Rates of (/sup 3/H)thymidine incorporation into thymidine triphosphate vs nuclear matrix DNA in vivo

Previous studies in vitro have indicated the possible existence of more than one thymidine pool accessible to DNA replication forks. To investigate this possibility in vivo, the incorporation of (/sup 3/H)thymidine (/sup 3/H-Thd) into matrix DNA and soluble thymidine triphosphate (TTP) was examined in regenerating rat liver. Nuclear matrix-attached DNA, although only 2% of total nuclear DNA, represents the most newly replicated DNA in eukaryotic cells, thereby providing a very sensitive means of examining precursor incorporation kinetics. Partially hepatectomized rats were injected with 200 ..mu..C /sup 3/H-Thd via the hepatic portal vein. After various pulse times, matrix DNA and/or soluble TTP were extracted and their specific activities determined. After only 1 min, the labeling of matrix DNA had already reached a maximum. Matrix DNA contained 27% of the label at 2 min, 11% at 5 min, and only 5% at 10 min with respect to total DNA. Contrary to the kinetics found for matrix DNA, /sup 3/H-Thd incorporation into TTP was still increasing at early pulse times and had not peaked even after a 10 min pulse. Five min after injection, TTP specific activity was 3050 dpm/nmole and was 7030 dpm/nmole after 10 min. Since the peak specific activity formore » (/sup 3/H)-Thd incorporation into matrix DNA precedes that for (/sup 3/H)-Thd into TTP, there may be channeling of exogenous thymidine directly to the site of DNA synthesis bypassing existing nucleotide pools.« less