Molecular spectrum of mutations induced by 5-hydroxymethyl-2'-deoxyuridine in (CHO)-PL61 cells

We have utilized (CHO)-PL61 cells to characterize the mutations produced in mammalian cells by exogenous treatment with the nucleoside 5-hydroxymethyl-2'-deoxyuridine (hmdUrd). HmdUrd is incorporated into DNA as a thymidine analogue and is removed by the repair enzyme hmUra-DNA glycosylase. PL61 cells are hprt(-) and contain adjacent single copies of the Escherichia coli gpt and ileo genes (gpt(+), neo(+)) separated by 2 kb, rendering the cells thioguanine sensitive (TG(s)) and geneticin resistant (G418(r)). Cells were exposed to hmdUrd and the colonies resistant to thioguanine or thioguanine and G418 were selected. Selection in thioguanine alone (TG(r)/gpt(-)) allows the growth of all gpt(-) mutants (small, intermediate and large deletions/insertions and point mutations) while selection in thioguanine and G418 (TG(r)/gpt(-), G418(r)/neo(+)) prevents survival of colonies containing vary large deletions of the gpt gene that include the neo gene. To confirm the types of mutation at the molecular level, the gpt gene was amplified from mutants' genomic DNA by PCR, and the amplified DNA was sequenced directly by the dideoxy method. Our study showed that 4 mu M hmdUrd induced mutations to TG(r)/gpt(-) at a rate 3-4 times that of control, but showed no marked increase in mutation to TG(r)/gpt(-), G418(r)/neo(+). The predominant type of hmdUrd induced mutation in the thioguanine resistant cells at the gpr locus was complete loss of the gpt gene resulting from a large deletion. Background mutations were generally point mutations or small insertion/deletion mutations. We propose that hmdUrd induces large/intermediate deletions as a major type of mutations in mammalian cells as a consequence of DNA repair, and not as a result of misincorporation or mispairing, suggesting that base excision repair by itself can lead to large deletion mutagenesis.