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Bifunctional alkylating agents and other drugs which produce DNA interstrand cross-links (ICLs) are among the most effective antitumor agents in clinical use. In contrast to agents which produce bulky adducts on only one strand of the DNA, the cellular mechanisms which act to eliminate DNA ICLs are still poorly understood, although nucleotide excision repair is known to play a crucial role in an early repair step. Using haploid Saccharomyces cerevisiae strains disrupted for genes central to the recombination, nonhomologous end-joining (NHEJ), and mutagenesis pathways, all these activities were found to be involved in the repair of nitrogen mustard (mechlorethamine)- and cisplatin-induced DNA ICLs, but the particular pathway employed is cell cycle dependent. Examination of whole chromosomes from treated cells using contour-clamped homogenous electric field electrophoresis revealed the intermediate in the repair of ICLs in dividing cells, which are mostly in S phase, to be double-strand breaks (DSBs). The origin of these breaks is not clear since they were still efficiently induced in nucleotide excision and base excision repair-deficient, mismatch repair-defective, rad27 and mre11 disruptant strains. In replicating cells, RAD52-dependent recombination and NHEJ both act to repair the DSBs. In contrast, few DSBs were observed in quiescent cells, and recombination therefore seems dispensable for repair. The activity of the Rev3 protein (DNA polymerase zeta) is apparently more important for the processing of intermediates in stationary-phase cells, since rev3 disruptants were more sensitive in this phase than in the exponential growth phase.

Original publication

DOI

10.1128/mcb.20.10.3425-3433.2000

Type

Journal article

Journal

Molecular and cellular biology

Publication Date

05/2000

Volume

20

Pages

3425 - 3433

Addresses

CRC Drug-DNA Interactions Research Group, Department of Oncology, Royal Free and University College Medical School, University College London, London W1P 8BT, United Kingdom. p.mchugh@ucl.ac.uk

Keywords

Saccharomyces cerevisiae, DNA Damage, Cisplatin, Mechlorethamine, Endodeoxyribonucleases, Exodeoxyribonucleases, Flap Endonucleases, DNA-Directed DNA Polymerase, DNA-Binding Proteins, Fungal Proteins, Saccharomyces cerevisiae Proteins, Antineoplastic Agents, Alkylating, Cross-Linking Reagents, DNA Repair, Recombination, Genetic, Haploidy, Mutation, Rad52 DNA Repair and Recombination Protein