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Compartmentation of dCTP pools. Evidence from deoxyliponucleotide synthesis.

1987; Elsevier BV; Volume: 262; Issue: 34 Linguagem: Inglês

10.1016/s0021-9258(18)49273-3

1083-351X

George M. Spyrou, Peter Reichard,

Advanced biosensing and bioanalysis techniques

The nucleotide fraction of cultured 3T6 and 3T3 mouse fibroblasts contains deoxy-CDP choline and deoxy-CDP ethanolamine as well as the corresponding riboliponucleotides.In permeabilized cells both deoxyliponucleotides were formed from dCTP.In intact cells they could be labeled from [B-"H]deoxycytidine or cytidine via transformation of the nucleosides to dCTP.Their turnover was slow compared to that of dCTP.When rapidly growing 3T3 cells were labeled during 90 min from deoxycytidine the specific activity of dCDP choline was 2.4 times higher than that of dCTP while after labeling from cytidine both nucleotides (and CTP) reached the same specific activity under steady state conditions.Also dCDP ethanolamine was labeled more rapidly from deoxycytidine than from cytidine.Our results suggest that the deoxyliponucleotides were synthesized from a dCTP pool that was labeled preferentially from deoxycytidine.Earlier work (Nicander, B., and Reichard, P. (1983) Proc.Natl.Acad.Sci.U. S. A. 80, 1347-1351) had demonstrated synthesis of DNA from a dCTP pool labeled preferentially from cytidine.Taken together our results suggest that deoxyliponucleotides and DNA are synthesized from separate dCTP pools.Compartmentation of deoxyribonucleoside triphosphate (dNTP) pools in mammalian cells is usually invoked to rationalize apparently conflicting data from experiments with intact cells involving isotope incorporation from labeled deoxynucleosides into DNA.One specific model for compartmentation (1, 2) suggests that dNTPs are synthesized and consumed for DNA replication within a large multienzyme complex (= replitase), protected from mixing with dNTPs outside the complex.The function of the outside pools is usually not considered.The concept of a channel for newly synthesized dNTPs, linked to DNA synthesis, originated from work with phage T4-infected Escherichia coli (3)(4)(5).Later work then formed the basis for the specific mammalian replitase model.While the evidence for compartmentation of at least some dNTPs is strong (6, 7), serious doubt exists concerning the replitase concept.Just to mention one major obstacle: ribonucleotide reductase is localized in the cytoplasm (8,9) while DNA polymerase is present in the cell nucleus (10).With two key enzymes localized in two separate cellular compartments