Are DNA precursors concentrated at replication sites?
We have asked whether the effective concentrations of deoxyribonucleotide 5'-triphosphates (dNTPs) at sites of DNA replication in vivo might be higher than the concentrations of dNTPs averaged over the entire cell volume. The approach involved determination of the dependence of DNA replication rate upon thymidine triphosphate concentration, both in vivo and in vitro system that closely approximates the intracellular replication apparatus. In T4 phage-infected Escherichia coli maximal rates of DNA synthesis were attained with dTTP pools of approximately 1.2 x 10(5) molecules per cell, corresponding to an average intracellular concentration of about 65 microM. When DNA synthesis was measured in the T4 purified protein system [Sinha, N. K., Morris, C. F. & Alberts, B. M. (1980) J. Biol. Chem. 255 4290--4303], maximal rates were observed at dTTP concentrations of 200--240 microM. This represents a minimal estimate, therefore, of dTTP concentration at replication sites and suggests that at least a 3- to 4-fold concentration gradient exists near these sites. We discuss why such concentration gradients might be needed and how they might be generated. We also discuss the implications of these results for understanding the relationship between intracellular dNTP pools and mutation rates. A by-product of our study was the finding that exogenous thymidine is used for T4 DNA synthesis in preference to endogenous pathways to thymidine nucleotides; at high thymidine concentrations in vivo the endogenous pathways can be completely bypassed.
D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis; D004279:DNA, Viral / Q000096:biosynthesis; D003854:Deoxyribonucleotides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D013604:T-Phages / Q000235:genetics; D013942:Thymine Nucleotides / Q000378:metabolism
D004269:DNA, Bacterial; D004279:DNA, Viral; D003854:Deoxyribonucleotides; D013942:Thymine Nucleotides; C024157:thymidine 5'-triphosphate