The Assay of 2’ Deoxyuridine-3’-Monophosphate (dUMP) by 3’ Phosphorylation and Two Dimensional Thin Layer Chromatography: A Potential Radiation Marker of DNA Damage

Abstract

Deoxyuridine is a stable pyrimidine adduct in DNA, and can be detected via 32P labeling cold DNA. Deoxyuridine (dU; monophosphate - MP) forms by the deamination of deoxycytidine, remains in DNA via deficient uracil glycosylase excision repair, or diminished scavenging by thymidylate synthetase, dUTPase and/or dihydropyrimidine dehydrogenase activities. The assessment of dUMP adduct formation and normal base composition is carried out by “shot-gun” 5′-phosphorylation of representative deoxyribonucleotides with all four standard 32p.alpha- triphosphates (dNMP’s as monophosphate) through nick translation. Subsequent 3′-monophosphate digest (micrococcal nuclease and spleen phosphodiesterase) “sister exchanges” a radioactive 32PO4= to the nearest neighbor cold nucleotide. Separation in two dimensional PEI-cellulose is carried out in acetic acid (1.0 M., pH 3.5 with NH4OH), and 5.6M (NH4)2SO4, 0.12M Na2EDTA, 0.035M (NH4)HS04 to pH 4. The technique was applied to human placental DNA, control and altered calf thymus DNA with cold stoichiometric replacement of dUMP; and adult Drosophila DNA, which lacks dUTPase and is deficient in uracil glycosylase, is heavily methylated, and surprisingly is devoid of dUMP. Scintillation detection of dUMP is more sensitive than densitometry, but densitometry more accurately measures adducts close to other dNMP’s. dUMP’s ubiquity in DNA is not completely explained by chemical deamination, since this forms slowly in vitro. Nor does enzymatic defense completely preclude dUMP’s presence. This technique easily and quickly quantifies the low molecular weight adduct dUMP in DNA both in vivo and in vitro.