Disulfide Cross-links Reveal Conserved Features of DNA Topoisomerase I Architecture and a Role for the N Terminus in Clamp Closure

Abstract

In eukaryotes, DNA topoisomerase I (Top1) catalyzes the relaxation of supercoiled DNA by a conserved mechanism of transient DNA strand breakage, rotation, and religation. The unusual architecture of the monomeric human enzyme comprises a conserved protein clamp, which is tightly wrapped about duplex DNA, and an extended coiled-coil linker domain that appropriately positions the C-terminal active site tyrosine domain against the Top1 core to form the catalytic pocket. A structurally undefined N-terminal domain, dispensable for enzyme activity, mediates protein-protein interactions. Previously, reversible disulfide bonds were designed to assess whether locking the Top1 clamp around duplex DNA would restrict DNA strand rotation within the covalent Top1- To assess the contribution of the N-terminal domain to the dynamics of Top1 clamping of DNA, the same disulfide bonds were engineered into full-length Top1 and truncated Topo70, and the activities of these proteins were assessed in vitro and in yeast. Here we report that the N terminus impacts the opening and closing of the Top1 protein clamp. We also show that the architecture of yeast and human Top1 is conserved in so far as cysteine substitutions of the corresponding residues suffice to lock the Top1-clamp. However, the composition of the divergent N-terminal/linker domains impacts Top1-clamp activity and stability in vivo.Eukaryotic Top1 (DNA topoisomerase I) is a highly conserved enzyme, which plays critical roles in cellular processes, such as DNA replication, transcription, recombination, and chromosome condensation (1-4). Top1 catalyzes changes in DNA topology or the linkage of the individual strands that comprise duplex DNA. The enzyme first binds double-stranded DNA as a protein clamp and then transiently cleaves a single strand forming a 3Ј-phosphotyrosyl linkage between the active site tyrosine and the nicked DNA. Torsional or flexural strain in the DNA then drives the rewinding or unwinding of the polynucleotide strands. In the subsequent transesterification reaction, the free 5Ј-OH acts as a nucleophile to resolve the phosphotyrosyl linkage and religate the phosphodiester backbone of the DNA. A random number of supercoils are removed in this process to effect changes in DNA linking number (5, 6).Top1 is also the cellular target of the camptothecin (CPT) 4 class of chemotherapeutic agents (3, 7-9). These drugs convert the enzyme into a cellular poison by reversibly stabilizing the Top1-DNA covalent complex. CPT intercalates into the protein-linked DNA complex, effectively displacing the 5Ј-OH and preventing DNA religation (10, 11). Although ternary CPTTop1-DNA complexes form throughout the cell cycle, the interaction of these complexes with the replication machinery during S-phase converts the readily reversible intermediate into irreversible DNA lesions that trigger cell death. Our recent in vivo studies and single molecule nanomanipulations revealed that CPT selectively slows Top1 uncoiling of positive supercoils, thereby inducing the accumulation of ...

DNA Topoisomerase I Domain Interactions Impact Enzyme Activity and Sensitivity to Camptothecin

“…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 yTop1 clamp constructs (20).…”

“…We previously demonstrated that locking the human Top1 clamp around the DNA prevents strand rotation within the Top1-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.…”

“…Placing the disulfide "lock" in close proximity to the active site Tyr 723 prevented DNA strand rotation within the covalent enzyme-DNA intermediate but did not affect CPT stabilization 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).…”

“…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 because of Top1 clamping of the DNA. Although the biochemical behavior of the (yL)-hTop1 did not suggest alterations in DNA binding, i.e.…”

“…The N terminus has also been implicated in numerous interactions with other proteins, including TATAbinding protein, nucleolin, and SV40 large T antigen (35)(36)(37)(38). Additional studies suggest a role for the N-terminal domain in the opening and closing of the Top1 clamp around DNA (20) and in CPT-induced alterations in the intracellular localization of Top1 (39).…”