Induction of sister-chromatid exchanges by the replication of 5-bromouracil-substituted DNA under conditions of nucleotide-pool imbalance

Abstract

The induction of sister-chromatid exchanges (SCEs) by the replication of 5-bromouracil(BrUra)-containing DNA under conditions of nucleotide-pool imbalance was investigated. A modification of a protocol developed for the induction of mutations under these conditions (.E.R. Kaufman, Mol. Cell. Biol., 4, 2449–2454, 1984) was used. To induce SCEs, Chinese hamster ovary cells were grown under non-mutagenic conditions which allowed the uniform incorporation of BrUra into their DNA at specific levels of substitution for thymine residues (25, 50 and 75% BrUra substitution). After 4 and 5 days of growth, the cells, which had incorporated BrUra into their DNA, were washed free of 5-bromodeoxyuridine (BrdUrd) and provided with fresh culture medium supplemented with various concentrations of thymidine (10 μM to 3 mM) and no BrdUrd. The cells were allowed to replicate their BrUra-containing DNA under these conditions, in the absence of BrdUrd, for two rounds of DNA synthesis to achieve sister-chromatid differentiation, and second-division metaphase were scored for SCEs. The results of these studies indicated that the SCEs observed (i) were proportional to the level of BrUra substituted for thymine in the cellular DNA, (ii) were induced by increasing concentrations of thymidine in the culture medium during replication of the BrUra-containing DNA, (iii) correlated well with the induction of mutations to thioguanine resistance and to ouabain resistance, (iv) correlated with increase in the intracellular levels of dTTP and dGTP generated by the high concentrations of thymidine. These findings provide direct evidence for the induction of SCEs by the replication of BrUra-containing DNA and for the importance of the pools of nucleotide triphosphate precursors for DNA replication in these processes. When the effects of 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) synthesis, were tested, it was found that 3-aminobenzamide significantly increased SCEs, but it had no effect on mutations induced.