Functional compartmentation of dCTP pools: Preferential utilization of salvaged deoxycytidine for DNA repair in human lymphoblasts

The utilization of dCTP derived from de novo synthesis through ribonucleotide reductase in exponentially growing CCRF-CEM cells was compared with the metabolic fate of dCTP produced by the salvage pathway. Exogenous dCyd was not effectively incorporated into replicating DNA; instead, dCTP derived from ribonucleotide reductase (labeled by [5-³H]Cyd) was the main precursor for that purpose, apparently because of functional compartmentation of the dCTP pool in these cells. Studies of the metabolic route of incorporation of exogenous [5-³H]dCyd into DNA of growing CCRF-CEM cells demonstrated that it was mainly incorporated through the DNA repair pathway. Incorporation of [5-³H]dCyd into DNA of synchronized cell populations was maximal in G₁ cells, whereas [³H] dThd incorporation occurred predominantly in S phase cells. When cellular DNA was density labeled by incubation with BrdUrd, repaired DNA, which was less dense than replicated DNA, was preferentially labeled by [5-³H]dCyd. In contrast, replicated DNA was labeled by both [³H]dThd and [5-³H]Cyd. The DNA-damaging agents methylmethanesulfonate, ultraviolet irradiation, and γ-irradiation inhibited [³H] dThd incorporation, whereas they stimulated the accumulation of [5-³H]dCyd in DNA. Based on these results, we propose that the dCTP pool is functionally compartmentalized in growing CCRF-CEM cells. dCTP derived from the salvage pathway is utilized predominantly for DNA repair, whereas the de novo pathway supplies dCTP for DNA replication.