The mutagenicity of 5-hydroxymethyluracil (HmUra) in the DNA of Chinese hamster V79 cells will be characterized. A HGPRT- V79 cell line containing the bacterial gpt gene (V79gpt) will be developed. The formation of gpt- cells following exposure to HmdUrd will be quantified, and representative gpt- clones will be isolated for genetic analysis. Southern analysis will be used to determine whether HmdUrd induced gpt- cells have large deletions in the gpt gene. Direct sequencing using the polymerase chain reaction will determine whether gpt- cells have point mutations. Our ability to introduce a single DNA base modification, the HmUra residue, will be further exploited to study the role of poly(ADP-ribose) in base excision repair. The effects of HmUra in DNA on poly(ADP-ribose) synthesis, NAD depletion, and poly(ADP-ribose) polymerase activity will be measured. It will be determined whether the toxic effects of 3- aminobenzamide (3AB) to cells containing HmUra in their DNA result from the inhibition of poly (ADP-ribose) synthesis. It will also be determined whether 3AB increases the mutagenicity of HmdUrd and whether 3AB alters the spectrum of mutations produced by HmdUrd. Finally, to test the hypothesis that the toxicity of HmUra residues in DNA results primarily from their repairability, V79 cells will be chemically mutagenized and HmUrd will be used to select HmdUrdr mutants. HmdUrdr cells which have lost HmUra-DNA glycosylase activity will be characterized 1) to determine whether the cells have an increased, decreased or unchanged spontaneous mutation rate at the gpt locus, as compared with the parent cell, 2) to determine whether these cells are more susceptible to mutagenesis by HmUrd or ionizing radiation and 3) to determine whether such cells are resistant to the synergistic toxic effects of HmdUrd and 3AB.
