Extreme dNTP pool changes and hypermutability in dcd ndk strains

Abstract

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.

Mutagen Synergy: Hypermutability Generated by Specific Pairs of Base Analogs

“…4 is similar to the respective figure we constructed for DCD-deficient cells (61). The spectrum of rpoB mutations in the 2AP plus ZEB com- bination does share one hot spot with the spectra of DCD-, NDK-, or DCD-and NDK-deficient strains, the A·T¡G·C mutation at position 1547 (36,41,42), although this site also represents a hot spot for spontaneous mutations in either a wild-type or mismatch repair-deficient background (35,39). What really stands out in Fig.…”

“…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).…”

“…Mutants defective in deoxycytidine deaminase (DCD) or nucleotide diphosphate kinase (NDK) have increased dCTP and dGTP (41,44,49,50; but also see reference 60), decreased dATP (41,44), and higher rates of certain base substitutions (36,41,44,50,51). The double mutant deficient in both DCD and NDK has a more extreme imbalance and a more extreme mutation rate increase (42). These findings are relevant, because ZEBtreated E. coli cells are likely to be phenotypically DCD deficient.…”

“…that is also seen in the 2AP spectrum, although here, it is 23-fold more frequent (see below). The hot spot at position 1547 is also seen in the spectra of spontaneous mutations in wild-type mismatch repair-deficient strains, as well as for NDK-and deoxycytidine deaminase (DCD)-deficient strains (35,36,39,41,42). However, the A·T¡G·C mutations at position 1598 are only rarely seen (see Discussion) and constitute only 5.5% of the mutations in the 2AP spectrum (5 of 90 mutations detected).…”

“…One explanation for the synergy between 2AP and ZEB involves altered dNTP pools, such as that seen in a number of E. coli mutants, for example DCD-deficient and NDK-deficient strains (41,42,44). We therefore decided to examine the 2AP plus ZEB combination in an NDK-deficient strain to determine whether the increased mutation frequency might be even further enhanced in this background.…”