Abstract

Camptothecin (CPT) and its clinically used congeners base their potency as anticancer drugs on the ability to stabilize topoisomerase I-cleaved DNA covalent complexes; such long-lived nucleoprotein complexes constitute a challenge to genomic integrity and cell viability as they can be converted in potentially lethal DNA damage upon collision with replication/transcription machineries [1]. Many DNA structural lesions have been shown to have a similar ‘poisoning’ effect [2]. The observation that PARP inhibitors potentiate the cytotoxicity of CPT in irradiated cells [3] suggests a correlation between poly(ADP-ribose) synthesis and cell’s ability to cope with topoisomerase I-induced DNA damage. Using an in vitro system we investigated whether the steady state level of topoisomerase I-DNA complexes could be modulated by poly(ADP-ribosyl)ation. We observed that both PARP-1 and PARP-2 in their automodified form reduced topoisomerase I-DNA covalent complexes, while they had little or no effect in their native state. The decrease in the number of nucleoprotein complexes occurred even in the presence of CPT and appeared to be specifically dependent on, and proportional to, the amount of PARP-bound poly(ADP-ribose). We found that poly(ADP-ribose) interacts noncovalently with topoisomerase I and identified two binding motifs at critical sites for the enzyme activity. Further experiments revealed that topoisomerase I in covalent complex with its substrate was a preferred target for poly(ADP-ribose). Such specific targeting resulted in a complete reversion of the cleavage complexes, in spite of the stabilizing effect of CPT. Work is in progress to evaluate the occurrence of such poly(ADP-ribose)-mediated repair of topoisomerase I-DNA covalent complexes in living cells. References: 1.Kohn KW, Pommier Y: Annals NY Acad Sci, 2000; 922: 11-26 2.Pourquier P, Waltman JL, Urasaki Y et al: Cancer Res, 2001; 61: 53-58 3.Bowman KJ, Newell DR, Calvert AH, Curtin NJ: Br J Cancer, 2001; 84: 106-112

Keywords: PARP-2, noncovalent interactions, DNA Repair