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Alteration of the Secondary Structure of Newly Synthesized DNA from Murine Bone Marrow Cells by 5-Fluorouracil¹

Department of Pharmacology and Comprehensive Cancer Center, Division of Clinical Pharmacology, University of Alabama at Birmingham, Birmingham, Alabama 35294 [J. D. S., H. J. W., R. B. D.], and Department of Biochemistry, Medical College of Virginia, Richmond, Virginia 23298 [J. M. C.]

The present study evaluates the effects of 5-fluorouracil (FUra) on the structure of newly synthesized DNA purified from bone marrow cells. DNA synthesis was decreased by 30 and 45% of control in the presence of 19 and 100 µM FUra, respectively. Furthermore at these concentrations of FUra, the DNA strand sizes were smaller as determined by alkaline sucrose gradients. Enzymatic digestion of the DNA demonstrated that most of the FUra (>90%) was localized in the internucleotide linkage and not at the chain terminus. As the concentration of FUra was varied, the percentage of FUra at the chain terminus was unchanged, suggesting that the decrease in chain size as well as inhibition of DNA synthesis was not due to chain termination. DNA that had been synthesized in the presence of FUra was shown to fragment after increasing time as demonstrated by alkaline sucrose gradient analysis. This time-dependent fragmentation was associated with an increased number of strand breaks as determined by neutral and alkaline sucrose gradient analysis. A parallel study demonstrated a time-dependent excision of FUra from DNA over this same time period. In summary, these studies demonstrate an association between the excision of FUra from DNA and the changes in secondary structure of newly synthesized DNA.

¹ This work was supported by Grants CA 23412 and CA 40530 from NIH.

² To whom requests for reprints should be addressed, at Division of Clinical Pharmacology, Box 186 Volker Hall, University of Alabama at Birmingham, Birmingham, AL 35294.

Received 5/17/85. Revised 9/23/85. Accepted 9/25/85.