Conversion of Topoisomerase I Cleavage Complexes on the Leading Strand of Ribosomal DNA into 5′-Phosphorylated DNA Double-Strand Breaks by Replication Runoff

Abstract

Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replicationmediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3 DNA ends are extended by DNA polymerase in vivo closely to the 5 ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5 ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5 kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA doublestrand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA.DNA topoisomerases are ubiquitous enzymes that regulate the topological state of DNA. They participate in essential cellular processes, including replication, transcription, chromosome segregation, and recombination (22,34,71). Eukaryotic DNA topoisomerase I (top1) acts as a monomer, and its catalytic activity can be divided into four steps (61): (i) binding of the enzyme to duplex DNA, (ii) single-stranded DNA cleavage by a transesterification reaction in which a top1 tyrosinehydroxyl group becomes covalently linked to the 3Ј phosphate of a DNA phosphodiester bond to generate a 5Ј-hydroxyl DNA terminus, (iii) DNA relaxation by controlled rotation around the intact DNA strand (61); and (iv) religation of the cleaved DNA by nucleophilic attack from the 5Ј-hydroxyl DNA end and dissociation of the top1 tyrosyl residue from the 3Ј end. The topoisomerase-linked DNA breaks are commonly referred to as cleavage complexes (22,34,71). Under physiological conditions, they are short-lived catalytic intermediates.A number of physiological and environmental DNA modifications can inhibit top1 by inducing top1 cleavage complexes. These include DNA mismatches or abasic sites (37, 48, 73), oxidative base damage (47), base alkylation and carcinogenic adducts (44, 66), UV photoproducts (50, 62), and DNA breaks (11, 45). Trapping of top1 cleavage complexes is also the primary mechanism of action of camptothecin (CPT), a potent anticancer agent which reversibly inhibits the religation step of the top1 catalytic cycle (25,29,39,40). The cytotoxicity of top1 cleavage complexes is atte...

Genetic evidence for a role of Saccharomyces cerevisiae Mph1 in recombinational DNA repair under replicative stress

“…We found viable mph1 srs2 segregants synergistically sensitive to MMS and 4-NQO, thought to form DNA lesions potently blocking replisome progression (Pegg, 1984;Ramotar et al, 1998) and, in particular, sensitive to camptothecin ( Figure 4A). Camptothecin is thought to induce replication fork collapse and the formation of double-strand ends (Pommier et al, 1998;Strumberg et al, 2000;Lin et al, 2000). The only way to restart replication in this case is probably via HR.…”

“…2. mph1 and srs2 single mutants show moderate sensitivity to camptothecin (Scheller et al, 2000;Mankouri et al, 2002), which is synergistically increased in viable mph1 srs2 mutants ( Figure 4A). This could indicate fork collapse, as induced by camptothecin (Pommier et al, 1998;Strumberg et al, 2000;Lin et al, 2000), to seriously compromise viability of mph1 srs2 cells. The models in Figure 6 could account for this observations.…”

Hyperphosphorylation of RNA Polymerase II in Response to Topoisomerase I Cleavage Complexes and Its Association with Transcription- and BRCA1-dependent Degradation of Topoisomerase I

“…18 To examine whether replication-induced DNA double-strand breaks induced Pol II hyperphosphorylation, we used the DNA polymerase inhibitor aphidicolin, which prevents CPT-induced replicationdependent DNA double-strand breaks. 30 Figure 2d shows that aphidicolin did not affect CPTinduced Pol II hyperphophorylation. In accordance with this observation, Pol II hyperphosphorylation was also induced by CPT in non-replicating human lymphocytes isolated from peripheral blood (Fig.…”

Tyrosyl-DNA Phosphodiesterase as a Target for Anticancer Therapy

“…This is especially the case when Top1 relaxes positive supercoiling [10]. Failure to reseal this normally transient break in the DNA results in the generation of a Top1-linked DNA single-strand break that can be transformed into a prolonged doublestranded break following collision of replication forks with the drug-stabilized cleavage complex [11][12][13][14]. In a similar manner, a number of naturally occurring DNA lesions, such as strand breaks [15], abasic sites, base mismatches, and certain oxidized or modified bases [16], have also been shown in vitro to physically block the Top1 religation reaction through the misalignment the 5′-hydroxyl with the tyrosyl-DNA phosphodiester backbone.…”