MOLECULAR ANALYSIS OF MUTATIONS IN THE HPRT GENE OF V79 HAMSTER FIBROBLASTS - EFFECTS OF IMBALANCES IN THE DCTP, DGTP AND DTTP POOLS

dCMP-deaminase-deficient V79/dC hamster cells have highly imbalanced d eoxyribonucleoside triphosphate (dNTP) pools, i.e. a 17-fold larger dC TP pool, a slightly reduced dTTP and a very low dGTP pool, compared to dCMP-deaminase-proficient V79/p cells. Nevertheless, the two lines sh owed the same rates of spontaneous mutation at the hprt and ouabain-re sistance loci. Analysis of spontaneous hprt mutations indicated an inc rease in misincorporation of C in V79/dC cells, although it was not st atistically significant. When the dCTP pool was further increased five fold by incubating V79/dC cells with cytidine, C misincorporation incr eased to 88%, but the mutation frequency remained unchanged. The dNTP pools of V79/dC cells were also altered by treatment with thymidine, o r with thymidine plus deoxycytidine. After incubation with thymidine a lone, the dCTP pool all but disappeared, whereas it maintained a norma l level in the presence of deoxycytidine. In both cases dTTP rose to n mol amounts, and dGTP accumulated. Incubation with 10 mM thymidine was the only treatment that increased the mutation frequency; T misincorp oration then accounted for 94% of the base substitutions. In the prese nce of deoxycytidine the cells had a dTTP/dCTP ratio of 0.04, but 86% of the base substitutions involved C misincorporation and most probabl y originated from G misincorporation caused by excess dGTP. Alteration s of RNA splicing and hot spots for base substitutions varied with the imbalance, the latter showed ''next-nucleotide effects''. Our results suggest that the fidelity of DNA replication in V79 cells is only aff ected by large changes in the pool and is more sensitive to changes in dGTP than in dCTP or dTTP. (C) 1995 Academic Press Limited