Effects of flanking base sequences on 5-bromodeoxyuridine mutagenesis in mammalian cells

Abstract

The molecular mechanisms of incorporation-dependent, 5-bromodeoxyuridine (BrdU)-induced mutagenesis were analyzed in murine A9 cells that possess a single copy of theEscherchia 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 integratedgpt 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 thegpt 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.