Are DNA precursors concentrated at replication sites?

Abstract

We have asked whether the effective concentrations of deoxyribonucleoside 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 an 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. ofdTTP 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 ofour 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.What are the effective concentrations of DNA precursors at replication sites? This question is ofinterest for several reasons: (i) The rate of DNA synthesis in vivo seems not to be limited by substrate availability (1), yet in vitro systems for DNA synthesis require concentrations of deoxyribonucleoside 5'-triphosphates (dNTPs) that are much higher than average intracellular concentrations as estimated from pool measurements.(ii) DNA precursors are compartmentalized, and several lines of investigation indicate that only a small fraction of the total dNTP in a cell is readily available to participate in DNA replication (2-5). (iii) The compartmentation may be brought about by the action of specific dNTP-synthesizing multienzyme complexes, which behave as though juxtaposed with the replication apparatus (6-11); such complexes seem able, in vitro, to maintain concentration gradients of deoxyribonucleotide intermediates (7). (iv) DNA precursor concentrations represent a determinant ofthe fidelity ofDNA replication. Deoxyribonucleotide pool imbalances, generated either in cell-free DNA replication systems or in vivo, lead to increased spontaneous mutation rates (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) seven-protein (p32, p41, p43, p44, p45, p61, and p62) system for DNA synthesis in vitro catalyzes DNA chain elongation at rates approaching those seen in vivo (22). The accuracy ofbase selection in this system is close to that achieved in normal DNA replication in vivo (17,19). In several other ways this system closely simulates the behavior ofT4 DNA replication forks in intact cells. If one can establish that dNTP levels are indeed saturating at replication sites in vivo, then the minimal dNTP concentrations that give maximal rates ofDNA synthesis in vitro should represent the minimal effective concentrations at replication sites in whole cells.When d...

Identification of a New Motif in Family B DNA Polymerases by Mutational Analyses of the Bacteriophage T4 DNA Polymerase

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