing the movement of the linker, either byphysically uncoupling the Top1 linker and core domains inreconstitution experiments (Fig. 8 and Ref. 15) or by mutationof Ala653 to Pro in hTop1 (26), decreases enzyme sensitivity toCPT. In contrast, a Top1D677G/V703I mutant has reducedlinker flexibility and is hypersensitive to CPT (29). In addition,replacing Gly721 with residues estimated to have a higher pro-pensity for � helix formation (Ala or Asp) induced alterations inyeast Top1 active site architecture that increased enzyme sen-sitivity to CPT (28). In co-crystal structures, the extension of ashort � helix within the active site of Topo70 to include Gly717

coincides with drug binding and a restriction of linker orienta-tion (7). Thus, we posited that this conserved Gly acts as aflexible hinge that allows for linker domain movement and thatsubstitutions of Gly721, which reduces linker flexibility, en-hance enzyme sensitivity to CPT.

A third strategy, taken here, was the design of yeast/humanchimeric enzymes to define the contribution of individual pro-tein domains to Top1 sensitivity to CPT and enzyme functionin vivo. Guided by crystallographic data, we previously usedhomologous recombination in yeast to design yeast/humanchimeras of the SUMO (small ubiquitin-like modifier)-conju-gating enzyme Ubc9 and in preliminary studies of Top1 (40).The biochemical activity of a hTop1/Plasmodium falciparumhybrid enzyme has also been described (53). Herein, our analy-ses of yeast/human Top1 chimera activity in vitro and in yeastcells establish that intrinsic enzyme sensitivity to CPT is dic-tated by the composition of the core and C-terminal domains.Independent of the origin of the N-terminal and/or linkerdomains (yeast or human), enzymes consisting of the humancore and C-terminal domains exhibited comparable specificenzyme activity and were �100-fold more sensitive to CPTthan yTop1 in vitro (Figs. 4, 5, 7, and 8 and Table 3).

However, additional aspects of chimera enzyme activity intop1� yeast cells are worth noting. First, in reciprocal swaps ofthe highly charged yeast and human N termini (at a juncturecorresponding to human residue 192/yeast residue 120), noalterations in cell viability or sensitivity to CPT were evident.Thus, any Top1-protein interactions mediated solely by theN-terminal domain failed to influence CPT-induced toxicity.Second, hTop1 residues 201–206 are required for enzymecatalysis (32). However, fusing hTop1 residues 1–209 with thecorresponding C-terminal residues of yTop1 also failed to alterthe CPT sensitivity of cells expressing these chimeras, relativeto cells expressing wild-type hTop1 (Fig. 3 and data not shown).These data suggest that the critical function of these humanresidues (KWKWWEEE) in enzyme catalysis is retained in thecorresponding residues of yeast Top1 (EYKWWEKE).

In contrast, the introduction of the longer yeast linkerdomain in hTop1 profoundly altered enzyme function in vivo.Expression of (yL)-hTop1 proved toxic even in the absence ofCPT, with no obvious alterations in enzyme catalysis (Fig. 4).This result was particularly surprising because Top1 is notrequired for yeast cell growth, and the linker domain is dispens-able for enzyme activity in vitro (23, 54). Indeed, (yL)-hTop1Y723F-induced toxicity was independent of DNA cleav-age (Fig. 6), which implicates toxic alterations in Top1-proteininteractions, instead of enzyme catalysis. However, becauseeither removal of the human N terminus [in (yL)-hTopo70] orthe introduction of the complementary yeast N-terminaldomain [in (yN,yL)-hTop1] suppressed chimera-inducedlethality, toxicity is not simply due to the presence of the longeryeast linker within the hTop1 protein. Rather, lethality appearsto derive from functional interaction between the extendedhuman N-terminal and yeast linker domains. Whether theseinteractions involve the direct physical association of thesedomains or indirect interactions mediated by DNA/other pro-teins has yet to be determined. In this context, it is noteworthythat the N-terminal domain, which has defied structural deter-mination, is absent from the human mtTop1 enzyme that istargeted to the mitochondria (55). Also, because both yeast andhuman N-terminal domains contain nuclear localization sig-nals (42), it is unlikely that the observed toxicity relates tochanges in nuclear import. Thus, the functional interaction ofN-terminal and linker domains appears to be restricted tonuclear protein-Top1 associations.

Expression of catalytically inactive yeast or human Top1clamps, locked in the closed conformation by the formation of adisulfide bond, was also toxic to yeast (20), presumably becauseof Top1 clamping of the DNA. Although the biochemicalbehavior of the (yL)-hTop1 did not suggest alterations in DNAbinding, i.e. there was not a shift in salt optimum or enzymeprocessivity in DNA relaxation assays using yeast cell lysates(Fig. 4C), we cannot exclude enhanced DNA binding in thecytotoxic mechanism of the chimera protein in yeast cells.However, our fragment complementation studies (Fig. 9B)demonstrated that the integrity of (yL)-hTop1 was required forcell lethality, because the reconstituted (yL)-Topo76/14 wasnot toxic when expressed in yeast cells. Although the lowenzyme activity of these reconstituted enzymes may well reflectdecreased DNA binding in cells, these data nevertheless dem-onstrate that the physical linkage of the core and linker domainsappears to be essential for maintaining the toxic protein and/orDNA interactions induced by (yL)-hTop1 expression. In con-trast, when the shorter human linker was inserted into the yeastenzyme (in (hL)-yTop1) and expressed in yeast, the cells wereviable and relatively resistant to CPT. In this case, our findingsare consistent with a defect in DNA binding, suggested by ear-lier studies of related yTop1 clamp constructs (20).

Together, these studies support a model whereby the intrin-sic CPT sensitivity of Top1 is dictated by the composition of theconserved core and C terminus. In contrast, the yeast celllethality induced by expression of (yL)-hTop1 chimera and theability of the yeast N-terminal domain to suppress this growthdefect imply the coordinated interplay between these poorlyconserved domains in regulating hTop1 interactions with DNA




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or other as yet unidentified proteins or protein complexes invivo. Further studies to elucidate the physiological context ofsuch protein domain and protein-protein associations, partic-ularly in the context of ongoing DNA replication, will undoubt-edly provide critical new insights into the cellular processes thatdictate the cytotoxic activity of chemotherapeutics that targetTop1.

Acknowledgments—We thank Francesco Giorgianni, Ana Otero, andAndrew Larkin for early efforts that led to the generation of these Top1chimeras and members of the Bjornsti lab, past and present, for help-ful discussion.

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Christine M. Wright, Marié van der Merwe, Amanda H. DeBrot and Mary-Ann BjornstiSensitivity to Camptothecin

DNA Topoisomerase I Domain Interactions Impact Enzyme Activity and

doi: 10.1074/jbc.M114.635078 originally published online March 20, 20152015, 290:12068-12078.J. Biol. Chem.

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