Effects of flanking base sequences on 5-bromodeoxyuridine mutagenesis in mammalian cells.
The molecular mechanisms of incorporation-dependent, 5-bromodeoxyuridine (BrdU)-induced mutagenesis were analyzed in murine A9 cells that possess a single copy of the Escherichia coli gpt gene integrated into the chromosomal DNA as part of a shuttle vector. Four independently derived GPT- mutants with single base changes within the integrated gpt gene were utilized in BrdU-induced reversion analyses to test the relative mutability of guanine residues in four different settings: the 5' and 3' guanine residues of a GG doublet, the 3' guanine residue of a GGGG quartet, and the middle guanine residue of a GGG triplet. Two of the mutant lines possessed GG doublet sequences in which a GC----AT transition at either guanine residue of the doublet leads to restoration of GPT enzyme activity without restoring wild-type DNA sequence. Both lines were shown to be effectively reverted by BrdU incorporation-dependent mutagenesis, and sequencing of the gpt genes from numerous independently derived revertants of both lines demonstrated that greater than 90% of the revertants arose due to GC----AT transitions at the 3' guanine residue of the doublet. BrdU-induced reversion of two additional GPT- mutant lines demonstrated that the 3' guanine residue of a GGGG quartet is efficiently mutated, while the middle guanine residue of a GGG triplet sequence is at least 10-fold less mutable by BrdU incorporation-dependent mutagenesis than the 3' guanine residue of a GG doublet or GGGG quartet. All four mutant lines tested were equally revertible by treatment with the alkylating agent ethyl methane sulfonate. The results from this study define a sequence-specific mechanism for BrdU-induced, incorporation-dependent mutagenesis and demonstrate the use of reversion analysis for the determination of sequence specific effects at precise sites within a gene.
Kresnak
M T
MT
Department of Genetics, University of Illinois College of Medicine, Chicago 60612.
Davidson
R L
RL
D000477:Alkylating Agents / Q000633:toxicity; D000818:Animals; D001482:Base Composition / Q000187:drug effects; D001483:Base Sequence / Q000187:drug effects; D001973:Bromodeoxyuridine / Q000633:toxicity*; D002460:Cell Line; D005020:Ethyl Methanesulfonate / Q000633:toxicity; D005786:Gene Expression Regulation / Q000187:drug effects; D007041:Hypoxanthine Phosphoribosyltransferase / Q000187:drug effects / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009153:Mutagens
D000477:Alkylating Agents; D009153:Mutagens; D005020:Ethyl Methanesulfonate; D007041:Hypoxanthine Phosphoribosyltransferase; D001973:Bromodeoxyuridine