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Reference: Palle K, et al. (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(41):27767-75

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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-DNA intermediate. The active site proximal disulfide bond in full-length Top1-Clamp534 restricted DNA rotation (Woo et al., Proc Natl Acad Sci USA 100, 13767), while the more distal disulfide bond of the N-terminally truncated Topo70-Clamp499 did not (Carey et al., Proc Natl Acad Sci USA 100, 5640). 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 activity 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.

Reference Type
Journal Article
Authors
Palle K, Pattarello L, van der Merwe M, Losasso C, Benedetti P, Bjornsti MA
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