Mutation of Gly721 Alters DNA Topoisomerase I Active Site Architecture and Sensitivity to Camptothecin*

DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA via a concerted mechanism of DNA strand cleavage and religation. Top1p is the cellular target of the anti-cancer drug camptothecin (CPT), which reversibly stabilizes a covalent enzyme-DNA intermediate. Top1p clamps around duplex DNA, wherein the core and C-terminal domains are connected by extended α-helices (linker domain), which position the active site Tyr of the C-terminal domain within the catalytic pocket. The physical connection of the linker with the Top1p clamp as well as linker flexibility affect enzyme sensitivity to CPT. Crystallographic data reveal that a conserved Gly residue (located at the juncture between the linker and C-terminal domains) is at one end of a short α-helix, which extends to the active site Tyr covalently linked to the DNA. In the presence of drug, the linker is rigid and this α-helix extends to include Gly and the preceding Leu. We report that mutation of this conserved Gly in yeast Top1p alters enzyme sensitivity to CPT. Mutating Gly to Asp, Glu, Asn, Gln, Leu, or Ala enhanced enzyme CPT sensitivity, with the acidic residues inducing the greatest increase in drug sensitivity in vivo and in vitro. By contrast, Val or Phe substituents rendered the enzyme CPT-resistant. Mutation-induced alterations in enzyme architecture preceding the active site Tyr suggest these structural transitions modulate enzyme sensitivity to CPT, while enhancing the rate of DNA cleavage. We postulate that this conserved Gly residue provides a flexible hinge within the Top1p catalytic pocket to facilitate linker dynamics and the structural alterations that accompany drug binding of the covalent enzyme-DNA intermediate.

reaction mechanism highly conserved from yeast to human. The active site tyrosine (Tyr 727 in yeast) acts as a nucleophile to cleave a single DNA strand, forming a covalent phosphotyrosyl linkage between the enzyme and the 3Ј-end of the DNA. The free 5Ј-OH end of the scissile strand then rotates about the uncleaved DNA strand in a manner dictated by torsional strain in the DNA. In a second transesterification reaction, the nucleophilic attack by the 5Ј-OH resolves the covalent Top1-DNA intermediate, and the enzyme is liberated from the religated DNA.
DNA cleavage by Top1p is, at least in part, rate-limiting, thus ensuring low steady state-levels of the covalent enzyme-DNA complex (4). However, increased concentrations of covalent complexes, either as a consequence of drug action, Top1p mutation, or the formation of DNA adducts, converts Top1p into a cellular poison (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). The camptothecin (CPT) class of chemotherapeutics reversibly stabilizes the covalent intermediate by intercalating into the protein-linked DNA nick and displacing the 5Ј-OH end of the DNA to prevent religation (5,10,12). Moreover, recent studies demonstrate that CPT binding preferentially impedes Top1p-catalyzed uncoiling of positively supercoiled DNA (17). Thus, during S-phase, either the ternary CPT-Top1p-DNA complex itself, or the positive supercoils that accumulate between the replication machinery and the ternary complex, present obstacles to advancing replication forks and trigger the irreversible DNA lesions that induce cell death. Mutation of conserved residues within the enzyme active site may also alter the DNA cleavage/religation equilibrium catalyzed by Top1p, leading to increased covalent complex formation and cell death (7,8,11). For example, as with CPT treatment of wild-type Top1p, substitution of Ala for Thr 722 reduces the rate of enzyme-catalyzed religation, while mutation of Asn 726 to His enhances the rate of DNA cleavage (6). The same substitutions of the corresponding residues in human Top1p also produce "self-poisoning" enzymes with similar effects on enzyme catalysis (8,18).
Co-crystal structures of a 70-kDa C-terminal fragment of human Top1p (Topo70) in noncovalent or covalent complexes with DNA revealed the unusual architecture of the monomeric enzyme (19 -21). Top1p forms a protein clamp around duplex DNA, where interaction of opposable "Lip" domains completes the circumscription of the tightly packed DNA by the protein core. We used molecular modeling to design a reversible disulfide bond between the lip domains, and demonstrated that locking the clamp prevents DNA rotation within the covalent Top1p-DNA complex (22). Moreover, expression of a catalytically inactive Top1Y 723 Fp-clamp proved toxic in the absence of DNA cleavage. The Top1p clamp consists of a conserved cen-tral core that includes all of the active site residues necessary for enzyme catalysis, except the active site Tyr 723 (19,21,23). A flexible linker physically connects the protein core with the conserved C-terminal active site tyrosine domain. The linker consists of a pair of ␣-helices, arranged in an antiparallel orientation, which extends out from the Top1p clamp and positions the C-terminal domain for enzyme catalysis. The poorly conserved N-terminal domain is missing in the Topo70 structures. Although these sequences have been implicated in DNA binding and in mediating protein-protein interactions, the N terminus is dispensable for catalytic activity (1, 24 -26).
Mutation of conserved residues in catalytically active yeast and human Top1 mutants has been reported to confer resistance to CPT (reviewed in Refs. 12,27,28). The majority of the mutated residues cluster along the Top1p-DNA interface or serve to alter the conformation of the protein clamp. Quantitative assays of drug binding are currently lacking; nevertheless, co-crystal structures of the CPT analog topotecan (TPT) bound to wild-type and mutant Topo70-DNA complexes suggest mutation-induced alterations in drug binding (20,27).
In the covalent Topo70-DNA structures, the linker domain extends ϳ56 Å from the enzyme active site at an oblique angle to the DNA when TPT is bound (see Fig. 1C). In the case of yeast Top1p, residues corresponding to the linker domain are predicted to form a similar structure that would extend beyond 110 Å. In the absence of drug, the structure of these ␣-helices in the covalent human Topo70-DNA complex was not resolved (Fig.  1A). Although present in Topo70, biochemical, genetic and molecular dynamic simulation data suggest that the flexibility of the linker in the absence of TPT would preclude structural determination (20, 29 -31). Indeed, the physical connection and/or flexibility of the linker contribute to the CPT sensitivity of Top1p. For instance, when Topo70 is reconstituted from separate polypeptides comprising the Top1p core and the linker/C-terminal domains, the enzyme is catalytically active yet exhibits reduced sensitivity to CPT (26). Within the N-terminal ␣-helix of the linker, molecular dynamic simulations suggest mutation of Ala 653 to Pro (A 653 P) increases the flexibility of this coiled-coil structure, to enhance the rate of DNA religation and Top1p resistance to CPT (30). More direct evidence for physical interactions between the Top1p linker and active site come from recent studies where the combination of the A 653 P mutation with the self-poisoning T 722 A mutation suppressed the lethal phenotype of Top1T 722 A (32). Taken together, these studies suggest alterations in the kinetics of Top1p-catalyzed DNA cleavage/religation may be induced by changes in linker flexibility or orientation.
This model raises several questions: How are alterations in the physical linkage of the coiled-coil transmitted to the active site to modulate enzyme-catalyzed DNA cleavage/religation? How does this impact CPT binding to the Top1-DNA covalent complex? Implicit in these considerations is that a dynamic organization of the active site is either directly or indirectly affected by the orientation or flexibility of the linker domain. To begin to address these questions, we mutated conserved residues that bridge the C terminus of the linker domain and the active site tyrosine domain, and asked what effect these substitutions had on yeast Top1p catalysis and CPT sensitivity. Our studies suggest that Gly 721 in yeast Top1p constitutes a flexible hinge between the linker domain and a short ␣-helix (delineated at the C terminus by the active site Tyr) that modulates the geometry of the active site during enzyme catalysis, which directly impacts Top1p sensitivity to CPT.

EXPERIMENTAL PROCEDURES
Chemicals, Yeast Strains, and Plasmids-Camptothecin purchased from Sigma was dissolved in Me 2 SO and stored at Ϫ20°C.
Yeast Cell Sensitivity to Camptothecin-To examine cell sensitivity to CPT, exponential cultures of top1⌬ yeast cells transformed with the indicated YCpGAL1-top1 vector, were adjusted to an A 595 ϭ 0.3, serially 10-fold diluted, and aliquots were spotted onto SC-uracil agar plates containing 25 mM HEPES pH 7.2, 2% dextrose or galactose and the indicated concentration of CPT in a final 0.125% Me 2 SO. Cell viability was scored following incubation at 30°C. In more quantitative assays of cell viability, transformants grown in SC-uracil containing 25 mM HEPES, pH 7.2, (SC-uracil-HEPES) and dextrose, were diluted into SC-uracil-HEPES supplemented with 2% raffinose. At an A 595 ϭ 0.3, galactose (2% final) was added to induce Top1 expression, along with 50 M CPT in a final 0.43% Me 2 SO or Me 2 SO alone. At the times indicated, aliquots were serially diluted and plated onto SC-uracil, dextrose plates, and the number of viable cells forming colonies was determined following incubation at 30°C.
DNA Topoisomerase I Expression, Purification, and Activity Assays-Extracts of galactose-induced cultures of EKY3 cells expressing plasmid encoded wild-type or mutant Top1 proteins were prepared essentially as described (35) except the glass beads were prechilled to Ϫ20°C. Purified Top1 preparations were obtained by successive ammonium sulfate fractionations followed by phosphocellulose chromatography, as described (35). Top1 protein fractions were adjusted to a final 30% glycerol and stored at Ϫ20°C.
To assess Top1p integrity and relative concentration, crude extracts, corrected for total protein concentration using a Bio-Rad protein assay, or purified proteins were resolved in 4 -12% BisTris gels (Invitrogen), transferred onto activated polyvinylidene difluoride membranes (PerkinElmer Life Sciences), immunostained with the M2-FLAG antibody (Sigma) and visualized by chemiluminescence. In the case of crude extracts, immunostaining with tubulin antibodies served as a loading control.
DNA topoisomerase I activity was assayed in plasmid DNA relaxation reaction as described (6,7). Briefly, serial 10-fold dilutions of Top1 proteins, corrected for concentration, were incubated at 30°C for 30 min with 0.3 g of negatively supercoiled plasmid pHC624 DNA in 20 mM Tris, pH 7.5, 10 mM MgCl 2 , 0.1 mM EDTA, 50 g/ml gelatin, and KCl concentrations ranging from 50 to 200 mM. Reactions products were resolved in agarose gels and visualized by ethidium bromide staining.
DNA Cleavage Assays-Wild-type and mutant enzyme sensitivity to CPT was determined in DNA cleavage assays as described (6,7). A single 3Ј 32 P-end-labeled DNA substrate was incubated with Top1 proteins in 50 l reaction volumes containing Cleavage buffer (20 mM Tris, pH 7.5, 10 mM MgCl 2 , 0.1 mM EDTA, 50 mM KCl, and 50 g/ml gelatin) and the indicated concentration of CPT in a final 4% (v/v) Me 2 SO. After 10 min at 30°C, reactions were terminated with 1% SDS at 75°C, treated with proteinase K, ethanol-precipitated, and resolved in 8% polyacrylamide/7 M urea gels. Cleavage products were visualized with a PhosphorImager.
Suicide Cleavage Reactions-Oligonucleotide-based assays were used to uncouple Top1p-catalyzed DNA cleavage and religation reactions. To assess the relative rates of DNA cleavage catalyzed by wild-type and mutant proteins, two suicide substrates (described in Ref. 6) were used. The first suicide substrate contains a truncated scissile strand with a high affinity cleavage site (5Ј-GATCTAAAAGACTT2GG-3Ј), where 2 indicates the phosphodiester bond cleaved by Top1p. The second suicide substrate is a complete duplex, where a longer scissile strand contains a bridging phosphorothiolate at the cleavage site (indicated by s in 5Ј-GATCTAAAAGACTTsGGAAAATTTTTA-AAAAAGATC-3Ј) to yield a 5ЈSH DNA end. The phosphorothiolates were the kind gift of Alex Burgin, Jr. (deCODE Genetics, Inc) and were synthesized as described (36). In both cases, the oligonucleotides were 5Ј-end-labeled with [␥-32 P]ATP and T4 polynucleotide kinase. Equimolar amounts of the labeled oligonucleotides were then annealed to an unlabeled, phosphorylated non-scissile strand (5Ј-ATCTTTTTTA-AAAATTTTTCCAAGTCTTTTAGATC-3Ј for the truncated oligo and 5Ј-GATCTTTTTTAAAAATTTTCCAAGTCTTT-TAGATC-3Ј for the phosphorothiolate oligo) in annealing buffer (10 mM Tris, pH 7.8, 100 mM NaCl, 1 mM EDTA) by heating to 90°C followed by cooling to room temperature over 3 h. The DNA substrates (3 pmol/reaction) were incubated with equal concentrations of Top1 proteins in Cleavage buffer at room temperature. At the times indicated, 10-l aliquots were quenched with 0.5% SDS at 75°C to terminate the reactions. Following ethanol precipitation, the covalently linked protein was hydrolyzed with trypsin in the presence or absence of 2 M urea and the labeled substrate and peptide-linked oligonucleotides were resolved in 16% polyacrylamide/7 M urea gels and visualized using a PhosphoImager.
DNA Religation Assays-Relative rates of Top1p-catalyzed DNA religation were assessed using oligonucleotide-based religation assays adapted from Colley et al. (6). In the post-annealed religation substrate reaction, the same 5Ј-end-labeled DNA suicide substrate described above, which contains the DNA scissile strand (5Ј-GATCTAAAAGACTT2GG-3Ј), was incubated with equal concentrations of Top1 proteins in Cleavage buffer at room temperature for 15 min to generate Top1-DNA cleavage complexes. Religation was then initiated (time ϭ 0) by the addition of a 10-fold excess of the religation oligonucleotide (5Ј-GGAAAAATTTTTAAAAAAGATC-3Ј). Alternatively, in reactions containing the pre-annealed religation substrate, the same DNA substrate was incubated with a 10-fold excess of the religation strand for 15 min prior to the addition of Top1 proteins. In both assays, aliquots were processed as described above for the 5Ј-end-labeled suicide cleavage reactions, except that when indicated, urea was omitted from the trypsin reaction to probe for alterations in active site architecture.

Substitution of Gly 721 and Leu 720 Alter Top1p Sensitivity to
Camptothecin-Within the active site pocket of yeast and human Top1p, residues immediately N-terminal to the active site tyrosine are highly conserved (Fig. 1E). Residues spanning Thr 722 to the active site Tyr 727 correspond to Thr 718 to Tyr 723 in human Top1p, which form a short ␣-helical structure highlighted in yellow in the co-crystal structures of human Topo70 covalently linked to DNA, either in the presence or absence of Topotecan ( Fig. 1, A-D) (20,37). Upon drug binding, however, this helical structure is extended to include conserved residues Gly 717 and Leu 716 (Gly 721 and Leu 720 in yeast Top1p). This rather subtle transition in active site structure coincides with a more dramatic restriction in linker domain flexibility, such that the linker is now oriented at an oblique angle to the DNA. Although these changes in structure might reflect differences in crystal packing, additional studies suggest that linker domain flexibility as well as the physical linkage of the linker to both the active site C-terminal domain and the protein clamp to which the C terminus is abutted constitute critical determinants of enzyme sensitivity to CPT (30,31). Given the flexibility of Gly residues within a polypeptide chain, and the structural transitions apparent in the co-crystal structures with drug bound, we propose that Gly 717 (Gly 721 in yeast) constitutes a flexible hinge and that alterations in active site architecture at this junction would affect the orientation and flexibility of the linker domain. To address this hypothesis, we examined the effects of single amino acid substitutions of yeast Gly 721 and the adjacent resi-due Leu 720 . These studies were initiated in yeast for several reasons: 1) the phenotypes of specific top1 mutants could be assessed in the absence of wild-type Top1 (in yeast top1⌬ strains) (5); 2) any confounding effects of protein-protein interactions due to the expression of human Top1p in yeast could be avoided and 3) mutant enzyme activity could be assayed in different genetic backgrounds in otherwise isogenic strains.
Individual Gly 721 and Leu 720 substitutions were isolated from a pool of yeast Top1p active site mutants generated by degenerate oligonucleotide mutagenesis. Two hydrophobic substituents of Gly 721 , (Val in G 721 V or Phe in G 721 F) led us to engineer a Leu mutation (G 721 L) to expand the analysis of amino acid shape at this position. In contrast, the isolation of a Leu 720 to Gln (L 720 Q) mutation, prompted considerations of charge, so an acidic residue Glu was also engineered at this position (L 720 E). The effect of these single amino acid substitutions on cell sensitivity to CPT was assessed in top1⌬ cells induced to express the indicated mutant allele from the GAL1 promoter on an ARS/CEN vector. As seen in Fig. 2A, substituting a basic or acidic residue for Leu 720 abolished the CPT sensitivity of cells expressing Top1L 720 Qp or Top1L 720 Ep, respectively. These proteins were expressed at slightly higher levels that wild-type Top1p; nevertheless, the specific activity of the two mutant enzymes was reduced by more than 100-fold (Fig. 2, B and C). These data suggest the CPT-resistant phenotype of cells expressing the L 720 Q or L 720 E mutant results from a decrease in Top1p activity, rather than specific alterations in enzyme sensitivity to CPT. Indeed, homologous recombination defective rad52⌬, top1⌬ cells expressing Top1L 720 E were sensitive to 10-fold higher CPT concentrations than cells expressing wild-type Top1 (data not shown), indicating the formation of lower levels of CPT-induced Top1L 720 E-DNA damage.
By contrast, substitution of hydrophobic residues for Gly 721 had distinct effects on enzyme activity and cell sensitivity to CPT. The G 721 L mutation has no obvious affect on CPT-induced toxicity, while cells expressing Top1G 721 Vp or  (20)). B and D, close up views of the active site of Topo70-DNA and Topo70-DNA-TPT, respectively. A ribbon diagram of the polypeptide backbone spanning residues Thr 718 to the active site Tyr 723 is colored yellow. The covalent linkage between the 3Ј-phosphoryl end of the DNA (green) and Tyr 723 is shown in yellow and red solid bond representation. The base pairs spanning the Topo70-linked DNA nick are also shown, and the conjugated ring structure of TPT is in magenta. Gly 717 (corresponds to Gly 721 in yeast Top1p) is in orange and Leu 716 in lavender. A and B, residues spanning the N-and C-terminal ends of the linker are highlighted in dark purple. C and D, the entire linker domain is shown in dark blue. All figures were made with PyMol (DeLano Scientific, San Carlos, CA). E, schematic representation of the domain structures of yeast and human Top1p, with residues preceding the active site tyrosine. Color coding of the protein clamp, linker, and C-terminal domains and conserved Leu and Gly residues correspond to that in C.
Top1G 721 Fp were CPT resistant ( Fig. 2A). All three mutant enzymes were expressed at levels comparable to wild-type Top1p (Fig. 2B). The specific activity of Top1G 721 L was comparable to wild-type Top1p, and the G 721 V and G 721 F mutants were slightly (ϳ5-fold) less active in plasmid DNA relaxation assays (Fig. 2C). However, expression of these mutants in a Rad9 DNA damage checkpoint defective (rad9⌬) strain revealed that the G 721 L mutant actually enhanced cell sensitivity to CPT, relative to cells expressing wild-type Top1p or the G 721 V and G 721 F mutants (Fig. 3A).
The side chain of Val is branched at the ␤ carbon, while the ␥ carbon branch of Leu is more distal to the polypeptide backbone. We then made additional substitutions of Gly 721 , including Asp and Asn (G 721 D and G 721 N, respectively), to probe differences in charge independent of side chain geometry, and Glu and Gln (G 721 E and G 721 Q, respectively), wherein the acidic and basic side chains are extended by a ␥-␦ carbon bond. As seen in Figs. 3A and 4, substituting the acidic residues for Gly 721 increased cell sensitivity to CPT by more than a factor of ten. However, as with the G 721 L mutant, a significant increase in cell sensitivity to CPT as a result of G 721 N or G 721 Q mutant expression was only evident in checkpoint-deficient strains, such as rad9⌬ or rad53⌬ cells (Fig. 3A and data not shown).
We previously described active site mutations, such as T 722 A and N 726 H, which enhance Top1p sensitivity to CPT in vitro (7,11). However, these mutant enzymes induced cell lethality in the absence of CPT and exhibited other alterations in enzyme catalysis. In contrast, the Gly 721 mutants that enhanced cell sensitivity to CPT, failed to induce growth defects in the absence of drug. In addition, as shown for Top1G 721 Lp (Fig. 2, B and C), there were no detectable differences in the levels of wild-type and mutant Top1 proteins in galactose-induced cells, and the specific activities of the purified G 721 D, G 721 E, G 721 N, and G 721 Q mutant proteins were indistinguishable from that of wild-type Top1p (Fig. 3B). Thus, this enhanced CPT sensitivity constitutes a unique phenotype for a single amino acid substitution in Top1p.
Gly 721 Mutations Increase Top1p Sensitivity to Camptothecin in Vitro-We next asked if the increased CPT sensitivity of cells expressing the G 721 D, G 721 E or G 721 N mutants was a direct effect of increased CPT-Top1p-DNA covalent complexes. First, the specific activity of these mutant enzymes exhibited the same salt optimum as wild-type Top1p; similar patterns of activity and DNA topoisomer distributions were observed for G 721 D, G 721 E and G 721 N mutant enzymes (Fig. 5A and data not shown). Equal concentrations of the proteins were then incubated with a 32 P-single end-labeled DNA and increasing concentrations of CPT, to assess the intrinsic CPT sensitivity of each mutant enzyme relative to wild-type Top1p. In such DNA FIGURE 2. Mutation of Leu 720 or Gly 721 in yeast Top1p induces CPT resistance in vivo. A, exponentially growing cultures of top1⌬ cells, transformed with the indicated YCpGAL1-top1constructs, were adjusted to an A 595 ϭ 0.3, serially 10-fold diluted, and aliquots were spotted onto SC-uracil plates containing 25 mM HEPES pH 7.2, 0.1% Me 2 SO, and either Dex, Gal or galactose plus 5 g/ml CPT. The viability of two individual transformants was assessed following incubation at 30°C. B, levels of galactose-induced wild-type and mutant Top1p in crude cell extracts were determined in immunoblots with the M2-FLAG antibody. Immunostaining with tubulin antibodies served as a loading control. C, the specific activity of wild-type and mutant Top1 enzymes was determined by incubating serial 10-fold dilutions of crude cell extracts (corrected for protein concentration) in plasmid DNA relaxation assays as described under "Experimental Procedures." After 30 min at 30°C, the reaction products were resolved in a 1.2% agarose gel and visualized after ethidium bromide staining. The relative positions of relaxed (R) and supercoiled (-) DNA topoisomers are indicated. C is plasmid DNA control.  FEBRUARY 8, 2008 • VOLUME 283 • NUMBER 6 cleavage assays (Fig. 5B), all three mutants exhibited higher levels of drug-stabilized Top1-DNA covalent complexes than that observed with wild-type Top1p: the acidic substituent Glu (G 721 E) conferred high levels of DNA cleavage across a wide range of CPT concentrations, while mutation to Asp (G 721 D) enhanced DNA cleavage to the greatest extent at higher drug concentrations. Indeed, these findings are consistent with the enhanced CPT sensitivity of cells expressing the G 721 D and G 721 E mutants (Figs. 3 and 4). Increased levels of covalent complexes were also evident with the G 721 N mutant, but not at lower drug concentrations (as with G 721 E) or to the same extent as G 721 D at higher concentrations. Together, these data suggest that the increased CPT sensitivity of cells expressing these mutants can be attributed to mutation-induced increases in drug poisoning of Top1p.

Gly 721 as a Flexible Hinge in Top1p
Replacing Gly 721 with Acidic Residues Affects Top1p Cleavage of DNA and Enzyme Architecture-Top1G 721 Dp and Top1G 721 Ep did not exhibit alterations in steady state levels of Top1-DNA covalent complexes in the absence of CPT, or specific enzyme activity in plasmid DNA relaxation assays, yet exhibited increased CPT sensitivity in vivo and in vitro (Figs. 4 and 5). We considered that changes in enzyme architecture induced by these substituents might enhance CPT binding and coincide with changes in linker domain flexibility. To address such dynamic interactions, we first used a series of oligonucleotide-based DNA substrates to uncouple Top1p catalyzed DNA cleavage from religation.
A common strategy to uncouple DNA cleavage from religation is to use a oligonucleotide-based substrate that contains a truncated scissile strand. For example, as depicted in Fig. 6A, Top1p cleavage of a high affinity site within a suicide DNA substrate liberates a dinucleotide and traps the covalent Top1p-DNA complexes (6,18,38). In this case, Top1-DNA covalent complex formation is monitored by the accumulation of a peptide-linked 14-mer, produced by trypsin digestion of the reaction products in the presence of urea (39). These data revealed a decreased rate of DNA cleavage by the G 721 D mutant, relative to that obtained with wild-type Top1p (Fig.  6A). We next asked if the extent of duplex DNA structure 3Ј to the site of DNA cleavage affected the relative rates of mutant and wild-type Top1 cleavage of DNA. One way to address this question is to use successively longer scissile strands. Such assays did demonstrate a proportional increase in the rate of DNA cleavage by Top1G 721 Dp (data not shown). However, in this case, the co-migration of the 14-mer ϩ peptide reaction product with the 20-mer substrate required the analysis of a 3Ј end-labeled substrate. So to avoid such complications, a second suicide substrate depicted in Fig. 6B was employed. In this duplex DNA substrate, a 5Ј-bridging phosphorothiolate at the preferred site of scission produces a 5Ј-SH in the Top1p-DNA covalent complex, which is ineffective in the transesterification that religates the DNA (6,36). As the substrate is 5Ј-end-labeled, the rate of DNA cleavage can be approximated by the accumulation of the same peptide-linked 14-mer as described in Fig. 6A. With this fully duplexed DNA, the G 721 D mutant exhibited an increased rate of DNA cleavage, relative to that  After incubation for 10 min at 30°C, covalent complexes were trapped with SDS at 75°C and treated with proteinase K. The reaction products were resolved in 8% polyacrylamide/7 M urea gels and visualized using a PhosphoImager. C is DNA alone, and the asterisk (*) indicates a high affinity Top1p cleavage site.
observed with wild-type Top1p (Fig. 6, B and C). Taken together, these findings indicate that in contrast to wild-type Top1p, the enhanced DNA scission catalyzed by Top1G 721 Dp required the presence of duplex DNA 3Ј to the site of cleavage.
We next considered the effects of Gly 721 mutations on the rate of Top1-catalyzed DNA religation, using the strategy depicted in Fig. 7A. Here, the dissociation of the GG dinucleotide facilitates the subsequent annealing of a complementary oligonucleotide, which provides the 5Ј-OH necessary to resolve the trapped covalent complex and produce a 35-mer. In the post-annealed religation assay, covalent Top1p-DNA complexes were first trapped with the 16-mer suicide substrate, then the complementary religation oligonucleotide is added (time ϭ 0). At the times indicated, the reactions were terminated with SDS at 75°C, and the relative levels of uncleaved 16-mer, religated 35-mer and covalent Top1-DNA intermediate were assessed in denaturing gels following trypsin digestion. These reactions were initially carried out in the absence of urea, because trypsin digestion of wild-type Top1 (Fig. 7B) efficiently liberated a 7-amino peptide (INY 727 IDPR) covalently linked to the radiolabeled 14-mer, which migrates slower than the 16-mer suicide oligonucleotide. However, a distinct pattern of bands was obtained in tryptic digests of covalent complexes formed by Top1G 721 D. As seen in Fig. 7B, two additional bands are observed at zero time, with the slower migrating band (labeled Ͻ Ͻ) being more prominent. Following addition of the religation oligonucleotide, the accumulation of the faster migrating band (labeled Ͻ) paralleled that of the religated 35-mer. Similar results were obtained in assays performed at 150 mM KCl (data not shown). These extra bands do not result from illegitimate religation events. Rather, they represent incomplete tryptic digests of the covalent Top1G 721 D-DNA complexes, as trypsin digestion of these complexes in the presence of urea produced a single 7 residue peptide linked to the radiolabeled 14-mer (Fig. 7C). Moreover, digestion with proteinase K, which cleaves at sites distinct from trypsin, failed to yield multiple bands (data not shown). Nevertheless, if one corrects for differences in the level of covalent complexes formed at t ϭ 0, then there was no difference in the rate of DNA religation (accumulation of 35-mer following the addition of the religation oligonucleotide) catalyzed by Top1 versus Top1G 721 Dp (Fig. 7C).
To determine if altered tryptic digests coincided with increased enzyme sensitivity to CPT, similar analyses of Top1G 721 Ep and Top1G 721 Np were performed. All three mutants produced lower levels of covalent enzyme-DNA intermediates at t ϭ 0, with no obvious defect in DNA religation (Fig.  7, B and D). However, only in the case of the more CPT sensitive Top1G 721 D and Top1G 721 E mutant enzymes were the longer peptide-linked oligonucleotides obtained; these bands were not  A and B, intact Top1p is indicated by F. Trypsin digestion of the covalent complexes in the presence of 2 M urea generates a 7-amino acid peptide covalently linked to a 14-mer. In these assays, equal concentrations of Top1p and Top1G 721 Dp were incubated with the suicide substrate and at the times indicated (minutes or s for seconds), reaction aliquots were terminated with 0.5% SDS at 75°C, ethanol-precipitated, and the trypsin products resolved in a denaturing gel and visualized by PhosphorImaging. C, quantitation of the % of DNA cleaved the reactions depicted in B. evident with the less CPT sensitive Top1G 721 Np (Fig. 7, B and  D). Thus, substituting acidic residues for Gly 721 appears to enhance CPT poisoning of Top1p and alter the geometry of the active site in the covalent complex.
We next asked if the presence of extended duplex DNA 3Ј to the site of DNA cleavage, which affected the rate of DNA cleavage by Top1G 721 D (Fig. 6B), also impacts enzyme active site architecture and/or rates of DNA religation. To address these questions, we took two approaches. First, as diagrammed for the "pre-annealed religation substrate in Fig. 8A, the religation oligonucleotide was annealed to the suicide DNA substrate prior to the addition of Top1p. Although this substrate contains a short DNA flap, the same suicide oligonucleotide used in the "post-annealed religation substrate" (Fig. 7A) was used to restrict the overlap to 2 nucleotides. In addition to the 14-mer ϩ peptide, trypsin digestion of the reaction products produced a longer Top1G 721 D-derived peptide covalently attached to the cleaved DNA (Ͻ in Fig. 8B), corresponding to the lower band obtained with the post-annealed religation substrate in Fig. 7B. Thus, the presence of DNA annealed to the non-scissile strand induced distinct alterations in mutant enzyme active site architecture when probed by trypsin digestion. Moreover, the accumulation of religated DNA products was also diminished in FIGURE 7. Distinct patterns of Top1p-DNA tryptic digests obtained in religation reactions suggest G 721 D-induced alterations in active site structure. A, in the post-annealed religation assay, the same suicide substrate diagrammed in Fig. 6A, is used to generate covalent [␥-32 P]DNA-Top1p complexes. At t ϭ 0, a 21-mer religation oligonucleotide (complementary to the 5Ј-end of the nonscissile strand) is added. As shown in B, C, and D, the resolution of the covalent Top1p-DNA complex to generate a 32 P-labeled 35-mer can be followed by the conversion of the tryptic 14-mer ϩ peptide to the 35-mer. B and D, equal concentrations of Top1p, Top1G 721 Dp, Top1G 721 Ep, and Top1G 721 Np were incubated with the suicide substrate for 15 min at room temperature. At t ϭ 0, the 21-mer religation oligonucleotide was added. At the times indicated (minutes or s for seconds), reaction aliquots were terminated with SDS at 75°C, treated with trypsin (in the absence of urea), resolved in 16% polyacrylamide/7 M urea gels visualized using a Phos-phoImager. Ͻ Ͻ and Ͻ indicate the position of the cleaved 14-mer covalently linked to Top1 peptides longer than 7 residues. C, as for the reactions shown in B, equal concentrations of Top1p and Top1G 721 Dp were incubated in the post-annealed religation assay. However, the reaction products were digested with trypsin in the presence of 2 M urea prior to 16% polyacrylamide/7 M urea gel electrophoresis.  Fig. 7A is annealed prior to the addition of enzyme. DNA cleavage by Top1p liberates a GG dinucleotide to allow formation of the 35-mer. B, equal concentration of Top1p and Top1G 721 Dp were added to pre-annealed religation assays at t ϭ 0. At the times indicated (minutes or s for seconds), reaction aliquots were quenched with SDS at 75°C. Covalently linked Top1 proteins were hydrolyzed with trypsin (in the absence of urea), and the reaction products were resolved in 16% polyacrylamide/7 M urea gels. C, tryptic digests of Top1G 721 Dp, incubated in pre-annealed or post-annealed religation assays, were resolved by 16% polyacrylamide/7 M urea gel electrophoresis. As in Fig. 7, Ͻ Ͻ and Ͻ indicate the position of the 14-mer covalently linked to Top1 peptides.
Top1G 721 D reactions containing the pre-annealed substrates, relative to that observed with wild-type Top1p (Fig. 8B) or Top1G 721 D incubated with post-annealed substrates (Fig. 8C). However, because the pre-annealed assay does not uncouple cleavage from religation, it is difficult to determine if the presence of the dinucleotide flap and/or duplex DNA also affects the rate of DNA cleavage.
To address these questions and avoid the potential complications of aberrant DNA structure, we asked if a similar analysis of Top1p-DNA complexes formed with the 5Ј bridging phosphorothiolate (diagrammed in Fig. 6B) would yield the same pattern of Top1G 721 D and Top1G 721 E-derived peptides linked to the 14-mer. Indeed, as shown in Fig. 9, the presence of duplex DNA 3Ј to the site of DNA cleavage precluded efficient trypsin digestion of Top1p-DNA complexes formed by the G 721 D and G 721 E mutants, but not wild-type Top1. Thus, independent of the presence of a DNA flap or religation, these findings support a model whereby the alterations in tryptic digests of the covalent enzyme-DNA intermediates, induced by the various DNA substrates, coincided with increased mutant enzyme sensitivity to CPT.
Based on these findings, we hypothesize that the increased CPT sensitivity of the Gly 721 mutant enzymes results from conformational changes within the Top1p catalytic pocket that limits linker domain flexibility and enhances CPT binding to the covalent enzyme complex. In particular, mutation of the highly flexible Gly might extend the ␣-helical structure of residues N-terminal to the active site Tyr, as seen in the structures shown in Fig. 1. One prediction of this model is that, independent of side chain charge, an increased tendency for ␣-helical structure at position 721 would augment CPT poisoning of Top1p. Estimates of the intrinsic helix-forming tendencies of various amino acid residues indicate that while Gly has a low propensity to form ␣-helices, Ala has the highest (40). Indeed, substituting Ala for Gly 721 (in Top1G 721 Ap) induced a 5-fold increase in cell sensitivity to CPT, relative to cells expressing wild-type Top1p, which was only slightly less than the 10-fold increase induced by the G 721 D mutation (Fig. 10).

DISCUSSION
The architecture of Top1p includes a flexible linker domain, comprised of an extended coiled-coil that positions the C-terminal active site Tyr domain within the catalytic pocket formed in concert with the central protein clamp. Biochemical, x-ray crystallographic and molecular dynamic simulation data suggest that the integrity and flexibility of the linker domain within the Top1p clamp are critical determinants of enzyme sensitivity to the CPT class of chemotherapeutics (20, 29 -31, 37). Increasing the flexibility of the human Top1p linker domain, either by mutation (A 653 P) (30) or by physically uncoupling the linker domain from the Top1 protein clamp (in reconstituted Topo70 preparations) (31), reduces enzyme sensitivity to CPT. In cocrystal structures of human Topo70 and DNA, the presence of CPT analogues appears to restrict the movement of the linker domain thereby enabling the structural determination of this coiled-coil. This contrasts with the mobility of the linker domain within the covalent Topo70-DNA complex in the absence of drug. Close inspection of active site architecture within these structures revealed an extension of a short ␣-helical structure, spanning Ser 719 through the active site Tyr (human Tyr 723 ) in the absence of drug, to include Leu 716 , Gly 717 , and Thr 718 in the presence of drug (see Fig. 1). These data suggest a dynamic interplay between active site ␣-helical structure and linker flexibility attends drug binding to the covalent Topo70-DNA complex. Such long range molecular interactions between the Top1 linker and active site in mediating enzyme sensitivity to CPT are also supported by recent studies where the cytotoxicity induced by a defect in human Top1pcatalyzed DNA religation (due to the active site mutation T 722 A) was suppressed by the increased linker flexibility of the A 653 P mutation (32). Based on these considerations, we hypothesized that the presence of this conserved Gly residue within the active site of the enzyme (Gly 717 in human Top1p; Gly 721 in yeast Top1p) functions as a flexible hinge to facilitate the alterations in active site geometry and linker domain flexibility that impact CPT poisoning of Top1p. Indeed, our analyses of Gly 721 mutations in yeast Top1p, in vitro and in vivo, support this model and suggest that the charge and geometry of amino acid side chains at this position directly impact active site architecture within the covalent enzyme-DNA complex and the intrinsic sensitivity of Top1p to CPT.
First, our findings indicate that the introduction of a bulky aromatic residue (Phe) or ␤ branched aliphatic side chain (Val) FIGURE 9. Alterations in Top1G 721 D and Top1G 721 E structure are detected in 5 bridging phosphorothiolate DNA suicide substrates. As in Fig. 6B, equal concentrations of Top1p, Top1G 721 Dp and Top1G 721 Ep were incubated with the 5Ј-end-labeled phosphorothiolate suicide substrate for 15 min. The reactions were quenched with SDS at 75°C, the covalently linked Top1 proteins were hydrolyzed with trypsin (in the absence of urea) and the reaction products were resolved in 16% polyacrylamide/7 M urea gels. Ͻ indicates a similar 14-mer-Top1 peptide product as that detected in Figs. 7 and 8. decreased the specific activity and CPT sensitivity of Top1p. By contrast, the introduction of an acidic side chain, with carboxyl groups at the ␤ or ␥ carbon in G 721 D and G 721 E, respectively, dramatically enhanced Top1p sensitivity to CPT, without any obvious alterations in specific activity in plasmid DNA relaxation assays. In this case, side chain charge was important, as the increased CPT sensitivity of cells expressing Top1 mutants engineered with the amide forms of these residues (G 721 N and G 721 Q) or a ␥ branched aliphatic side chain (G 721 L) was only evident in the absence of the DNA damage checkpoint. However, the tendency of residues at this position to form ␣-helices also appears to be a critical determinant of Top1p sensitivity to CPT, as mutating Gly 721 to Ala (estimated to have the highest propensity for helix formation (40)) also increased cell sensitivity to CPT. Together, our data support the following rank order of mutant-induced increased in Top1p sensitivity to CPT: (G 721 E, G 721 D) Ͼ G 721 A Ͼ (G 721 N, G 721 Q, G 721 L) Ͼ wildtype Ͼ G 721 V Ͼ G 721 F.
Second, our studies also suggest mutation-induced alterations in enzyme active site architecture coincide with increased CPT sensitivity. The G 721 D, G 721 E mutants exhibited the greatest CPT sensitivity in vivo and in vitro, as well as specific alterations in tryptic digests of covalent enzyme-DNA complexes formed with distinct suicide substrates DNA. A comparison of potential trypsin cleavage sites in yeast and human Top1p with the pattern of DNA-bound tryptic fragments obtained with yeast and human Top1p mutants (Figs. 7-9 and data not shown) allows for an accurate determination of the alterations in trypsin digestion. In the residues spanning the active site tyrosine of yeast Top1p, K 712 EENSQYSLG 721 TSK 724 INY 727 IDPR, trypsin digestion of SDS-denatured wild-type Top1p-DNA covalent complexes typically yields a 7 residue peptide (INY 727 IDPR) covalently attached to a 5Ј-end-labeled 14-mer oligonucleotide via a 3Ј-phosphotyrosyl linkage. Mutation of Gly 721 to Asp or Glu (G 721 D or G 721 E, respectively) limits trypsin digestion at K 724 to yield longer peptides linked to the DNA. One possible explanation is that the G 721 D and G 721 E mutations alter the ␣-helical structure of the active site so as to restrict trypsin digestion of the partially denatured proteins. The presence of an acidic side chain may also preclude efficient trypsin digestion at K 724 . Nevertheless, the changes in enzyme active site geometry suggested by the altered tryptic digests coincided with increased Top1p sensitivity to CPT. Alterations in trypsin digestion were only observed with the more CPT sensitive G 721 D or G 721 E mutants and not with the less CPT sensitive G 721 N mutant. Moreover, these alterations were observed in the absence of DNA religation or alterations in DNA helical structure (Fig. 9).
The presence of duplex DNA 3Ј to the site of DNA scission also altered the rates of Top1G 721 Dp and Top1G 721 Ep catalyzed DNA cleavage, as well as the pattern of tryptic digests. These data suggest the enhanced CPT sensitivity of these mutants derives from the increased cleavage of duplex DNA and that downstream protein-DNA contacts impact dynamic interactions between the active site and the linker. Indeed, the linker of human Top1p is 10-fold more resistant to limited proteolysis when the enzyme is noncovalently bound to duplex DNA, consistent with linker-DNA contacts 3Ј to the site of DNA scission (23). Recent single molecule analyses of individual human Top1p-DNA complexes using magnetic tweezers also determined that the binding of TPT selectively impede enzyme uncoiling of positively supercoiled DNA (17). While mutation-induced alterations in Top1p structure await x-ray structure determination, our studies implicate the conserved Gly 721 residue as a flexible hinge within the active site of Top1p that enable linker domain flexibility and the structural alterations that accompany drug binding of the covalent enzyme-DNA complex.