Repair of intermediate structures produced at DNA interstrand cross-links in Saccharomyces cerevisiae

Bifunctional alkylating agents and other drugs which produce DNA interstran d cross-links (ICLs) are among the most effective antitumor agents in clini cal use. In contrast to agents which produce bulky adducts on only one stra nd of the DNA, the cellular mechanisms which act to eliminate DNA ICLs are still poorly understood, although nucleotide excision repair is known to pl ay a crucial role in an early repair step. Using haploid Saccharomyces cere visiae strains disrupted for genes central to the recombination, nonhomolog ous 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 held electrophoresis revealed the inte rmediate in the repair of ICLs in dividing cells, which are mostly in S pha se, to be double-strand breaks (DSBs), The origin of these breaks is not cl ear since they mere still efficiently induced in nucleotide excision and ba se excision repair-deficient, mismatch repair defective, rad27 and mre11 di sruptant strains. In replicating cells, RAD52-dependent recombination and N HEJ both act to repair the DSBs. In contrast, few DSBs were observed in qui escent cells, and recombination therefore seems dispensable for repair. The activity of the Rev3 protein (DNA, polymerase zeta) is apparently more imp ortant for the processing of intermediates in stationary phase cells, since rev3 disruptants were more sensitive in this phase than in the exponential growth phase.