Localization of 5-bromodeoxyuridine distribution in rat DNA as determined by single-strand specific nucleases

Summary Secondary cultures of rat embryo cells were synchronized by a double thymidine block. Following release from synchrony, the first entire S phase (7.5 hrs) was pulsed with either 10 −7 M [ 3 H]thymidine (TdR) or 10 −7 M [ 3 H]5-bromodeoxyuridine (BrdU) in order to determine the localization and distribution of the thymidine analog in DNA. The DNA was extracted, purified by hydroxyapatite column chromatography, and dialyzed against a sodium acetate buffer at pH 4.5. Each DNA sample was then reacted with the single-strand specific nuclease of Aspergillus oryzae (S1 nuclease) and assayed over hydroxy-apatite. Nearly 25% of the DNA was physically lost from all the samples following nuclease treatment. However, 21% of the [ 3 H]TdR moieties were digested, as compared to 62% of the [ 3 H]BrdU residues. Reaction of native DNA samples with the Neurospora crassa endonuclease at pH 8.0 resulted in little degradation. Dialysis of [ 3 H]guanosine-labeled rat embryo DNA against the acetate buffer alone caused a 24% level of depurination. These results suggest that mild depurination creates artifactual single-stranded regions in rat DNA which are apparently enriched in [ 3 H]BrdU as compared to [ 3 H]TdR.