Importance of pyrimidine nucleotide salvage pathways for DNA synthesis in skin

Split-thickness rabbit skins were minced and incubated in vitro with radioactive precursors selected to measure do novo and salvage pathways for pyrimidine nucleotide synthesis. In this system, both the salvage precursors [3H]thymidine and [14C]cytidine were incorporated actively into skin DNA and only [14C]cytidine into skin RNA. In contrast, the de novo precursor [14C]orotic acid labeled skin RNA extensively but did not significantly label skin DNA. An unusually high ratio of specific activities of UMP:CMP in RNA was observed when [14C]orotic acid was used as a nucleotide precursor. Methotrexate effectively blocked thymidylate synthesis do novo but did not inhibit DNA synthesis as determined by [3H]thymidine incorporation into DNA thymidylate residues or by [14C]deoxycytidine incorporation into DNA deoxycytidylate residues. Azauridine inhibited labeling of RNA by [14C]orotic acid to a greater extent than labeling of DNA by [3H]thymidine. These results suggest that in rabbit skin, cytidine nucleotides are utilized more effectively than uridine nucleotides for deoxypyrimidine biosynthesis, that DNA synthesis is primarily dependent upon salvage mechanisms to supply deoxypyrimidine nucleotides, and that inhibition of thymidylate synthesis by methotrexate does not inhibit DNA synthesis in this system.