Seminars

In Escherichia coli, the DNA damage response entails several processes such as stalling of the replication fork, activation of the SOS response and induction of dNTP synthesis. Following UV-irradiation, the ribonucleotide reductase (RNR) NrdAB that catalyzes the limiting step in dNTP synthesis, is up-regulated, leading to an increase in dNTP levels. In the present study, we investigate the effect of increased dNTP levels on the process of translesion synthesis (TLS) under DNA damage conditions. We present direct evidence that an elevated dNTP pool level falicitates translesion synthesis (TLS). TLS is a two-step mechanism involving the insertion of a nucleotide opposite the lesion and the subsequent extension steps. Although Pol III, the replicative DNA polymerase, can insert a nucleotide across certain lesion, its proofreading activity usually recognizes the inserted base across the lesion site as a mispair and removes it, thus preventing the subsequent extension steps. Our results suggest that high dNTP levels bolster the polymerase activity of Pol III thus in turn reducing its exonuclease activity. The shift in the equilibrium from the exonuclease activity towards the polymerase activity of Pol III favors the production of a key TLS intermediate, i.e. the intermediate that contains a base inserted across from the lesion site. This intermediate is subsequently elongated by the specialized TLS polymerases. In support of the hypothesis of proofreading activity attenuation of Pol III, we show a robust increase in spontaneous mutagenesis under conditions of elevated dNTP levels referred to as the spontaneous dNTP mutator phenotype.