Polynucleotide phosphorylase (PNP) plays a central role in RNA degradation, generating a pool of ribonucleoside diphosphates (rNDPs) that can be converted to deoxyribonucleoside diphosphates (dNDPs) by ribonucleotide reductase. We report here that spontaneous mutations resulting from replication errors, which are normally repaired by the mismatch repair (MMR) system, are sharply reduced in a PNP-deficient Escherichia coli strain. This is true for base substitution mutations that occur in the rpoB gene leading to Rif r and the gyrB gene leading to Nal r and for base substitution and frameshift mutations that occur in the lacZ gene. These results suggest that the increase in the rNDP pools generated by polynucleotide phosphorylase (PNP) degradation of RNA is responsible for the spontaneous mutations observed in an MMR-deficient background. The PNP-derived pool also appears responsible for the observed mutations in the mutT mutator background and those that occur after treatment with 5-bromodeoxyuridine, as these mutations are also drastically reduced in a PNP-deficient strain. However, mutation frequencies are not reduced in a mutY mutator background or after treatment with 2-aminopurine. These results highlight the central role in mutagenesis played by the rNDP pools (and the subsequent dNTP pools) derived from RNA degradation. Elucidating the pathways that lead to mutations resulting from replication errors, arising either spontaneously or induced by chemical agents, has intrigued molecular biologists ever since the elucidation of the structure of DNA allowed one to pose this question in molecular terms (57,58). What is the actual source of these mutations? Different tautomeric forms of the four bases in DNA or their analogs have been considered which provoke errors directly (18,24,28,29,57) or even indirectly, for instance by affecting the pools of nucleoside triphosphates (28,29,34). Also, the field has defined a myriad of repair systems aimed at preventing or repairing DNA damage (24) and also aimed at correcting errors of DNA replication (30,47). In humans, defects in one of a number of repair systems leads to inherited cancer susceptibilities (e.g., see references 1 and 22). In Escherichia coli, the replicating DNA polymerase (Pol III) contains an editing subunit that corrects numerous replication errors (14,19,41). Directly after replication, the mismatch repair (MMR) system recognizes still-uncorrected mismatches and repairs them using the pattern of methylation to distinguish the template strand from the newly synthesized strand (30, 47). Mutants lacking any one of the components of this system (e.g., MutH, MutL, MutS, UvrD) have sharply elevated mutation rates that involve transitions (A:T¡G:C or G:C¡A:T) (11, 36, 54) or short indels (insertion/deletions) at repeated sequences, such as monotonous runs of G's or A's on the same strand (10, 54). The size and balance of the nucleoside triphosphate (NTP) pools are important for replication fidelity (33). Not only do unbalanced pools provoke an increase in m...

SUMMARY Cells lacking deoxycytidine deaminase (DCD) have been shown to have imbalances in the normal dNTP pools that lead to multiple phenotypes, including increased mutagenesis, increased sensitivity to oxidizing agents, and to a number of antibiotics. In particular, there is an increased dCTP pool, often accompanied by a decreased dTTP pool. In the work presented here, we show that double mutants of E. coli lacking both DCD and NDK (nucleoside diphosphate kinase) have even more extreme imbalances of dNTPs than mutants lacking only one or the other of these enzymes. In particular, the dCTP pool rises to very high levels, exceeding even the cellular ATP level by several-fold. This increased level of dCTP, coupled with more modest changes in other dNTPs, results in exceptionally high mutation levels. The high mutation levels are attenuated by the addition of thymidine. The results corroborate the critical importance of controlling DNA precursor levels for promoting genome stability. We also show that the addition of certain exogenous nucleosides can influence replication errors in DCD-proficient strains that are deficient in mismatch repair.