CELL-CYCLE REGULATION AND INVITRO HYBRID ARREST ANALYSIS OF THE MAJOR HUMAN URACIL-DNA GLYCOSYLASE

Uracil-DNA glycosylase (UDG) is the first enzyme in the excision repair pathway for removal of uracil in DNA. In vitro transcription/translation of a cloned human cDNA encoding UDG resulted in easily measurable UDG activity. The apparent size of the primary translation product was 34 kD. Two lines of evidence indicated that this cDNA encodes the major nuclear UDG. First, in vitro translation of human fibroblast mRNA isolated from S-phase cells resulted in measurable UDG activity and this UDG translation was specifically inhibited 90% by an anti-sense UDG mRNA transcript. Secondly, cell cycle analysis revealed an 8 - 12 fold increase in transcript level late in the G1-phase preceding a 2 - 3 fold increase in total UDG activity in the S-phase. UDG degradation was found to be very slow (T1/2 almost-equal-to 30h), therefore, the rate of UDG synthesis could be derived from the rate of UDG accumulation, and was found to correlate temporarily and quantitatively with the transcript level. Inhibitor studies showed that RNA and protein synthesis was required for induction of UDG. However, specific inhibition of DNA replication with aphidicolin indicated that entrance of fibroblasts into the S-phase was not required for UDG accumulation.