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Nucleic Acids Research, 1991, Vol. 19, No. 12 3337-3343
© 1991

CHEMISTRY

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

Dan D. Levy1,+ and George W. Teebor1,2,*

1Departments of Environmental Medicine and Rita and Stanley Kaplan Cancer Center, New York University Medical Center New York, NY 10016, USA 2Departments of Pathology and the Rita and Stanley Kaplan Cancer Center, New York University Medical Center New York, NY 10016, USA

* To whom correspondence should be addressed

Received March 6, 1991. Revised May 14, 1991. Accepted May 14, 1991.

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.

+ Present address: Bulding 37 Room 3D06, National Cancer Institute, NIH, Bethesda, MD 20982, USA


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