While investigating the novel anticancer drug ecteinascidin 743 (Et743), a natural marine product isolated from the Caribbean sea squirt, we discovered a new cell-killing mechanism mediated by DNA nucleotide excision repair (NER). A cancer cell line selected for resistance to Et743 had chromosome alterations in a region that included the gene implicated in the hereditary disease xeroderma pigmentosum (XPG, also known as Ercc5). Complementation with wild-type XPG restored the drug sensitivity. Xeroderma pigmentosum cells deficient in the NER genes XPG, XPA, XPD or XPF were resistant to Et743, and sensitivity was restored by complementation with wild-type genes. Moreover, studies of cells deficient in XPC or in the genes implicated in Cockayne syndrome (CSA and CSB) indicated that the drug sensitivity is specifically dependent on the transcription-coupled pathway of NER. We found that Et743 interacts with the transcription-coupled NER machinery to induce lethal DNA strand breaks.

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1-␤-D-Arabinofuranosylcytosine (Ara-C) is a nucleoside analog commonly used in the treatment of leukemias. Ara-C inhibits DNA polymerases and can be incorporated into DNA. Its mechanism of cytotoxicity is not fully understood. Using oligonucleotides and purified human topoisomerase I (top1), we found a 4-to 6-fold enhancement of top1 cleavage complexes when ara-C was incorporated at the ؉1 position (immediately 3) relative to a unique top1 cleavage site. This enhancement was primarily due to a reversible inhibition of top1-mediated DNA religation. Because ara-C incorporation is known to alter base stacking and sugar puckering at the misincorporation site and at the neighboring base pairs, the observed inhibition of religation at the ara-C site suggests the importance of the alignment of the 5-hydroxyl end for religation with the phosphate group of the top1 phosphotyrosine bond. This study also demonstrates that ara-C treatment and DNA incorporation trap top1 cleavage complexes in human leukemia cells. Finally, we report that camptothecin-resistant mouse P388͞ CPT45 cells with no detectable top1 are crossresistant to ara-C, which suggests that top1 poisoning is a potential mechanism for ara-C cytotoxicity.camptothecin ͉ DNA repair ͉ DNA damage ͉ nucleoside analog D NA topoisomerases I (top1) are essential and ubiquitous enzymes (1, 2). They are critical for DNA replication and transcription by regulating the topological state of DNA by means of reversible transesterification reactions (3-5). Eukaryotic top1 reversibly cleaves one strand of the DNA by binding covalently to the 3Ј end of the broken DNA (6). This intermediate is referred to as the top1 cleavage complex. Top1 also mediates religation of the DNA. Under normal conditions, the religation step of the equilibrium is favored and only a small fraction of the DNA is cleaved (4). Top1 inhibitors, such as camptothecin (CPT) and its derivatives, stabilize (trap) the cleavage complexes by inhibiting top1-mediated DNA religation (7,8). Trapping of cleavage complexes by CPT converts the top1 enzyme into a cellular poison, and top1-mediated DNA damage probably results from replication or transcription complex collisions with CPT-arrested top1-DNA covalent complexes (7,8).DNA damages, such as base mismatches, abasic sites, UV photo-lesions, and ethenoadenine adducts, can also stabilize top1 cleavage complexes by inhibiting top1-mediated DNA religation (9-11). Oxidized bases, and benzo[a]pyrene adducts, on the other hand, enhance top1 cleavage complexes by enhancing the cleavage step (forward rate) of the nicking-closing reaction (12, 13).