Locking the DNA topoisomerase I protein clamp: A novel mechanism of cytotoxicity.

Proc Amer Assoc Cancer Res, Volume 47, 2006 5523 DNA topoisomerase I (Top1) plays a critical role in DNA replication, transcription and recombination. Top1 forms a protein clamp around duplex DNA to catalyze changes in DNA topology. The enzyme transiently cleaves a single DNA strand to generate a covalent Top1-DNA complex, which is reversibly stabilized by camptothecin (CPT), resulting in S-phase dependent cell killing. Crystallographic studies of a 70 kDa fragment of human Top1 (Topo70) complexed with DNA demonstrate a lone salt bridge between opposable “Lip” domains in Top1 completes the circumferential binding of DNA. The tight packing of the DNA within the central protein pore predicted substantial movement of domains would be necessary for DNA binding and strand rotation within the covalent complex. Using molecular modeling, we reported that cysteine residues substituted for Gly365 and Ser534 formed a spontaneous disulfide bond to lock the Top1-clamp534 closed, which inhibited DNA rotation (Woo et al., 2003 PNAS 100:13767). In contrast, crosslinking Lys369 and Glu497 (salt bridge) loops in the context of truncated Topo70 (Topo70-clamp499), DNA rotation was not inhibited (Carey et al, 2003 PNAS 100:5640). As the G365C–S534C loops are more proximal to the active site than the salt bridge loops, a disulfide bond at this position may have more profound effects in impeding DNA rotation. Alternatively, the Top1-clamp534 was full-length, while the Topo70-clamp499 crosslink lacked the N-terminus. To address these issues, the activity of both clamps were examined in yeast. In contrast to Top1-clamp534, expression of Top1-clamp499 did not induce yeast cell lethality, even in the more oxidizing environment of a glr1Δ strain. These data suggest that locking of the full length Top1-clamp499 around duplex DNA does not readily occur in vivo. A defect in Top1-clamp499 DNA binding was further evident in the distributive mode of DNA relaxation evident in vivo, and the low salt optimum for catalytic activity in vitro. Although both Top1-clamp499 and Top1-clamp534 enzymes were sensitive to CPT in DNA cleavage assays, genetic analyses of replication and DNA damage checkpoint mutants indicated that the defects in DNA binding by Top1-clamp499 also diminished the production of CPT-induced DNA lesions in vivo. The N-terminus also affected Top1-clamp activity. Topo70-clamp534 was less toxic than Top1-clamp534, while in a glr1Δ strain, deletion of the N-terminus completely suppressed the cytotoxicity of a catalytically inactive Top1-clamp. These results demonstrate that the N-terminal domain is critical for effective clamping of Top1 around duplex DNA and for the cytotoxic activity of this novel Top1 poisons. This work was supported by NIH grant CA58775 and ALSAC (MAB) and MIUR cofin 2003 (PB).