Polynucleotide Phosphorylase Plays an Important Role in the Generation of Spontaneous Mutations in Escherichia coli

Abstract

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...

Rate and molecular spectrum of spontaneous mutations in the bacteriumEscherichia colias determined by whole-genome sequencing

“…Table 2 gives mutation rates for the wild-type strain estimated using classical fluctuation tests, scoring for resistance to rifampicin (Rif R ) and to nalidixic acid (Nal R ). When normalized to the number of nucleotides that, when mutated, give the resistance phenotypes (22)(23)(24), the two mutation rates are 0.33 and 0.21 × 10 −10 mutations per nucleotide per generation, respectively. The mean rate for BPS mutations from the MA experiment was 1.99 × 10 −10 per nucleotide per generation, six-to nine-fold higher than that obtained from fluctuation tests.…”

Mutagen Synergy: Hypermutability Generated by Specific Pairs of Base Analogs

“…Long-standing work in both microbial and mammalian cells has shown that alterations of the dNTP pools can affect mutation rates (31,41,42,44,(50)(51)(52)(53)(54). More recently, studies in E. coli have shown that perturbations in the absolute levels of all four dNTPs, even when the ratios are not altered, leads to changes in mutation rates (26,55,56). Increases in the levels raise mutation rates (55,56), and decreases in the levels lead to lower mutation rates (26).…”

“…5AZ and ZEB have been used in combination in chemotherapy (20). 2-Aminopurine results principally in G·C¡A·T and A·T¡G·C changes (e.g., see references 18 and 21 to 25), as does 5BrdU (24)(25)(26). 2AP has been used to enhance the oncolytic activity of E1b-deleted adenovirus in hepatocellular carcinoma cells (27), and 5BrdU has been used in human glioma cell combination treatments with 1-nitrosourea and cisplatin (28).…”

“…It is now clear that there are several routes to mutagenesis other than direct mispairing that could emanate from the presence of some of the four compounds studied here. This includes partial induction of the SOS system in E. coli and related bacteria (6), the partial or complete saturation of the mismatch repair system (7,26,29,30), and potential effects on altering dNTP pools, as the integrity of these pools is crucial to replication fidelity (e.g., see references 24, 25, and 31; see Discussion). Here, we report that strong synergy between certain pairs of these mutagens results in hypermutation.…”

The small RNA SraG participates in PNPase homeostasis

“…It can also be noted that PNPase is necessary for bacterial virulence in some well-known pathogens, including the Salmonellae and the Yersiniae (Clements et al 2002;Rosenzweig et al 2007;Numata et al 2014). In addition, it has been proposed that the pool of nucleoside diphosphates generated by PNPase-mediated degradation of RNA plays an important role in the appearance of spontaneous mutations in E. coli (Becket et al 2012).…”