Endonuclease cleavage of blocked replication forks: An indirect pathway of DNA damage from antitumor drug–topoisomerase complexes

Abstract

The cytotoxicity of several important antitumor drugs depends on formation of the covalent topoisomerase-DNA cleavage complex. However, cellular processes such as DNA replication are necessary to convert the cleavage complex into a cytotoxic lesion, but the molecular mechanism of this conversion and the precise nature of the cytotoxic lesion are unknown. Using a bacteriophage T4 model system, we have previously shown that antitumor drug-induced cleavage complexes block replication forks in vivo. In this report, we show that these blocked forks can be cleaved by T4 endonuclease VII to create overt DNA breaks. The accumulation of blocked forks increased in endonuclease VII-deficient infections, suggesting that endonuclease cleavage contributes to fork processing in vivo. Furthermore, purified endonuclease VII cleaved the blocked forks in vitro close to the branch points. These results suggest that an indirect pathway of branched-DNA cleavage contributes to the cytotoxicity of antitumor drugs that target DNA topoisomerases. S everal important classes of antitumor drugs, as well as the antibacterial quinolones, inhibit type II topoisomerases by a common mechanism of action (1-3). These drugs alter the equilibrium of the reaction cycle to favor an otherwise transient reaction intermediate called the cleavage complex. Within the cleavage complex, the enzyme is covalently linked, by means of phosphotyrosine bonds, to the cleaved DNA at two staggered positions on the opposing strands of the helix. Camptothecin and related anticancer drugs attack eukaryotic topoisomerase I by a related mechanism, trapping a cleavage complex with a single cleaved phosphodiester bond (4).Various results demonstrate that cytotoxicity of type II topoisomerase inhibitors depends on formation of the cleavage complex, and not just inhibition of enzyme activity (2, 5, 6). However, the cleavage complex itself is reversible, and is thus unlikely to be the cytotoxic lesion (7). Instead, cellular processes apparently convert the cleavage complex into a cytotoxic lesion (8,9). DNA replication appears to play an important role in this conversion because S phase cells are more sensitive to topoisomerase inhibitors than G 1 cells, and because the replication inhibitor aphidicolin can abrogate drug sensitivity (8-11). Despite the implication that DNA replication is involved in cytotoxicity, the exact nature of the cytotoxic lesion and the pathway of its formation are not clear.A drug-induced cleavage complex with a type II topoisomerase contains a latent double-strand DNA break (DSB), but in vitro this break is revealed only when the protein is denatured with a strong detergent such as SDS (see ref. 5). Nonetheless, overt DNA breaks are apparently generated from the druginduced cleavage complexes in vivo, and these breaks are probably important in cytotoxicity. Several types of indirect evidence support this view. First, mutants deficient in DSB repair are hypersensitive to topoisomerase inhibitors that induce the cleavage complex (12-15). Second, cleava...

5-Azacytidine–Induced Methyltransferase-DNA Adducts Block DNA Replication In vivo

“…Replication fork blockage can allow a branch-specific endonuclease to cleave one arm of a fork (30,42), and then two general models for RC replication are possible. In one model, RC replication ensues after gap repair/ligation at the broken fork and replication restart at the unbroken fork ( Supplementary Fig.…”

Hypersensitivity of Nonhomologous DNA End-joining Mutants to VP-16 and ICRF-193

“…Recently, topo II poison-induced cleavable complexes have been shown to be proteolytically degraded by the ubiquitin/26 S proteasome pathway, and a model is suggested in which the repair of cleavable complexes may involve transcription-dependent topo II proteolysis to reveal the protein-concealed DSBs (41). It is also suggested that the collision of DNA replication machinery with cleavable complexes leads to generation of overt DSBs (42). Such protein-free DSBs would then be repaired by the DSB repair pathway(s).…”

Regression supports two mechanisms of fork processing in phage T4

“…This decrease is consistent with EndoVII-mediated cleavage of the regressed fork Holliday junction. However, previous reports have suggested that EndoVII also cleaves Y-shaped molecules, such as the origin fork, in vivo (14,15). Direct cleavage of the origin fork junction by EndoVII would decrease the number of origin forks available for regression, indirectly decreasing the accumulation of regressed origin forks.…”

“…Several reports have described the appearance of DSEs (and/or broken chromosomes) in association with replication fork stalling (15,(25)(26)(27)(28). DSEs could be created from stalled forks by several possible mechanisms, including fork breakage during inactivation, fork cleavage by endonucleases, head-to-tail fork collisions, and fork regression.…”

“…The major function of EndoVII appears to be the resolution of branched molecules before packaging of DNA into phage heads. In contrast to some other resolvases, such as RuvC, EndoVII can act on a broad range of DNA structures in vitro, including fork-shaped structures (14)(15)(16).…”