Increase in dNTP pool size during the DNA damage response plays a key role in spontaneous and induced-mutagenesis in Escherichia coli

Abstract

Exposure of Escherichia coli to UV light increases expression of NrdAB, the major ribonucleotide reductase leading to a moderate increase in dNTP levels. The role of elevated dNTP levels during translesion synthesis (TLS) across specific replication-blocking lesions was investigated. Here we show that although the specialized DNA polymerase PolV is necessary for replication across UV-lesions, such as cyclobutane pyrimidine dimers or pyrimidine(6-4)pyrimidone photoproduct, Pol V per se is not sufficient. Indeed, efficient TLS additionally requires elevated dNTP levels. Similarly, for the bypass of an N-2-acetylaminofluorene-guanine adduct that requires Pol II instead of PolV, efficient TLS is only observed under conditions of high dNTP levels. We suggest that increased dNTP levels transiently modify the activity balance of Pol III (i.e., increasing the polymerase and reducing the proofreading functions). Indeed, we show that the stimulation of TLS by elevated dNTP levels can be mimicked by genetic inactivation of the proofreading function (mutD5 allele). We also show that spontaneous mutagenesis increases proportionally to dNTP pool levels, thus defining a unique spontaneous mutator phenotype. The so-called "dNTP mutator" phenotype does not depend upon any of the specialized DNA polymerases, and is thus likely to reflect an increase in Pol III's own replication errors because of the modified activity balance of Pol III. As up-regulation of the dNTP pool size represents a common physiological response to DNA damage, the present model is likely to represent a general and unique paradigm for TLS pathways in many organisms.

Fig. 1.

dNTP levels increase upon UV light exposure but are restricted by dATP feedback inhibition in E. coli. (A) Equal amounts of total cellular proteins from wild-type MG1655 strain grown to midexponential phase and subjected (+) or not (−) to UV light exposure (40 J/m²) were separated by SDS-10% PAGE. NrdB protein and control TrxA protein were detected by Western blotting. (B) dNTP pools were measured in a wild-type strain (MG1655) treated or not by UV light (40 J/m²) or containing a plasmid carrying either nrdAB, nrdEF, or nrdA_H59AB genes. The RNR genes under the control of the ptrc promoter were induced by addition of 1 mM of IPTG at mid-exponential growth. The relative fold-increase in dNTP levels relative to untreated cells is indicated on top of each bar. (C) Increased dNTP levels enhance spontaneous mutagenesis. The spontaneous mutation frequencies to rifampicin (Rif^R) is determined and plotted against the average dNTP concentration in strains expressing the different RNR genes as indicated B. (Inset) The relative fold-increase in spontaneous mutagenesis relative to untreated cells is indicated on top of each bar.

Fig. 2.

Translesion synthesis pathways. (A) Effect of increased dNTP pool size on TLS pathways. For all three lesions, G-AAF, TT-CPD, and TT(6-4), we have compared the efficiency of TLS under the following conditions: (i) Strains expressing normal levels of dNTP (designated “low”) in the presence of the cognate specialized DNA polymerase. For G-AAF, Pol II is expressed from the chromosomal polB gene (∼50 molecules per cell); for TT-CPD and TT(6-4), Pol V is expressed from a low copy number pRW134 that produces 50–60 Pol V molecules per cell. (ii) Strains expressing NrdAB from a low copy-number plasmid, leading to ∼threefold higher than normal concentrations of dNTP (designated “high”) without the cognate specialized DNA. (iii) Strains expressing NrdAB from a low copy-number plasmid in the presence of the cognate specialized DNA polymerase. For all three lesions, a similar trend can be observed: efficient TLS requires, simultaneously, the presence of the cognate DNA polymerase and high dNTP levels. (B) Effect of proofreading inactivation (mutD5 allele) on TLS pathways. For all three lesions we have compared the efficiency of TLS under the following conditions: (i) Proofreading-proficient strains in the presence of the cognate specialized DNA polymerase. For G-AAF, Pol II is expressed from the chromosomal polB gene (∼30 molecules per cell); for TT-CPD and TT(6-4), Pol V is expressed from a low copy number pRW134 that produces 50–60 Pol V molecules per cell. (ii) Proofreading-deficient strains (mutD5) without the cognate specialized DNA. (iii) Proofreading-deficient strains in the presence of the cognate specialized DNA polymerase. For all three lesions a similar trend can be observed: efficient TLS requires, simultaneously, the presence of the cognate DNA polymerase and the inactivation of proofreading function of Pol III. Overall, the effect of proofreading inactivation mimics high dNTP levels (as seen in A).

Fig. 3.

How does dNTP pool size expansion increase spontaneous and induced mutagenesis? (A) Spontaneous dNTP mutator pathway. Under normal dNTP concentrations, if Pol III makes a misinsertion error, the mispaired intermediate is removed by the proofreading function and T is correctly inserted leading to high-fidelity replication. In response to DNA damage, the increase in dNTP concentration modifies the activity of Pol III holoenzyme by shifting the balance between the exonuclease and polymerase functions into a more elongation/less proofreading mode (Pol III^TLS hatched oval). The arrow lengths represent the relative pol/exo activities of Pol III. Pol III^TLS represents a transient modification of Pol III's activity and not a distinct biochemical form of Pol III. In this mode, the rates of insertion errors and subsequent mispair elongation are increased compared with the normal mode of Pol III. (B) Induced mutagenesis. The cartoon pertains to TLS across a replication blocking lesion that requires Pol V [for example: TT-CPD or TT(6-4)]. For the sake of simplicity, neither the β-clamp nor the RecA filament are represented (51, 57). The thick dotted line represents the limited patch of DNA synthesis made by Pol V (TLS patch). Under normal dNTP conditions, the TLS patch generated by Pol V is too short to support elongation by Pol III. Thus, upon rebinding Pol III, exo degrades the TLS patch leading to an aborted TLS event. Under high dNTP conditions, the attenuated proofreading activity associated with Pol III^TLS (hatched oval) contributes to successful TLS by allowing elongation, rather than degradation, of short TLS patches. The overall scheme also applies for Pol II-mediated TLS across G-AAF adduct. In that case, Pol III^TLS specifically acts at the level of the first switch by inserting a C across the lesion site. The resulting replication intermediate undergoes a slippage reaction before Pol II association and elongation (–60).