Characterization of antibodies to dihydrothymine, a radiolysis product of DNA

• Published in: Biochemistry (Easton) Vol. 28; no. 10; pp. 4382 - 4387
• Main Authors: Hubbard, Karen, Ide, Hiroshi, Erlanger, Bernard F, Wallace, Susan S
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 16.05.1989
• Subjects: 560120 - Radiation Effects on Biochemicals, Cells, & Tissue Culture; ANTIBODIES; Antibody Specificity; AZINES; BACTERIA; BIOASSAY; BIOSYNTHESIS; CHEMICAL COMPOSITION; CHEMICAL RADIATION EFFECTS; CHEMISTRY; DNA; DNA - radiation effects; ELECTROMAGNETIC RADIATION; ENZYME IMMUNOASSAY; ESCHERICHIA COLI; Escherichia coli - radiation effects; HETEROCYCLIC COMPOUNDS; HYDROXY COMPOUNDS; IMMUNOASSAY; Immunochemistry; IONIZING RADIATIONS; MICROORGANISMS; Nucleic Acid Denaturation; NUCLEIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; PYRIMIDINES; RADIATION CHEMISTRY; RADIATION EFFECTS; RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT; RADIATIONS; SPECIFICITY; SYNTHESIS; THYMINE; Thymine - analogs & derivatives; Thymine - immunology; Thymine - radiation effects; URACILS; X RADIATION
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00436a039

Antibodies to dihydrothymine were elicited by immunizing rabbits with dihydrothymidine monophosphate conjugated by carbodiimide to BSA. By use of an ELISA assay, the antibodies produced were found to be specific for dihydrothymine. Hapten inhibition studies showed that dihydrothymidine monophosphate was 3 orders of magnitude more effective as an inhibitor than thymidine monophosphate and 4 orders of magnitude more effective than thymidine glycol monophosphate. With DNA containing dihydrothymine, antibody reactivity was observed at 20 fmol of dihydrothymine, which is approximately 0.1 dihydrothymine per 10,000 bases. Thus, the assay is very sensitive. The antibody reacted with denatured DNA containing dihydrothymine but not with native DNA containing this lesion. The antibody was used for measurement of in vivo incorporation of dihydrothymidine in wild-type Escherichia coli or mutants defective in their ability to remove dihydrothymine from DNA or in the de novo synthesis of thymidylate. Lastly, antibodies to dihydrothymine were use to quantitate the formation of dihydrothymine in DNA X-irradiated under N2. Production of dihydrothymine in irradiated DNA correlated with the level of reducing species produced by X-rays, and dihydrothymine was produced preferentially in irradiated single-stranded or denatured DNA as compared to irradiated duplex DNA.