DNA topoisomerase I domain interactions impact enzyme activity and sensitivity to camptothecin.

JBC Papers in Press. Published on March 20, 2015 as Manuscript M114.635078 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M114.635078 Activity of Human / yeast DNA topoisomerase I chimeras DNA TOPOISOMERASE I DOMAIN INTERACTIONS IMPACT ENZYME ACTIVITY AND SENSITIVITY TO CAMPTOTHECIN* JBC/2014/635078

Christine M. Wright1,4, Marié van der Merwe2,3,4, Amanda H. DeBrot1 and Mary-Ann Bjornsti1 1

From the Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35294 and 2 Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, TN 38105 3 Current address: Department of Health and Sport Sciences, University of Memphis, Memphis, TN, 38152. Running Title: Activity of human / yeast DNA topoisomerase I chimeras Address correspondence to: Mary-Ann Bjornsti, Department of Pharmacology and Toxicology, University of Alabama at Birmingham, 1670 University Blvd, Birmingham, AL 35294 Tel: (205) 9344579 Fax: (205) 934-8240; E-mail: [email protected]

Background: Despite similarities in mechanism and architecture, human DNA topoisomerase I (Top1) is ~100-fold more sensitive 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 enzyme catalysis. SUMMARY During processes such as DNA replication and transcription, DNA topoisomerase I (Top1) catalyzes the relaxation of DNA supercoils. The nuclear enzyme is also the cellular target of camptothecin (CPT) chemotherapeutics. Top1 contains four domains: the highly conserved core and C-terminal domains involved in catalysis, a coiled-coil linker domain of variable length, and a poorly conserved N-terminal domain. Yeast and human Top1 share a common reaction mechanism and domain structure. Yet, the human Top1 is ~100-fold more sensitive to CPT. Moreover, substitutions of a conserved Gly717 residue, which alter intrinsic enzyme sensitivity to CPT, induce distinct phenotypes in yeast. To address the structural basis for these differences, reciprocal

swaps of yeast and human Top1 domains were engineered in chimeric enzymes. Here we report that intrinsic Top1 sensitivity to CPT is dictated by the composition of the conserved core and C-terminal domains. However, independent of CPT, biochemically similar chimeric enzymes produced strikingly distinct phenotypes in yeast. Expression of a human Top1 chimera containing the yeast linker domain proved toxic, even in the context of a catalytically inactive Y723F enzyme. Lethality was suppressed either by splicing the yeast Nterminal domain into the chimera, deleting the human N-terminal residues or in enzymes reconstituted by polypeptide complementation. These data demonstrate a functional interaction between the N-terminal and linker domains, which, when mispaired between yeast and human enzymes, induces cell lethality. As toxicity was independent of enzyme catalysis, the inappropriate coordination of N-terminal and linker domains may induce aberrant Top1:protein interactions to impair cell growth. Eukaryal DNA topoisomerase I (Top1) catalyzes the relaxation 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 monomeric enzyme that forms an asymmetric protein clamp, which encircles double-stranded DNA. During the reaction cycle, the active site tyrosine (Tyr723 in human Top1) attacks a single

1

Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Downloaded from http://www.jbc.org/ by guest on April 7, 2015

Keywords: DNA topoisomerase, protein domain, protein chimera, Sacccharomyces cerevisiae, camptothecin

Activity of Human / yeast DNA topoisomerase I chimeras

2

linker domain (21,22). In addition, fragment complementation experiments demonstrate that polypeptides comprising only the human Top1 core and C-terminal domains can reconstitute an active enzyme in vitro (23), albeit with reduced specific activity. In the co-crystal structure of the covalent Topo70-DNA intermediate, the linker is not resolved (7). However, when the Topo70-DNA intermediate is bound by CPT or the analog topotecan, the linker becomes extended at an oblique angle to the DNA helix, as in the noncovalent structure in Fig. 1 (7,15-17). Although the role of the linker domain in mediating enzyme sensitivity to CPT is not clear, the “linker-less” poxvirus Top1 enzymes are resistant to CPT (24). Moreover, the in vitro CPT resistance of the two subunit Leishmania Top1, and reconstituted human Top1, demonstrates that the physical linkage of the linker to the core domain is necessary for CPT sensitivity (23,25). Studies of mutant yeast and human enzymes further suggest a correlation of linker flexibility with CPT resistance (7,26-30). The function of the N-terminal domain also remains poorly understood, in part due to a lack of structural information. In vitro, residues 1-190 of the nuclear enzyme are dispensable for enzyme activity and CPT sensitivity. However, amino acids 191-206 are required for DNA binding and enzyme processivity (31-34). The N-terminus has also been implicated in numerous interactions with other proteins, including TATA Binding Protein, nucleolin, and SV40 large T antigen (35-38). Additional studies suggest a role for the Nterminal domain in the opening and closing of the Top1 clamp around DNA (20) and in CPTinduced alterations in the intracellular localization of Top1 (39). Despite the considerable conservation of yeast (yTop1) and human (hTop1) enzyme mechanism, structure and function, there are notable differences. Importantly, wild-type yTop1 and hTop1 exhibit an ~ 100-fold difference in intrinsic sensitivity to CPT in vitro. Moreover, mutant-induced alterations in enzyme sensitivity to CPT are not always manifested in cells. For instance, substitution of Asp for a conserved Gly residue, which is posited to form a flexible hinge with the linker domain, just N-terminal to the active site Tyr in yeast and human Top1, renders these mutant enzymes intrinsically more sensitive to CPT in vitro. Yet, expression of yTop1G721D in yeast enhanced cell sensitivity to CPT, while cells


strand of duplex DNA to form a 3’ phosphotyrosyl intermediate. Within this covalent Top1-DNA complex, DNA strand rotation allows for the relaxation of DNA supercoils. The 5’ OH of the cleaved DNA strand then acts in a second transesterification to resolve the phosphotyrosyl bond and religate the DNA. Top1 is the cellular target of the camptothecin (CPT) class of chemotherapeutics (5-7). These drugs reversibly stabilize the covalent Top1-DNA intermediate by intercalating into the protein-linked DNA nick. During S-phase, the interaction of the replication machinery with the ternary CPT-Top1-DNA complex itself, or with the positive supercoils that accumulate ahead of the fork, create irreversible DNA lesions that induce cell death (5-8). Increased concentrations of the covalent intermediate can also be induced with Top1 mutants, where altered rates of DNA cleavage / religation effectively convert the enzyme into a cellular poison (9-12). Biochemical and X-ray crystallographic data reveal an unusual architecture of monomeric Top1. The human nuclear enzyme consists of four distinct domains: a charged N-terminus, a conserved core domain, a linker domain formed by an extended pair of alpha-helices (636-712), and a conserved C-terminal domain that contains the active site Tyr (1,7,13-17). Fig. 1A depicts the cocrystal structure of human Topo70, 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 Top1 share only 17% and 12% amino acid identity, respectively, while the conserved core and C-terminal domains are 58% and 62% identical, respectively (43).The conserved core domain of Top1 forms a protein clamp around duplex DNA. We previously demonstrated that locking the human Top1 clamp around the DNA prevents strand rotation within the Top-DNA intermediate (19). Similar impediments to DNA rotation were observed with the analogous studies of yeast Top1 (20), suggesting a conservation of protein clamp enzyme architecture and catalytic mechanism. The core domain of Top1 provides most of the catalytic residues. However, Tyr723 is in the conserved C-terminal domain, and is positioned within the catalytic pocket of the core domain by the extended coiled-coil linker domain. Yet, the linker domain does not appear to be required for activity. Top1 enzymes found in some bacteria and the poxviruses lack both the N-terminus and

Activity of Human / yeast DNA topoisomerase I chimeras 717

EXPERIMENTAL PROCEDURES Yeast strains and plasmids. Saccharomyces cerevisiae strain EKY3 (MATα, ura3-52, his3Δ200, leu2Δ1, trp1Δ63, top1::TRP1) was previously described (11). Herein, “h” and “y” designations distinguish human and yeast TOP1, respectively. Plasmids YCpGAL1-yTOP1 and YCpGAL1-hTOP1 express wild-type yeast TOP1 and human TOP1 cDNA from the galactoseinducible GAL1 promoter in an ARS/CEN, URA3 vector (11,19), while YCpGAL1-hTopo70 expresses an N-terminal truncated human Top1 (residues 175-765), referred to as Topo70. All of the constructs used in these studies contained an N-terminal Flag epitope (DYKDDD) recognized by the M2 monoclonal antibody (Sigma). Yeasthuman chimeras were generated using homologous recombination of PCR-generated chimeric junctions as described in (40) and diagrammed in Fig. 2A. See Table 1 and Fig. 2B for primer design and chimera junctions. Plasmids were recovered from individual yeast colonies as in (40), amplified in E. coli and sequenced to confirm yTop and hTop sequences. Vector pESC-ura (Stratagene) contains the bicistronic, galacatose-inducible GAL1-10 promoter cassette with FLAG and myc tags, and terminator sequences in a 2µm, URA3 vector. The entire GAL1-10 cassette flanking polylinker 3

sequences were PCR amplified and used to replace the polylinker sequences of pRS416, by homologous recombination, as in (40), to create YCpGAL1-10. Reconstituted Top1 protein expression vectors were generated as described in Table 2. As an example, the linker and C-terminus of hTop1 were PCR amplified from YCpGAL1hTOP1 and inserted 3’ to the myc epitope (EQKLISEEDL) sequences under the GAL1 promoter in YCpGAL1-10 to create YCpGAL1-10 hTopo14. The hTop1 N-terminus and core domain sequences were then PCR amplified from YCpGAL1-hTOP1 and inserted 3’ to the FLAG epitope sequences of the GAL10 promoter to create YCpGAL1-10 hTopo76/14. Bicistronic constructs were also created without the myc tag. In all cases, mutant sequences were confirmed by DNA sequencing. Primer sequences are available upon request. Yeast cell sensitivity to CPT. To assay yeast cell sensitivity to CPT, cultures of EKY3 (top1Δ) yeast cells, transformed with the indicated YCpGAL1-TOP1 vector, were serially 10-fold diluted. Aliquots (4 µL) were spotted onto synthetic complete agar lacking uracil (SC-ura) media containing 25 mM Hepes (pH 7.2), 2% dextrose or galactose, and the indicated concentration of CPT in a final 0.125% DMSO. Cell growth was assessed after incubation at 30˚C. DNA topoisomerase I expression and purification. Galactose-induced cultures of yeast top1Δ 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 complete protease inhibitors (Roche). Lysates were normalized for total protein and Top1 protein levels were verified by immunoblotting and chemiluminescence using the M2-FLAG antibody (Sigma). Wild type and chimeric Top1 were partially purified by phosphocellulose chromatography or purified to homogeneity by phosphocellulose and FLAG affinity chromatography, as described (12,28). Top1 protein fractions were adjusted to a final concentration of 30% glycerol and stored at -20˚C. Protein integrity was assayed by immunoblot analysis (28,41). Plasmid relaxation assays. The specific activity of the Top1 preparations was measured in plasmid DNA relaxation reactions as described (9,41). Briefly, serial 10-fold dilutions of purified Top1 protein or cell lysates (corrected for protein


expressing the corresponding hTop1G D mutant were no more sensitive to CPT than cells expressing wild-type hTop1 5(28). Such results prompted us to ask if differences in intrinsic CPT sensitivity might be attributed to the domain composition of Top1, and if the different phenotypes induced by mutations of conserved residues within the catalytic pocket of yTop1 and hTop1 were due to distinct architectural features of these enzymes. To address these questions, crystallographic and biochemical data were used to design a series of chimeric enzymes that comprise reciprocal swaps of the yeast and human N-terminal and linker domains. Here, we report that the composition of the conserved core and C-terminal domains dictate intrinsic Top1 enzyme sensitivity to CPT; while the functional interaction of the N-terminus with the linker domain impacts cell viability, independent of CPT and enzyme catalysis. Thus, intramolecular interactions between poorly conserved protein domains dictate the critical conserved function of Top1.


RESULTS N-terminal human/yeast Top1 chimeras. To address the function of Top1 protein domains in regulating enzyme activity and sensitivity to CPT in cells, homologous recombination [as described (40) and diagrammed in Fig. 2A] was used to generate reciprocal swaps of the N-terminal and linker domains of yTop1 and hTop1. The poorly conserved N-terminal domains have been shown to contain nuclear localization signals (42), and to mediate Top1 interactions with other proteins (3537). Hence, N-terminal domain chimeras were first engineered to ask if yeast protein interactions with the N-terminal domains of yTop1 or hTop1 could account for the observed alterations in enzyme activity in cells. hTopo70 (residues 175-765) has 4

been extensively used for biochemical and structural studies of hTop1. However, available structural information is restricted to the core, linker and C-terminal domains (residues 215-765) (see Fig. 1A and B). Within the N-terminal domain, residues 1-190 are dispensable for hTop1 catalysis, while residues 191-206 are required for efficient DNA binding (32). Based on sequence homology and codon usage, we engineered two sets of reciprocal swaps that spanned these critical residues (summarized in Fig. 2, and Table 1). In h120yTop1, human N-terminal residues 1-191 were fused to yeast residues 120-769, while y192hTop1 contains yeast N-terminal residues 1119 fused to human residues 192-765 (Figs. 2B and 3A). For ease of discussion, hereafter these chimeras are referred to as (hN)-yTop1 and (yN)hTop1, respectively. As shown in Fig. 3B and Table 3, exchanging these portions of the Nterminal domains had no effect on the pattern of in vivo CPT sensitivity in yeast. CPT-induced toxicity of cells expressing (hN)-yTop1 mirrored that of wild-type yTop1, and was only observed at high drug concentrations. Conversely, cells expressing either wild-type hTop1 or (yN)-hTop1 were sensitive to 100-fold lower concentrations of drug (compare cell viability at 5 vs. 0.05 µg/ml in Fig. 3B and in Table 3). The levels of chimera protein expressed, and the specific activity of the purified N-terminal chimeric proteins were similar to that of the wild-type enzymes (Fig. 3C,D). Similar results were obtained with additional constructs: (1) a second set of N-terminal chimeras (y210hTOP1 and h138yTOP1) that contained junctions at human residue 210 or yeast residue 138, respectively, just N-terminal to the core domain, and (2) a yeast/human chimera y∆201hTOP1, where deletion of 8 residues was engineered in the yeast sequences just upstream of the human 201 chimera junction (Fig. 2B, Table 1, Fig. 3D, and data not shown). In all cases, the Nterminal composition of the chimeras had no effect on enzyme catalysis in vitro or on yeast cell sensitivity to CPT (Fig. 3 and data not shown). Thus, it is unlikely that yeast protein interactions with N-terminal Top1 domain residues alone dictate enzyme sensitivity to CPT in yeast cells. Introducing the yeast linker domain into hTop1 induces yeast cell lethality. The other nonconserved domain of Top1 is the coiled-coil linker, whose flexibility has been implicated in altering enzyme sensitivity to CPT (7,26-28). We therefore engineered reciprocal exchanges of the linker domains. To accomplish this, the linker and


concentration) were incubated in 20 µl reaction volumes with 0.3 µg of negatively supercoiled pHC624 plasmid DNA in 20 mM Tris (pH 7.5), 10 mM MgCl2, 0.1 mM EDTA, 50 µg/ml gelatin and 50-200 mM KCl (as indicated), at the temperatures indicated, for 30 minutes. DNA products were extracted with phenol/chloroform, resolved by agarose gel electrophoresis, and visualized following staining with ethidium bromide or GelRed (Phenix). DNA cleavage assays. Intrinsic enzyme sensitivity to CPT was assessed in DNA cleavage assays, as previously described (12), using a 480 base-pair DNA fragment that contained a high affinity Top1 cleavage site and was 32P-endlabeled at a single 3’ end. The cleavage reaction products were treated with SDS at 75˚C, digested with proteinase K, ethanol precipitated, resolved in denaturing 8% polyacrylamide/7 M urea gels and visualized by PhosphorImage analysis. Co-immunoprecipitations. Lysates of galactose-induced cultures of EKY3 cells expressing the indicated Top1 reconstitution vectors, prepared as above, were split into two 400 µL aliquots and Triton X-100 was added to a final concentration of 1%. 20 µL Protein A/G PLUS Agarose (Santa Cruz) was added and the samples were rotated end-over-end for several hours at 4°C. Following centrifugation, the supernatant was added to 30 µL Protein A/G PLUS Agarose and 5 µg of either anti-FLAG or anti-myc antibody and rotated end-over-end overnight at 4°C. The samples were washed five times with 1xTEEG containing 200 mM KCl, 1% Triton, and protease inhibitors. The proteins were resolved by SDSPAGE and visualized by immunoblotting.


5

4C). Thus, (yL)-hTop1-induced toxicity could not be attributed to gross alterations in enzyme catalysis. Second, comparable levels of hTop1, (yL)-hTop1 and (yN,yL)-hTop1 proteins were expressed in cells (Fig. 7B, and data not shown). Moreover, in DNA cleavage assays performed with equal concentrations of purified yTop, hTop1 and the (yL)-hTop1 and (yN,yL)-hTop1 chimeras, the intrinsic CPT sensitivity of the chimeras were indistinguishable from that of intact hTop1. As seen in Fig. 5, at least 100-fold higher concentrations of CPT were required to induce similar levels of covalent yTop1-DNA intermediates as that observed for wild-type hTop1. Thus, independent of N-terminal/linker domain composition, the intrinsic CPT sensitivity of the (yL)-hTop1 and (yN,yL)-hTop1 chimeras mirrored that of wild-type hTop1. Moreover, subtle differences in sequence specificity between the yeast and human enzymes, (as indicated by the arrow heads in Fig. 5), were also retained by the hTop1 chimeras. Thus, the N-terminal and linker domain composition of hTop1 does not impact the intrinsic pattern of enzyme sensitivity to CPT. Since the toxic phenotype of (yL)-hTop1 expressing cells could not be attributed to any obvious alterations in enzyme binding to DNA, sequence specificity, or shift in cleavage-religation in the absence of CPT, we next investigated the role of DNA cleavage in (yL)-hTop1 toxicity. The active site Tyr723 is essential for enzyme catalyzed DNA cleavage and formation of the covalent phosphotyrosyl intermediate, but not DNA binding by the Top1 protein (1-3,16). In (yL)-hTop1Y723F, the active site Tyr723 was mutated to Phe to produce a catalytically inactive 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). These findings demonstrate that the lethal phenotype induced by (yL)-hTop1 was not a consequence of alterations in enzyme catalysis. Since the appropriate pairing of the yeast Nterminal domain with the yeast linker partially suppressed the (yL)-hTop1 lethal phenotype (Fig. 4B), we then asked if N-terminal/ linker domain interactions were required for (yL)-hTop1-induced toxicity. Here, we simply deleted the first 174 amino acid residues (upstream of the chimera junction at human residue 192) to generate (yL)hTopo70. The hTop1 NLS (KPKKIKTED) (44) was also inserted after the N-terminal FLAG epitope to ensure nuclear localization of all


C-terminus of each enzyme was first replaced with the corresponding residues of the other. These chimeras were then used in reciprocal swaps to reintroduce 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 conserved residues found at the N- and C-terminal ends of the coiled-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, and yTop1 residues 561 – 716 (see Table 1). Introducing the shorter hTop1 linker into yeast Top1, in (hL)-yTop1, induced a >10-fold reduction in specific enzyme activity in vitro, and induced a CPT resistant phenotype in yeast (data not shown). In contrast, replacing the shorter hTop1 linker with the longer yeast linker, in (yL)hTop1, induced a surprising phenotype in yeast. As shown in Fig. 4B and Table 3, galactoseinduced expression of (yL)-hTop1 inhibited cell growth, even in the absence of CPT. Thus, the presence of the longer yeast linker in the human enzyme is toxic to yeast. However, when the corresponding yeast N-terminal domain was introduced into this chimera [to generate (yN,yL)hTop1], such that the conserved core and Cterminal domains were hTop1, while the divergent N-terminal and linker domains were of yeast Top1 origin, this toxic phenotype was largely suppressed (in Fig. 4B and Table 3). Moreover, CPT-induced lethality of cells expressing the (yN,yL)-hTop1 chimera was evident at concentrations sufficient to sensitize cells expressing wild-type hTop1, but not yTop1 (Fig. 4B and Table 3). Similar results were obtained with other N-terminal chimera junctions (Table 1 and data not shown). These finding suggest that the CPT sensitivity of Top1 is dictated by the composition of the conserved core and C-terminal domains, while alterations in N-terminal – linker domain interactions adversely impact enzyme function in cells. This model was further supported by assays of chimeric enzyme catalysis. First, in cell lysates corrected for total protein, the specific activity of the (yN,yL)-hTop1 chimera was similar to that of wild-type hTop1 over a range of salt concentrations. The (yL)-hTop1 chimera exhibited a similar pattern of optimal activity at 150-200 mM KCl, yet was slightly more active (~5-fold) than comparable levels of wild-type hTop1 (Fig.


6

Champoux lab. The corresponding hTopo56/14 and hTopo58/12 reconstituted enzymes each lacked the N-terminal 174 residues of Topo70. As shown in Fig. 9B, cells co-expressing hTopo76/14 (the hTop1 N-terminus+core and linker+C-teminus) or hTopo56/14 (hTop1 core and linker+C-terminus) were resistant to CPT. Yet, the catalytic activity of the reconstituted enzymes was detectable in cell extracts, albeit only under optimized reaction conditions of low salt and temperature (Fig. 9C). Similar results were obtained with co-expression of hTopo78/12 and hTopo58/12 (data not shown). The association of the hTopo76 and 14 polypeptides to form a reconstituted enzyme was also evident in yeast. As shown in Fig. 9D, myc-tagged hTopo14 was detectable in immunoprecipitates of FLAG-tagged hTopo76, and vice versa. Thus, consistent with the in vitro results of Champoux and colleagues (23), the physical linkage of the core and linker domains also appears to be required for CPT sensitivity in yeast, and may reflect the low enzyme 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 cells expressing the intact (yL)-hTop1 chimera, was not induced by coexpression of (yL)-hTopo76/14. However, in contrast to the CPT 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 longer yeast linker, rather than any detectable alterations in enzyme catalysis. The specific activity of (yL)-hTopo76/14 was comparable to that of hTopo76/14 (Fig. 9C), as was the association of the (yL)-hTopo76/14 fragments in co-immunoprecipitation experiments (Fig. 9D). These data suggest that despite the physical disconnect between the core and linker domain, the yeast linker (in the context of full length hTop1) has a modest impact on Top1 sensitivity to CPT. Taken together, these findings support a model whereby the intrinsic CPT sensitivity of Top1 is dictated by the composition of the core and C-terminus, while the functional interaction of hTop1 with other proteins/ protein complexes in yeast is affected by linker composition. In the context of hTop1, the ability of the yeast Nterminal domain to suppress the growth defect induced by the yeast linker, further implies the coordinated function of these poorly conserved domains in regulating hTop1 interactions with


hTopo70 proteins. As shown in Fig. 7 and Table 3, removal of the N-terminal 1-174 residues completely suppressed the toxicity of (yL)-hTop1. Indeed, yeast cells expressing (yL)-hTopo70 grew equally well as cells expressing hTopo70 in the absence of CPT, and exhibited the same sensitivity to low concentrations of CPT as wild-type hTop1 and hTopo70 expressing cells (Fig. 7A and Table 3). In cell lysates, the levels of (yL)-hTop1 protein were comparable to hTop1, while hTopo70 and (yL)-hTopo70 levels were ~2-fold higher (Fig. 7B). As in Fig. 4B, (yL)-hTop1 was slightly more active than hTop1. However, the specific activities of hTop1 and (yL)-hTop1 were slightly lower than that observed with hTopo70, and to a lesser extent, with (yL)-hTopo70 (Fig. 7C). As with the full length enzymes shown in Fig. 5, the intrinsic CPT sensitivity of hTopo70 and (yL)-hTopo70 also mirrored that of full length hTop1, in DNA cleavage assays performed with equal concentrations of the purified proteins (Fig. 8). CPT-induced stabilization of covalent Top1DNA intermediates was observed at low drug concentrations, with the same alterations in 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 consequence of the presence of the yeast linker domain, but requires a functional interaction with the first 174 residues of the hTop1 N-terminus. Reconstituted human/yeast chimeras. Champoux and colleagues (23) demonstrated that an active enzyme could be reconstituted from separate polypeptides comprising the core and linker-C-terminal domains of hTop1. However, the intrinsic CPT resistance of such reconstituted enzymes argued that a physical tethering of the core and linker domains was required for drug sensitivity. Consequently, we wondered if a physical connection between the core and linker domain was also required for the (yL)-hTop1 chimera lethal phenotype. A library of plasmids was engineered to coordinately express Top1 fragments from a galactose-inducible, bicistronic GAL10-1 promoter (Fig. 9A and Table 2). The inclusion of distinct Flag and myc tags eased detection and immunoprecipitation of the expressed polypeptides. The constructs used to reconstitute hTopo76/14 or (yL)-hTopo76/14 were based on the same linker domain junction used to generate the chimeric enzymes (see Tables 1 and 2), while the hTopo78/12 construct recapitulated the polypeptide sequences reported by the

Activity of Human / yeast DNA topoisomerase I chimeras other, as yet unidentified, proteins or protein complexes.

7


DISCUSSION The formation of clamp structures about a DNA strand or duplex is a common feature of proteins that function in DNA replication, repair and transcription. Examples include the DNA polymerase processivity factor PCNA, the replicative Mcm2-7 helicase, DNA polymerases, the 9-1-1 checkpoint complex and DNA topoisomerases (45-50). Eukaryal nuclear Top1 possesses some unique architectural features. First, the protein clamp, formed by the highly conserved core domain of this monomeric enzyme, tightly encircles duplex DNA (7,13,18). Second, Top1 comprises a highly asymmetric structure. An extended coiled-coil of the linker domain connects the core with the conserved C-terminal domain, which contains the active site Tyr723. The positioning of residue Tyr723 within the protein clamp completes the catalytic pocket of the enzyme. This nuclear enzyme also contains a highly charged N-terminal domain, for which little structural information is available, but has been implicated in regulating the intracellular localization of Top1, Top1-protein interactions and the catalytic activity of Top1 (32,42,51). The movement and functional interactions of Top1 protein domains during enzyme catalysis, and how the structural features of this enzyme impact drug sensitivity, have been of considerable interest. One strategy used to address these questions is the use of molecular modeling to design reversible disulfide bonds that lock the hTop1 clamp around duplex DNA (7,13,1820,52). Placing the disulfide “lock” in close proximity to the active site Tyr723 prevented DNA strand rotation within the covalent enzymeDNA intermediate, but did not affect CPTstabilization of the Top1-DNA complex (19). Parallel studies with yTop1 demonstrated a conservation of clamp architecture and a role for the N-terminal domain in Top1 clamp binding of duplex DNA (20). Structural and biochemical studies also implicate the flexibility of the linker domain as a determinant of enzyme sensitivity to CPT. Increasing the movement of the linker, either by physically uncoupling the Top1 linker and core domains in reconstitution experiments [Fig. 8 and (15)], or by mutation of Ala653 to Pro in hTop1 (26), decreases enzyme sensitivity to CPT. In contrast, a Top1D677G-V703I mutant has reduced

linker flexibility and is hypersensitive to CPT (29). In addition, replacing Gly721 with residues estimated to have a higher propensity for α helix formation (Ala or Asp), induced alterations in yeast Top1 active site architecture that increased enzyme sensitivity to CPT (28). In co-crystal structures, the extension of a short α helix within the active site of Topo70 to include Gly717 coincides with drug binding and a restriction of linker orientation (7). Thus, we posited that this conserved Gly acts as a flexible hinge that allows for linker domain movement, and that substitutions of G721, which reduce linker flexibility, enhance enzyme sensitivity to CPT. A third strategy, taken here, was the design of yeast /human chimeric enzymes to define the contribution of individual protein domains to Top1 sensitivity to CPT and enzyme function in vivo. Guided by crystallographic data, we previously used homologous recombination in yeast to design yeast/human chimeras of the SUMO (Small Ubiquitin-like MOdifier) conjugating enzyme Ubc9, and in preliminary studies of Top1 (40). The biochemical activity of a hTop/ Plasmodium falciparum hybrid enzyme has also been described (53). Herein, our analyses of yeast /human Top1 chimera activity in vitro and in yeast cells establish that intrinsic enzyme sensitivity to CPT is dictated by the composition of the core and Cterminal domains. Independent of the origin of the N-terminal and/or linker domains (yeast or human), enzymes consisting of the human core and C-terminal domains exhibited comparable specific enzyme activity and were ~100-fold more sensitive to CPT than yTop1 in vitro (Figs. 4,5,7,8 and Table 3). Yet, additional aspects of chimera enzyme activity in top1∆ yeast cells are worth noting. First, in reciprocal swaps of the highly charged yeast and human N-termini (at a juncture corresponding to human residue 192 / yeast residue 120), no alterations in cell viability or sensitivity to CPT were evident. Thus, any Top1protein interactions mediated solely by the Nterminal domain failed to influence CPT-induced toxicity. Second, hTop1 residues 201-206 are required for enzyme catalysis (32). However, fusing hTop1 residues 1-209 with the corresponding C-terminal residues of yTop1 also failed to alter the CPT sensitivity of cells expressing these chimeras, relative to cells expressing wild-type hTop1 (Fig. 3 and data not shown). These data suggest that the critical

Activity of Human / yeast DNA topoisomerase I chimeras Top1 clamping of the DNA. Although the biochemical behavior of the (yL)-hTop1 did not suggest alterations in DNA binding, i.e., there was not a shift in salt optimum or enzyme processivity in DNA relaxation assays using yeast cell lysates (Fig. 4C), we cannot exclude enhanced DNA binding in the cytotoxic mechanism of the chimera protein in yeast cells. However, our fragment complementation studies (Fig. 9B) demonstrated that the integrity of (yL)-hTop1 was required for cell lethality, as the reconstituted (yL)-Topo76/14 was not toxic when expressed in yeast cells. Although the low enzyme activity of these reconstituted enzymes may well reflect decreased DNA binding in cells, these data nevertheless demonstrate that the physical linkage of the core and linker domains appears to be essential for maintaining the toxic protein and/or DNA interactions induced by (yL)-hTop1 expression. In contrast, when the shorter human linker was inserted into the yeast enzyme [in (hL)-yTop1] and expressed in yeast, the cells were viable and relatively resistant to CPT. In this case, our findings are consistent with a defect in DNA binding, suggested by earlier studies of related yTop-clamp constructs (20). Together, these studies support a model whereby the intrinsic CPT sensitivity of Top1 is dictated by the composition of the conserved core and C-terminus. In contrast, the yeast cell lethality induced by expression of (yL)-hTop1 chimera, and the ability of the yeast N-terminal domain to suppress this growth defect, imply the coordinated interplay between these poorly conserved domains in regulating hTop1 interactions with DNA or other, as yet unidentified, proteins or protein complexes in vivo. Further studies to elucidate the physiological context of such protein domain and protein-protein associations, particularly in the context of on-going DNA replication, will undoubtedly provide critical new insights into the cellular processes that dictate the cytotoxic activity of chemotherapeutics that target Top1.

REFERENCES 1. Champoux, J. J. (2001) DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem 70, 369-413. 2. Chen, S. H., Chan, N. L., and Hsieh, T. S. (2013) New mechanistic and functional insights into DNA topoisomerases. Annu Rev Biochem 82, 139-170 3. Vos, S. M., Tretter, E. M., Schmidt, B. H., and Berger, J. M. (2011) All tangled up: how cells direct, manage and exploit topoisomerase function. Nat Rev Mol Cell Biol 12, 827-841 4. Wang, J. C. (2002) Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol 3, 430-440. 5. Bjornsti, M. A. (2002) Cancer therapeutics in yeast. Cancer cell 2, 267-273 8


function of these human residues (KWKWWEEE) in enzyme catalysis is retained in the corresponding residues of yeast Top1 (EYKWWEKE). In contrast, the introduction of the longer yeast linker domain in hTop1 profoundly altered enzyme function in vivo. Expression of (yL)hTop1 proved toxic even in the absence of CPT, with no obvious alterations in enzyme catalysis (Fig. 4). This result was particularly surprising as Top1 is not required for yeast cell growth and the linker domain is dispensable for enzyme activity in vitro (23,54). Indeed, (yL)-hTop1Y723F-induced toxicity was independent of DNA cleavage (Fig. 6), which implicates toxic alterations in Top1protein interactions, instead of enzyme catalysis. However, as either removal of the human Nterminus [in (yL)-hTopo70] or the introduction of the complementary yeast N-terminal domain [in (yN,yL)-hTop1] suppressed chimera-induced lethality, toxicity is not simply due to the presence of the longer yeast linker within the hTop1 protein. Rather, lethality appears to derive from functional interaction between the extended human N-terminal and yeast linker domains. Whether these interactions involve the direct physical association of these domains or indirect interactions mediated by DNA /other proteins has yet to be determined. In this context, it is noteworthy that the N-terminal domain, which has defied structural determination, is absent from the human mtTop1 enzyme that is targeted to the mitochondria (55). Also, as both yeast and human N-terminal domains contain nuclear localization signals (NLS) (42), it is unlikely that the observed toxicity relates to changes in nuclear import. Thus, the functional interaction of N-terminal and linker domains appears to be restricted to nuclear protein-Top1 associations. Expression of catalytically inactive yeast or human Top1-clamps, locked in the closed conformation by the formation of a disulfide bond, was also toxic to yeast (20), presumably due to

Activity of Human / yeast DNA topoisomerase I chimeras 6. 7. 8. 9. 10. 11. 12.

14. 15. 16.

17. 18. 19. 20. 21. 22. 23. 24.

9


13.

Pommier, Y., Leo, E., Zhang, H., and Marchand, C. (2010) DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem & Biol 17, 421-433 Staker, B. L., Hjerrild, K., Feese, M. D., Behnke, C. A., Burgin, A. B., Jr., and Stewart, L. (2002) The mechanism of topoisomerase I poisoning by a camptothecin analog. Proc Natl Acad Sci U S A 99, 15387-15392. Koster, D. A., Palle, K., Bot, E. S., Bjornsti, M. A., and Dekker, N. H. (2007) Antitumour drugs impede DNA uncoiling by topoisomerase I. Nature 448, 213-217 Fertala, J., Vance, J. R., Pourquier, P., Pommier, Y., and Bjornsti, M. A. (2000) Substitutions of Asn-726 in the active site of yeast DNA topoisomerase I define novel mechanisms of stabilizing the covalent enzyme-DNA intermediate. J Biol Chem 275, 15246-15253 Fiorani, P., Amatruda, J. F., Silvestri, A., Butler, R. H., Bjornsti, M. A., and Benedetti, P. (1999) Domain interactions affecting human DNA topoisomerase I catalysis and camptothecin sensitivity. Mol Pharm 56, 1105-1115 Megonigal, M. D., Fertala, J., and Bjornsti, M. A. (1997) Alterations in the catalytic activity of yeast DNA topoisomerase I result in cell cycle arrest and cell death. J Biol Chem 272, 1280112808 Woo, M. H., Vance, J. R., Marcos, A. R., Bailly, C., and Bjornsti, M. A. (2002) Active site mutations in DNA topoisomerase I distinguish the cytotoxic activities of camptothecin and the indolocarbazole, rebeccamycin. J Biol Chem 277, 3813-3822. Redinbo, M. R., Stewart, L., Kuhn, P., Champoux, J. J., and Hol, W. G. J. (1998) Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA. Science 279, 1504-1513 Stewart, L., Ireton, G. C., and Champoux, J. J. (1996) The domain organization of human topoisomerase I. J Biol Chem 271, 7602-7608 Chrencik, J. E., Staker, B. L., Burgin, A. B., Pourquier, P., Pommier, Y., Stewart, L., and Redinbo, M. R. (2004) Mechanisms of camptothecin resistance by human topoisomerase I mutations. J Mol Biol 339, 773-784 Ioanoviciu, A., Antony, S., Pommier, Y., Staker, B. L., Stewart, L., and Cushman, M. (2005) Synthesis and mechanism of action studies of a series of norindenoisoquinoline topoisomerase I poisons reveal an inhibitor with a flipped orientation in the ternary DNA-enzyme-inhibitor complex as determined by X-ray crystallographic analysis. J Med Chem 48, 4803-4814 Laco, G. S., and Pommier, Y. (2008) Role of a tryptophan anchor in human topoisomerase I structure, function and inhibition. Biochemical J 411, 523-530 Stewart, L., Redinbo, M. R., Qiu, X., Hol, W. G. J., and Champoux, J. J. (1998) A model for the mechanism of human topoisomerase I. Science 279, 1534-1541 Woo, M. H., Losasso, C., Guo, H., Pattarello, L., Benedetti, P., and Bjornsti, M. A. (2003) Locking the DNA topoisomerase I protein clamp inhibits DNA rotation and induces cell lethality. Proc Natl Acad Sci U S A 100, 13767-13772 Palle, K., Pattarello, L., van der Merwe, M., Losasso, C., Benedetti, P., and Bjornsti, M. A. (2008) Disulfide cross-links reveal conserved features of DNA topoisomerase I architecture and a role for the N terminus in clamp closure. J Biol Chem 283, 27767-27775 Krogh, B. O., and Shuman, S. (2000) Catalytic mechanism of DNA topoisomerase IB. Mol Cell 5, 1035-1041 Krogh, B. O., and Shuman, S. (2002) A poxvirus-like type IB topoisomerase family in bacteria. Proc Natl Acad Sci U S A 99, 1853-1858 Stewart, L., Ireton, G., and Champoux, J. (1997) Reconstitution of human topoisomerase I by fragment complementation. J Mol Biol 269, 355-372 Shuman, S., Golder, M., and Moss, B. (1988) Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli. J Biol Chem 263, 16401-16407

Activity of Human / yeast DNA topoisomerase I chimeras 25. 26. 27. 28. 29.

30.

32.

33. 34.

35. 36. 37. 38. 39. 40.

10


31.

Villa, H., Otero Marcos, A. R., Reguera, R. M., Balana-Fouce, R., Garcia-Estrada, C., PerezPertejo, Y., Tekwani, B. L., Myler, P. J., Stuart, K. D., Bjornsti, M. A., and Ordonez, D. (2003) A novel active DNA topoisomerase I in Leishmania donovani. J Biol Chem 278, 3521-3526 Fiorani, P., Bruselles, A., Falconi, M., Chillemi, G., Desideri, A., and Benedetti, P. (2003) Single mutation in the linker domain confers protein flexibility and camptothecin resistance to human topoisomerase I. J Biol Chem 278, 43268-43275 Stewart, L., Ireton, G. C., and Champoux, J. J. (1999) A functional linker in human topoisomerase I is required for maximum sensitivity to camptothecin in a DNA relaxation assay. J Biol Chem 274, 32950-32960. van der Merwe, M., and Bjornsti, M. A. (2008) Mutation of gly721 alters DNA topoisomerase I active site architecture and sensitivity to camptothecin. J Biol Chem 283, 3305-3315 D'Annessa, I., Tesauro, C., Fiorani, P., Chillemi, G., Castelli, S., Vassallo, O., Capranico, G., and Desideri, A. (2012) Role of Flexibility in Protein-DNA-Drug Recognition: The Case of Asp677Gly-Val703Ile Topoisomerase Mutant Hypersensitive to Camptothecin. J Amino Acids 2012, 206083 Gongora, C., Vezzio-Vie, N., Tuduri, S., Denis, V., Causse, A., Auzanneau, C., Collod-Beroud, G., Coquelle, A., Pasero, P., Pourquier, P., Martineau, P., and Del Rio, M. (2011) New Topoisomerase I mutations are associated with resistance to camptothecin. Mol Cancer 10, 64 Bjornsti, M.-A., and Wang, J. C. (1987) Expression of Yeast DNA Topoisomerase I can Complement a Conditional-lethal DNA Topoisomerase I Mutation in Esherichia coli. Proc Natl Acad Sci U S A 84, 8971-8975 Lisby, M., Olesen, J. R., Skouboe, C., Krogh, B. O., Straub, T., Boege, F., Velmurugan, S., Martensen, P. M., Andersen, A. H., Jayaram, M., Westergaard, O., and Knudsen, B. R. (2001) Residues within the N-terminal domain of human topoisomerase I play a direct role in relaxation. J Biol Chem 276, 20220-20227 Frohlich, R. F., Andersen, F. F., Westergaard, O., Andersen, A. H., and Knudsen, B. R. (2004) Regions within the N-terminal domain of human topoisomerase I exert important functions during strand rotation and DNA binding. J Mol Biol 336, 93-103 Frohlich, R. F., Veigaard, C., Andersen, F. F., McClendon, A. K., Gentry, A. C., Andersen, A. H., Osheroff, N., Stevnsner, T., and Knudsen, B. R. (2007) Tryptophane-205 of human topoisomerase I is essential for camptothecin inhibition of negative but not positive supercoil removal. Nucleic Acids Res 35, 6170-6180 Albor, A., Kaku, S., and Kulesz-Martin, M. (1998) Wild-type and mutant forms of p53 activate human topoisomerase I: a possible mechanism for gain of function in mutants. Cancer Res. 58, 2091-2094 Bharti, A. K., Olson, M. O., Kufe, D. W., and Rubin, E. H. (1996) Identification of a nucleolin binding site in human topoisomerase I. J Biol Chem 271, 1993-1997 Shykind, B. M., Kim, J., Stewart, L., Champoux, J. J., and Sharp, P. A. (1997) Topoisomerase I enhances TFIID-TFIIA complex assembly during activation of transcirption. Genes & Dev 11, 397-407 Czubaty, A., Girstun, A., Kowalska-Loth, B., Trzcinska, A. M., Purta, E., Winczura, A., Grajkowski, W., and Staron, K. (2005) Proteomic analysis of complexes formed by human topoisomerase I. Biochim Biophys Acta 1749, 133-141 Girstun, A., Kowalska-Loth, B., Czubaty, A., Klocek, M., and Staron, K. (2008) Fragment responsible for translocation in the N-terminal domain of human topoisomerase I. Biochem Biophys Res Commun 366, 250-257 van Waardenburg, R. C., Duda, D. M., Lancaster, C. S., Schulman, B. A., and Bjornsti, M. A. (2006) Distinct functional domains of Ubc9 dictate cell survival and resistance to genotoxic stress. Mol Cell Biol 26, 4958-4969

Activity of Human / yeast DNA topoisomerase I chimeras 41. 42. 43. 44. 45. 46. 47.

49. 50. 51. 52. 53. 54. 55.

Acknowledgments--We thank Francesco Giorgianni, Ana Otero and Andrew Larkin for early efforts that led to the generation of these Top1 chimeras, and members of the Bjornsti lab, past and present, for helpful discussion. FOOTNOTES *This work was supported in part by National Institutes of Health Grants CA57855 (to M-AB) and the UAB Cancer Center Core Grant P30CA013148. 4

These authors contributed equally to the work.

5

van der Merwe, Wright, DeBrot and Bjornsti, unpublished data.

11


48.

Colley, W. C., van der Merwe, M., Vance, J. R., Burgin, A. B., Jr., and Bjornsti, M. A. (2004) Substitution of conserved residues within the active site alters the cleavage religation equilibrium of DNA topoisomerase I. J Biol Chem 279, 54069-54078 Christensen, M. O., Barthelmes, H. U., Boege, F., and Mielke, C. (2002) The N-terminal domain anchors human topoisomerase I at fibrillar centers of nucleoli and nucleolar organizer regions of mitotic chromosomes. J Biol Chem 277, 35932-35938 D'Arpa, P., Machlin, P. S., Ratrie, H., 3rd, Rothfield, N. F., Cleveland, D. W., and Earnshaw, W. C. (1988) cDNA cloning of human DNA topoisomerase I: catalytic activity of a 67.7-kDa carboxyl-terminal fragment. Proc Natl Acad Sci U S A 85, 2543-2547 Alsner, J., Svejstrup, J. Q., Kjeldsen, E., Sorensen, B. S., and Westergaard, O. (1992) Identification of an N-terminal domain of eukaryotic DNA topoisomerase I dispensable for catalytic activity but essential for in vivo function. J Biol Chem 267, 12408-12411 Corbett, K. D., and Berger, J. M. (2004) Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases. Annu Rev Biophys Biomol Struct 33, 95-118 Georgescu, R. E., Kim, S. S., Yurieva, O., Kuriyan, J., Kong, X. P., and O'Donnell, M. (2008) Structure of a sliding clamp on DNA. Cell 132, 43-54 Jansen, J. G., Fousteri, M. I., and de Wind, N. (2007) Send in the clamps: control of DNA translesion synthesis in eukaryotes. Mol Cell 28, 522-529 Johnson, A., and O'Donnell, M. (2005) Cellular DNA replicases: components and dynamics at the replication fork. Annu Rev Biochem 74, 283-315 Pape, T., Meka, H., Chen, S., Vicentini, G., van Heel, M., and Onesti, S. (2003) Hexameric ring structure of the full-length archaeal MCM protein complex. EMBO Rep 4, 1079-1083 Patel, S. S., and Picha, K. M. (2000) Structure and function of hexameric helicases. Annu Rev Biochem 69, 651-697 Haluska, P. J., and Rubin, E. H. (1998) A role for the amino terminus of human DNA topoisomerase I. Adv Enzyme Regul 38, 253-262 Carey, J. F., Schultz, S. J., Sisson, L., Fazzio, T. G., and Champoux, J. J. (2003) DNA relaxation by human topoisomerase I occurs in the closed clamp conformation of the protein. Proc Natl Acad Sci U S A 100, 5640-5645 Arno, B., D'Annessa, I., Tesauro, C., Zuccaro, L., Ottaviani, A., Knudsen, B., Fiorani, P., and Desideri, A. (2013) Replacement of the human topoisomerase linker domain with the plasmodial counterpart renders the enzyme camptothecin resistant. PLoS One 8, e68404 Frohlich, R. F., Juul, S., Nielsen, M. B., Vinther, M., Veigaard, C., Hede, M. S., and Andersen, F. F. (2008) Identification of a Minimal Functional Linker in Human Topoisomerase I by Domain Swapping with Cre Recombinase. Biochem 47, 7127-7136 Zhang, H., Barcelo, J. M., Lee, B., Kohlhagen, G., Zimonjic, D. B., Popescu, N. C., and Pommier, Y. (2001) Human mitochondrial topoisomerase I. Proc Natl Acad Sci U S A 98, 1060810613


The abbreviations used are: Top1, DNA topoisomerase I; Topo 70, N-terminally truncated 70kd version of DNA topoisomerase I; CPT, camptothecin; NLS, nuclear localization signal; OD, optical density; DMSO, dimethyl sulfoxide. FIGURE LEGENDS FIGURE 1. The structure of a C-terminal 70 kD fragment of human DNA topoisomerase I (Topo70) in a noncovalent complex with a 22 base pair DNA duplex. (A) Ribbon diagram of PDB file 1A36(18), viewed perpendicular to helical axis of the DNA duplex (backbone is in orange), the core domain of Topo70 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-terminal domain is in green. The active site Tyr is mutated to Phe (indicated in magenta). (B) Based on the structure in (A) and the alignment of yeast Top1 (yTop1) and human Top1 (hTop1) sequences (43), the % of identical and (similar) amino acid residues in the four domains are indicated. The same domain designations are depicted in Figs. 3A, 4A and 9A.

FIGURE 3. Reciprocal swaps of the N-terminal domains of yeast and human Top1 do not alter enzyme activity or yeast cell sensitivity to CPT. (A) Scheme of N-terminal residues exchanged between yeast and human Top1 via homologous recombination. The structurally and biochemically defined domain junctions are labeled. (hN)-yTOP1 encodes the yeast enzyme with a human N-terminal domain; (yN)hTOP1 encodes human Top1 with the yeast N-terminus. (B) Exponential cultures of top1Δ cells, transformed with the indicated YCpGAL1-TOP1 constructs, were serially diluted and were spotted onto SC –uracil agar containing dextrose (Dex) or galactose (Gal) and the indicated concentration of CPT in 0.125% DMSO. The viability of two independent transformants was assessed following incubation at 30˚C. (C) Equal concentrations of purified Top1 proteins were serially 10-fold diluted and incubated in a plasmid DNA relaxation assay. The reaction products were resolved by agarose gel electrophoresis and visualized with ethidium bromide. The relative positions of relaxed (R) and supercoiled (-) DNA topoisomers are indicated. C indicates DNA alone. (D) Extracts of galactose-induced top1∆ cells, expressing the indicated FLAG-tagged Top1 proteins, were corrected for protein concentration, resolved in SDS acrylamide gels and immunoblotted with an anti-FLAG antibody. The asterick (*) indicates the relative position of an unrelated yeast protein also recognized by the FLAG antibody. Immunostaining of GAPDH served as a loading control. Data are representative of n=3 experiments. FIGURE 4. The introduction of the yeast N-terminal domain suppresses the lethal phenotype of the (yL)hTop1chimera. (A) A scheme of the N-terminal and linker domains used to generate the indicated chimeras. (yL)-hTOP1 encodes human Top1 with a yeast linker domain; (yN, yL)-hTOP1 encodes human Top1 with a yeast N-terminal and yeast linker domain. (B) Serial dilutions of exponential cultures of 12


FIGURE 2. Generation of yeast/human Top1 chimeras in yeast. (A) Strategy for generating chimeric human/yeast Top1 proteins using homologous recombination in yeast. In this example, sequences encoding the N-terminal domain of human Top1 were PCR amplified with primers containing an additional 40 nucleotides at the 5' end, which were complementary to sequences in the GAL1 promoter (black) or sequences flanking the N-terminal domain of yeast 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 be exchanged. The PCR amplified DNA and linearized plasmid were co-transformed into yeast cells. Homologous recombination of the DNA ends regenerated a circular plasmid and the resulting transformants were selected for uracil prototrophy. The plasmid DNA was extracted and the identity of the chimeric sequences confirmed by DNA sequencing. (B) Amino acid sequence alignment of the portion of human and yeast Top1 N-terminal domains used in the chimeras. Purple represents the first six residues of the Topo70 constructs. Red and green indicate the junctions of (yN)-hTop1 and (yN137)hTop1, respectively. The corresponding residues surrounding the junction lines designate (hN)-yTop1 and (hN209)-yTop1. The grey box denotes the beginning of the Top1 core domain.

Activity of Human / yeast DNA topoisomerase I chimeras top1Δ cells, transformed with the indicated YCpGAL1-TOP1 constructs, were spotted onto SC –uracil agar containing dextrose (Dex) or galactose (Gal) and CPT (as indicated). (C) Equal concentrations of yeast lysates expressing hTop1 and chimeric proteins were serially 10-fold diluted and incubated in a plasmid DNA relaxation assay containing the indicated concentration of KCl. The relative positions of relaxed (R) and supercoiled (-) DNA topoisomers are indicated. For B,C, data are representative of at least n=3 experiments. FIGURE 5. The core and C-terminal domains of Top1 dictate CPT sensitivity and sequence specificity. Equal concentrations of the purified Top1 proteins (described in Fig. 4A) were incubated with a single 3’ 32 P-end labeled DNA substrate in a DNA cleavage reaction containing 0, 0.5, 2, 10 or 50 µM CPT in a final 0.125% DMSO. After incubation for 10 min at 30°C, the covalent Top1-DNA complexes were trapped with SDS at 75°C and treated with proteinase K. The reaction products were resolved in 8% polyacrylamide/7M urea gels and visualized using a PhosphorImager. C is DNA alone and the asterisk (*) indicates a high affinity Top1 cleavage site. Arrowheads indicate cleavage products that differ between yTop1 and hTop1. (n=3 experiments)

FIGURE 7. The presence of the hTop1 N-terminal domain is essential for the lethal phenotype seen with the (yL)-hTop1chimera. (A) The hTop1 and hTopo70 constructs were transformed into exponentially growing top1∆ cells. Liquid cultures of the transformed cells were diluted to an OD 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, extracts of galactose-induced top1∆ cells, expressing the indicated FLAG-tagged Top1 proteins, were subjected to immunoblot analysis with an anti-FLAG antibody, with GAPDH as a loading control. Images are of different lanes from the same gel. (C) Equal concentrations of yeast cells 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 visualized with ethidium bromide. The relative positions of relaxed (R) and supercoiled (-) DNA topoisomers are indicated. C indicates DNA alone. (n ≥ 3 experiments). FIGURE 8. The N-terminal domain of hTop1 does not impact enzyme sensitivity to CPT in vitro. As in Fig 5, equal concentrations of partially purified hTop1, hTopo70, and (yL)-hTopo70 proteins were assessed for sensitivity to 0.5 and 5 µM CPT sensitivity. The reaction products were resolved in 8% polyacrylamide/7M urea gels and visualized using a PhosphorImager. C is DNA alone and the asterisk (*) indicates a high affinity Top1 cleavage site. As in Fig. 5, arrowheads indicate cleavage products specific for hTop1, not yTop1. FIGURE 9. The physical connection between the core and linker domain is required for chimera-induced toxicity. (A) The bicistronic YCpGAL1-10 vector was used to express the Top1 N-terminal and core domains from the GAL10 promoter and the linker and C-terminus from the GAL1 promoter (see Table 2). (B) Exponential cultures of top1∆ cells transformed with the indicated constructs were serially 10-fold diluted, spotted onto the indicated plates and incubated at 30°. (C) Equal concentrations of lysates of galactose-induced yeast cells expressing the indicated Top1 constructs were 10-fold serially diluted and incubated in a plasmid DNA relaxation assay with a final 50 mM KCl for 30 min at 30°C. The reaction products were resolved by agarose gel electrophoresis and visualized with GelRed. The relative positions of relaxed (R) and supercoiled (-) DNA topoisomers are indicated. C indicates DNA alone. (D) Lysates

13


FIGURE 6. Catalytically inactive hTop1 containing a yeast linker is toxic to yeast cells. Exponentially growing cultures of top1∆ cells, transformed with the indicated YCpGAL1-TOP1 constructs, were adjusted to an OD = 0.3, serially 10-fold diluted, and aliquots were spotted onto SC-ura agar containing 25 mM Hepes (pH 7.2), 0.125% DMSO and either dextrose (Dex), galactose (Gal) or galactose plus 0.5 µM CPT. Viability was assessed following incubation at 30°C. (n=3 experiments)

Activity of Human / yeast DNA topoisomerase I chimeras from (C) were incubated with precleared protein A/G beads and either anti-FLAG or anti-myc antibody overnight. Input lysates and the bead-bound immunoprecipitates (α FLAG IP, α myc IP) were resolved by SDS-PAGE and visualized by immunoblotting with the indicated antibodies. Immunostaining of tubulin served as a loading control for lysate samples. Data are representative of n=4 experiments..


14


Table 1. Composition of yeast/human Top1 chimerasa Compositionc

Junction sequenced

(hN)-yTop1

hTop1(1-191)-yTop1(120-769)

DKKVPEPDNK / EDKKAKEE

h138yTop1

hTop1(1-209)-yTop1(138-769)

EQKWKWWEEE / NEDDTIKW

(yN)-hTop1

yTop1(1-119)-hTop1(192-765)

EKKKREEEEEE / KKKPKKEEEQKW

y210-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

hTop1(1-634)-yTop1(561-716)QLKDKEENS / QIALGTSKLNY723 hTop1(713-765) a Chimeric 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. b The heterologous Top1 protein domains are in parenthesis 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-terminus and core domains, with the yeast linker and C-terminus. c 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 to generate (hL)-yTop1, while the (yL,yC)-hTop1 chimera was used to generate (yL)-hTop1. d Residues spanning the chimera junctions, indicated by a /. Y727 and Y723 are the active site tyrosine residues of yTop1 and hTop1, respectively. (yL)-hTop1

15


Chimerab

Activity of Human / yeast DNA topoisomerase I chimeras Table 2. Composition of reconstituted Top1 enzymesa Reconstituted enzymeb

GAL10 expressed constructc

GAL1 expressed constructd

Table 3. Viability and CPT sensitivity of top1∆ cells expressing yeast/human chimeras TOP1 allele

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

a

No drug

0.05µg/ml CPT

5.0 µg/ml CPTc

vector

1.0

1.0 ± 0.0024

1.0 ± 0.0098

yTOP1

1.1 ± 0.2

1.0 ± 0.087

Y-box binding protein 1 enhances DNA topoisomerase 1 activity and sensitivity to camptothecin via direct interaction.

Poly(ADP-ribose) polymers regulate DNA topoisomerase I (Top1) nuclear dynamics and camptothecin sensitivity in living cells.

Camptothecin inhibits both the cleavage and religation reactions of eukaryotic DNA topoisomerase I.

Synthesis of water-soluble (aminoalkyl)camptothecin analogues: inhibition of topoisomerase I and antitumor activity.

Mutations of human DNA topoisomerase I at poly(ADP-ribose) binding sites: modulation of camptothecin activity by ADP-ribose polymers.

Inhibition of Escherichia coli DNA topoisomerase I activity by phospholipids.

Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites.

Effects of camptothecin, an inhibitor of DNA topoisomerase I on ribosomal gene structure and function in TG cells.

Catalytic core of human topoisomerase IIα: insights into enzyme-DNA interactions and drug mechanism.

Topoisomerase I as a target of erlotinib and gefitinib: efficacy of combined treatments with camptothecin.

Targeting of Topoisomerase I for Prognoses and Therapeutics of Camptothecin-Resistant Ovarian Cancer.

Monoclonal antibodies to human DNA topoisomerase I and the two isoforms of DNA topoisomerase II: 170- and 180-kDa isozymes.

Histone H1 inhibits eukaryotic DNA topoisomerase I.

Doxorubicin inhibits human DNA topoisomerase I.

Mitochondrial DNA topoisomerase I from human platelets.

Drosophila topoisomerase II-DNA interactions are affected by DNA structure.

The natural inhibitor of DNA topoisomerase I, camptothecin, modulates HIF-1α activity by changing miR expression patterns in human cancer cells.

Activity of a DNA topoisomerase (nicking-closing enzyme) during sea urchin development and the cell cycle.

Camptothecin resistance is determined by the regulation of topoisomerase I degradation mediated by ubiquitin proteasome pathway.

Topoisomerase I inhibitor, camptothecin, induces apoptogenic signaling in human embryonic stem cells.

How Trypanosoma cruzi handles cell cycle arrest promoted by camptothecin, a topoisomerase I inhibitor.

The topoisomerase I inhibitor, camptothecin, inhibits equine infectious anemia virus replication in chronically infected CF2Th cells.

Autonomous parvovirus DNA replication requires topoisomerase I and its activity is increased during infection.

Purification and properties of DNA topoisomerase I from broccoli.