The inefficiency of incisions of ecteinascidin 743-DNA adducts by the UvrABC nuclease and the unique structural feature of the DNA adducts can be used to explain the repair-dependent toxicities of this antitumor agent.
BACKGROUND
Ecteinascidin 743 (Et 743), a natural product derived from a marine tunicate, is a potent antitumor agent presently in phase II clinical trials. Et 743 binds in the minor groove of DNA and alkylates N2 of guanine via a unique mechanism involving catalytic activation. The sequence selectivity of Et 743 is governed by different patterns of hydrogen-bonding to DNA, which results in differential reversibility of the covalent adducts. As determined by nuclear magnetic resonance spectroscopy, the preferred sequences 5'-PuGC and 5'-PyGG are stabilized by a hydrogen-bonding network, while the non-preferred sequences 5'-NG(A/T) are much less stabilized due to the lack of a key hydrogen bond to the GC base pair on the 3'-side of the alkylated guanine.


RESULTS
Mammalian cell lines (XPB, XPD, XPF, XPG, and ERCC1) deficient in the nucleotide excision repair (NER) gene products show resistance to Et 743. The recognition and subsequent incision of Et 743-DNA adducts by the bacterial multisubunit endonuclease UvrABC were used to evaluate DNA repair-mediated toxicity as a rationale for the resistance of NER-defective cell lines and the antitumor activity of Et 743. The Et 743-DNA adducts are indeed recognized and incised by the UvrABC repair proteins; however, the pattern of incision indicated that the non-preferred, and less stable, sequences (i.e. 5'-NG(A/T)) modified with Et 743 are generally incised at a much higher efficiency than the preferred, more stable sequences (i.e. 5'-PuGC or 5'-PyGG). In addition, within the same Et 743 recognition sequence, the level of incision varies, indicating that flanking regions also contribute to the differential incision frequency.


CONCLUSIONS
The inefficient repair incision by the UvrABC nuclease of Et 743-DNA adducts provides a basis for rationalizing the observed repair-dependent cytotoxicities of these DNA adducts, if other associated structural properties of Et 743-DNA adducts are taken into account. In particular, the wedge-shaped Et 743, which forces open the minor groove of DNA, introducing a major groove bend, and the extrahelical protrusion of the C-subunit of Et 743 provide unique characteristics alongside the hydrogen-bonding stabilization of a covalent DNA adduct, which we propose traps an intermediate in NER processing of Et 743-DNA adducts. This trapped intermediate protein-Et 743-DNA adduct complex can be considered analogous to a poisoned topoisomerase I- or topoisomerase II-DNA complex. In the absence of an intact NER nuclease complex, this toxic lesion is unable to form, and the Et 743-DNA adducts, although not repaired by the NER pathway, are less toxic to cells. Conversely, elevated levels of either of these nucleases should lead to enhanced Et 743 toxicity.
Zewail-Foote
M
M
Department of Chemistry and Biochemistry, The University of Texas at Autin, 78712, USA.
Li
V S
VS
Kohn
H
H
Bearss
D
D
Guzman
M
M
Hurley
L H
LH
D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism; D004260:DNA Repair; D004149:Dioxoles / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D016503:Drug Delivery Systems; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods; D006801:Humans; D007546:Isoquinolines / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods; D044005:Tetrahydroisoquinolines; D000077606:Trabectedin
D018906:Antineoplastic Agents, Alkylating; D018736:DNA Adducts; D004149:Dioxoles; D029968:Escherichia coli Proteins; D007546:Isoquinolines; D044005:Tetrahydroisoquinolines; D004247:DNA; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC; D000077606:Trabectedin