Are DNA precursors concentrated at replication sites?

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Abstract
Whether the effective concentrations of deoxyribonucleoside 5''-triphosphates (dNTP) at sites of DNA replication in vivo might be higher than the concentrations of dNTP averaged over the entire cell volume was investigated. The approach involved determination of the dependence of DNA replication rate on TTP concentration, both in vivo and in an in vitro system that closely approximates the intracellular replication apparatus. In phage T4-infected Escherichia coli maximal rates of DNA synthesis were attained with dTTP pools of .apprx. 1.2 .times. 105 molecules/cell, corresponding to an average intracellular concentration of about 65 .mu.M. When DNA synthesis was measured in the T4 purified protein system, maximal rates were observed at dTTP concentrations of 200-240 .mu.M. 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. Why such concentration gradients might be needed and how they might be generated was discussed. The implications of these results for understanding the relationship between intracellular dNTP pools and mutation rates were discussed. A by-product of this study was the finding that exogenous thymidine is used for T4 DNA synthesis in preference to endogenous pathways to thymine nucleotides; at high thymidine concentrations in vivo the endogenous pathways can be completely bypassed.