Site directed substitution of 5-hydroxymethyluracil for thymine in replicating øX-M4am3 DNA via synthesis of 5-hydroxymethyl-2'-deoxyuridine-5'-triphosphate

Abstract

5-hydroxymethyluracil (HmUra) is formed in DNA as a product of oxidatlve attack on the methyl group of Thy. It Is removed from DNA by HmUra-DNA glycosylase. To determine whether the replacement of Thy by HmUra is mutagenic, which might explain the repairabillty of HmUra, a HmUra residue was substituted for Thy In a target (amber) codon by in vitro extension of an oligonucleotlde primer annealed to øX-174am3 virion DNA. This was accomplished by synthesizing HmdUTP and using DNA polymerase to effect primer extension. E.coll spheroplasts were transfected with the HmUra-containlng DNA and the yield of revertant phage determined following replication In the bacterial host. Since E. coli do not express HmUra-DNA glycosylase activity, mutagenesis could be assessed In the absence of repair. X2c analysis showed that replacing Thy with HmUra did not result in an Increase In revertant phage. These data indicate that the oxidation of Thy to HmUra in cellular DNA probably does not result in substantial mutagenesis. © 1991 Oxford University Press.