DNA relaxation by human topoisomerase I occurs in the closed clamp conformation of the protein

Abstract

In cocrystal structures of human topoisomerase I and DNA, the enzyme is tightly clamped around the DNA helix. After cleavage and covalent attachment of the enzyme to the 3' end at the nick, DNA relaxation requires rotation of the DNA helix downstream of the cleavage site. Models based on the cocrystal structure reveal that there is insufficient space in the protein for such DNA rotation without some deformation of the cap and linker regions of the enzyme. Alternatively, it is conceivable that the protein clamp opens to facilitate the rotation process. To distinguish between these two possibilities, we engineered two cysteines into the opposing loops of the "lips" region of the enzyme, which allowed us to lock the protein via a disulfide crosslink in the closed conformation around the DNA. Importantly, the rate of DNA relaxation when the enzyme was locked on the DNA was comparable to that observed in the absence of the disulfide crosslink. These results indicate that DNA relaxation likely proceeds without extensive opening of the enzyme clamp.

Figure 1

Schematic showing the key structural features of human topoisomerase I. (A) The crystal structure of human topoisomerase I (residues 215–765) in complex with a 22-bp DNA (5, 6). Core subdomains I and II form the upper lobe or cap of the enzyme (magenta) and contain the nose cone helices. The lower lobe of the enzyme, shown in cyan, comprises subdomain III, the linker region, and the C-terminal domain. (B) The predicted structure for the disulfide bond formed between Cys-367 and Cys-499 in topo70^2XCys. The predicted distance between the Cβ atoms of the two cysteines is 4.6 Å. The two loops (Arg-362–Met-370 and Lys-493–Thr-501) follow the same color scheme as in A. (C) An end view of the cocrystal structure of human topoisomerase I looking down the axis of the DNA helix. The two opposing loops shown in B contact each other in a region referred to as “lips.” (D) Model of a hypothetical open clamp form of the protein (3).

Figure 2

Assays of sucrose gradient fractions for plasmid DNA and topoisomerase I proteins. Plasmid pRc/CMV DNA was incubated at 4°C with topo70^WT under oxidizing conditions (GSSG, A), with topo70^2XCys under reducing conditions (DTT, B), or with topo70^2XCys under oxidizing conditions (GSSG, C). After centrifugation through sucrose gradients, aliquots of the indicated fractions were subjected to immunoblot assays for the presence of topoisomerase I protein. The lane marked C for A–C contained purified topo70, which provided a mobility standard for the immunoblot analysis. In D, additional aliquots of the same fractions were treated with proteinase K and analyzed by agarose gel electrophoresis to locate the pRc/CMV DNA in the gradient. The DNA profiles were very similar for all three gradients; only the fractions from the gradient for topo70^2XCys incubated with the DNA in the presence of GSSG are shown here. Lane C in D contained purified pRc/CMV DNA that had been relaxed by topo70 under the same conditions as used for the crosslinking experiments.

Figure 3

Assay for the cosedimentation of topoisomerase I activity with pRc/CMV DNA. Supercoiled pBluescript KS II⁺ DNA (pKS II) was incubated under standard relaxation conditions with fraction 8 from each of the sucrose gradients shown in Fig. 2 (70^WT/GSSG, 70^2XCys/DTT, and 70^2XCys/GSSG) at 23°C in the absence of DTT (lanes 1, 3, and 5) to detect any nonspecific association of topoisomerase I with the sedimented pRc/CMV DNA. Similar incubations with exogenous pBluescript KS II⁺ DNA were carried out in the presence of DTT to detect any cosedimenting activity that could be released by reduction of disulfide bonds (lanes 2, 4, and 6). The reactions were stopped with proteinase K and analyzed by agarose gel electrophoresis in the absence (A, no chloroquine in gel) or presence (B, chloroquine in gel) of 1.5 μg/ml chloroquine. Supercoiled and relaxed pBluescript KS II⁺ (pKS II) are shown in lanes 7 and 8, respectively. The mobilities of the nicked, relaxed, and supercoiled forms of pBluescript KS II⁺ DNA are indicated on the right.

Figure 4

Assay for covalent complex formation by topo70^2XCys on the endogenous pRc/CMV DNA. Aliquots of fraction 8 from the sucrose gradient purification of topo70^2XCys–pRc/CMV complexes were incubated under standard reaction conditions at 37°C in the presence (lanes 3 and 5) or absence (lanes 2 and 4) of 10 μM camptothecin (CPT) without (lanes 2 and 3) or with (lanes 4 and 5) DTT. The reactions were stopped with SDS to trap enzyme–DNA covalent complexes and treated with proteinase K before agarose gel electrophoresis. Another aliquot of fraction 8 that had been stored at 4°C was treated with proteinase K and analyzed in lane 1 to establish the baseline level of nicked DNA in the sample. The mobilities of the nicked and topoisomer forms of pRc/CMV DNA are indicated on the left side.

Figure 5

Relaxation of pRc/CMV DNA by the cosedimenting topo70^2XCys enzyme. (A) An aliquot of fraction 8 from the sucrose gradient purification of topo70^2XCys–pRcCMV DNA complexes that had been stored at 4°C was added directly to proteinase K to establish the initial topoisomer distribution for the DNA (lane 1). Additional aliquots were incubated in the absence (lanes 2 and 4) or the presence (lanes 3 and 5) of DTT in reaction buffer at both 23°C and 37°C for 60 min as indicated in the figure, and the reactions were stopped with proteinase K. The agarose gel analysis was carried out in the presence of 0.3 μg/ml chloroquine to enhance the resolution of the topoisomers and separate the topoisomers from the nicked DNA. The mobilities of the various forms of pRc/CMV DNA are labeled as for Fig. 4. (B) A series of reactions containing a sample of fraction 8 from a sucrose gradient similar to one used for the analyses shown in A were initially incubated in the absence (odd numbered lanes) or presence (even numbered lanes) of DTT at 4°C for 60 min. For the zero time control, one of the reactions from each set was stopped by the addition of proteinase K (lanes 1 and 2). The slightly reduced mobility for the topoisomer population in lane 2 compared with lane 1 reflects the fact that the incubation conditions here are slightly different from those before purification through sucrose and, after release by DTT, the enzyme can act on the entire population of topoisomers. The remaining reactions were transferred to 37°C and the incubation was continued for the indicated lengths of time before being stopped by the addition of proteinase K (lanes 3–12). The agarose gel analysis was the same as for A.