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Are DNA precursors concentrated at replication sites?

C K Mathews, N K Sinha

Proceedings of the National Academy of Sciences of the United States of America

|January 1, 1982

Summary

DNA replication sites may have higher deoxyribonucleotide triphosphate (dNTP) concentrations than average cell levels. This study investigated dNTP concentration gradients during DNA synthesis in T4 phage-infected E. coli.

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Area of Science:

Molecular Biology
Biochemistry
Genetics

Background:

Intracellular deoxyribonucleotide triphosphate (dNTP) concentrations are crucial for DNA replication fidelity and efficiency.
Previous studies have averaged dNTP levels across the cell, potentially masking localized variations.

Purpose of the Study:

To investigate whether effective dNTP concentrations at DNA replication sites in vivo differ from bulk intracellular concentrations.
To determine the relationship between dNTP pool sizes and DNA synthesis rates.

Main Methods:

Measured DNA replication rates in T4 phage-infected Escherichia coli.
Determined the dependence of DNA synthesis rate on thymidine triphosphate (dTTP) concentration in vivo and in an in vitro replication system.
Compared dTTP concentrations required for maximal DNA synthesis in vivo versus in vitro.

Main Results:

Maximal DNA synthesis rates in T4 phage-infected E. coli were achieved with dTTP pools of approximately 1.2 x 10^5 molecules per cell (approx. 65 microM average intracellular concentration).
In a purified T4 replication system, maximal rates required 200-240 microM dTTP, suggesting a 3- to 4-fold higher concentration at replication sites.
Exogenous thymidine was preferentially used for T4 DNA synthesis over endogenous pathways.

Conclusions:

A significant concentration gradient for dTTP likely exists at DNA replication sites, being higher than the average intracellular concentration.
These findings have implications for understanding the regulation of dNTP pools and their impact on mutation rates.
The preferential utilization of exogenous thymidine highlights metabolic regulation during viral DNA replication.

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