Thymidine-induced mutations in mammalian cells: sequence specificity and implications for mutagenesis in vivo

Imbalances in the intracellular nucleotide precursor pools in mammalian cells can result in the induction of mutations during the DNA replication process. By using a shuttle vector system developed in our laboratory, we have analyzed the sequence specificity of mutations induced in mouse A9 cells by exposure of the cells to a high concentration of thymidine. The target for mutagenesis in these studies was the bacterial gpt gene stably integrated into the chromosomal DNA of the mouse cells. Previous studies in this laboratory had generated a large panel of xanthine guanine phosphoribosyl-transferase (EC 2.4.2.22)-negative mutant lines that possess single-base mutations within the gpt coding sequence. This study utilized four xanthine guanine phosphoribosyltransferase-negative mutant lines to assess the frequency of mutation induced by thymidine at guanine residues in four sequence contexts: the 5' and 3' guanine residues of a GG doublet, the middle guanine residue of a GGG triplet, and the 3' guanine residue of a GGGG quartet. The results of this study demonstrate that treatment of cultured cells with a high concentration of thymidine can result in G.C----A.T transition mutations that occur preferentially at the 3' guanine residue of a run of two or more adjacent guanines. Guanine residues flanked on their 3' side by other guanine residues are severalfold less mutable by thymidine than are guanine residues flanked on their 3' side by a different base. This study demonstrates a sequence-specific mode for thymidine-induced mutations and suggests implications for mutagenesis in vivo.