Increased spontaneous mutation frequency in human cells expressing the phage PBS2-encoded inhibitor of uracil-DNA glycosylase

The Ugi protein inhibitor of uracil-DNA glycosylase encoded by bacteriophag e PBS2 inactivates human uracil-DNA glycosylases (UDG) by forming a tight e nzyme:inhibitor complex. To create human cells that are impaired for UDG ac tivity, the human glioma U251 cell line was engineered to produce active Ug i protein. In vitro assays of crude cell extracts from several Ugi-expressi ng clonal lines showed UDG inactivation under standard assay conditions as compared to control cells, and four of these UDG defective cell Lines were characterized for their ability to conduct in vivo uracil-DNA repair. Where as transfected plasmid DNA containing either a U:G mispair or U:A base pair s was efficiently repaired in the control lines, uracil-DNA repair was not evident in the lines producing Ugi. Experiments using a shuttle vector to d etect mutations in a target gene showed that Ugi-expressing cells exhibited a S-fold higher overall spontaneous mutation frequency compared to control cells, due to increased C:G to T:A base pair substitutions. The growth rat e and cell cycle distribution of Ugi-expressing,a cells did not differ appr eciably from their parental cell counterpart. Further in vitro examination revealed that a thymine DNA glycosylase (TDG) previously shown to mediate U gi-insensitive excision of uracil bases from DNA was not detected in the pa rental U251 cells. However, a Ugi-insensitive UDG activity of unknown origi n that recognizes U:G mispairs and to a lesser extent U:A base pairs in dup lex DNA, but which was inactive toward uracil residues in single-stranded D NA, was detected under assay conditions previously shown to be efficient fo r detecting TDG. (C) 2000 Elsevier Science B.V. All rights reserved.