Abstract— Substitution of bromodeoxyuridine for thymidine in the DNA of mammalian cells sensitizes them to a range of wavelengths of ultraviolet light. Cells are also sensitized to photochemical reactions involving dyes such as Hoechst 33258, which is used to produce differential staining of chromatids according to their bromodeoxyuridine content. Irradiation with 313 nm light of human and hamster cells containing bromodeoxyuridine in their DNA produced single‐strand breaks, detectable by alkaline elution, but no DNA‐protein cross‐links. Irradiation with 360 nm light in the presence of Hoechst 33258 produced extensive DNA‐protein cross‐linkage as well as single‐strand breaks. These cross‐links were observed in DNA containing bromodeoxyuridine incorporated by either semiconservative or repair replication, and may provide a method for identification of proteins in close proximity to replication forks or repair sites. When the protein was removed with proteinase K, bromodeoxyuridine in repair patches after irradiation by doses of ultraviolet (254 nm) light as low as 0.26 J/m2 could readily be detected. Hoechst 33258‐mediated photolysis, therefore, provides a sensitive new technique for measuring repair replication after ultraviolet light irradiation.
An inhibitor of polyadenosine diphosphoribose polymerase, 3-aminobenzamide, had no effect on survival or mutagenesis in Chinese hamster ovary cells exposed to X-rays or u.v. light. After exposure to methyl methanesulfonate, 3-aminobenzamide increased cell killing and mutagenesis at the 6-thio-guanine-resistance locus, but had no effect on mutagenesis at the ouabain-resistance locus. These results are consistent with the greater role played by polyadenosine diphosphoribose in cells damaged by alkylating agents rather than by radiations.
Poly(ADP-ribose) is synthesized in response to DNA strand breaks and covalently modifies numerous intracellular proteins. We have proposed that this modification regulates, i.e., inhibits, the activity of these enzymes, e.g., topoisomerases and proteases, which could otherwise cause additional DNA damage or alterations in chromatin structure. Inhibition of poly(ADP-ribose) polymerase by 3-amino-benzamide (3AB) in cells exposed to DNA-damaging agents would, according to this proposal, eliminate the regulatory role of ADP-ribosylation. When Chinese hamster ovary cells are cultured with methyl methanesulfonate (MMS) and 3AB, a synergistic increase in sister chromatid exchange frequency is observed. We investigated the regulatory role of poly(ADP-ribose) polymerase to see if topoisomerases or proteases are involved in this synergistic increase. Cells were exposed to MMS or the intercalating agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), 3AB, and either the topoisomerase inhibitor novobiocin or the protease inhibitor antipain. Neither novobiocin nor antipain affected the synergistic response of MMS and 3AB or the additive response of m-AMSA and 3AB. These results suggest that topoisomerases or proteases do not account for the effect of 3AB on sister chromatid exchange frequency after DNA damage.
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