Abstract
Human topoisomerase I is a 765-residue protein composed of four major domains as follows: the unconserved and highly charged NH(2)-terminal domain, a conserved core domain, the positively charged linker region, and the highly conserved COOH-terminal domain containing the active site tyrosine. Previous studies of the domain structure revealed that near full topoisomerase I activity can be reconstituted in vitro by fragment complementation between recombinant polypeptides approximating the core and COOH-terminal domains. Here we demonstrate that deletion of linker residues Asp(660) to Lys(688) yields an active enzyme (topo70DeltaL) that purifies as both a monomer and a dimer. The dimer is shown to result from domain swapping involving the COOH-terminal and core domains of the two subunits. The monomeric form is insensitive to the anti-tumor agent camptothecin and distributive during in vitro plasmid relaxation assays, whereas the dimeric form is camptothecin-sensitive and processive. However, the addition of camptothecin to enzyme/DNA mixtures causes enhancement of SDS-induced breakage by both monomeric and dimeric forms of the mutant enzyme. The similarity of the dimeric form to the wild type enzyme suggests that some structural feature of the dimer is providing a surrogate linker. Yeast cells expressing topo70DeltaL were found to be insensitive to camptothecin.
Highlights
The free DNA 5Ј-hydroxyl that reseals the phosphodiester bond and releases the enzyme from the DNA [1]
Unlike topo70 that elutes as a single species from the column [34], the topo70⌬L proteins eluted as two distinct peaks at ϳ140 and ϳ170 mM KPO4, irrespective of the status of the active site residue 723
The crystal structure of human topoisomerase I reveals that the 77-residue linker region connecting the core domain to the COOH-terminal domain consists of an antiparallel coiled-coil that protrudes from the remainder of the protein [2]
Summary
Plasmids were constructed using conventional cloning techniques [42] and propagated using the Escherichia coli strain TOP10FЈ (mcrA, ⌬(mrr-hsdRMS-mcrBC), 80⌬lacZ⌬M15, ⌬lacX74, deoR, recA1, araD139, ⌬(ara, leu)7697, galU, galK, rpsL, endA1, nupG (FЈ: lacIq Tn10 tetr)) (Invitrogen). PADH1-topoI—In a three-fragment ligation reaction, the plasmid pADH1-topoI was constructed by replacing the GAL4 DNA binding domain sequence in GBT9 (HindIII411 (end-filled) to BamHI880) [44] with sequences encoding the human topoisomerase I cDNA. The resulting plasmid was subjected to oligonucleotide-directed mutagenesis in order to restore a BamHI site to the 5Ј end of the topoisomerase I cDNA. PGB-topo58 —An ϳ2.9-kbp BamHI (end-filled) to PstI fragment from pAc-topo58 [34] was inserted into the polylinker of pGBT9 to generate pGB-topo which encodes a fusion protein comprised of the Gal DNA binding domain fused to topo. The resulting plasmid was subjected to oligonucleotide-directed mutagenesis in order to restore a BamHI site to the 5Ј end of the topo cDNA. The purification of topo by factor Xa cleavage of the GST fusion protein and subsequent purification by Mono S column (5H/R) chromatography were described previously [41]
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