Hypoxanthine Incorporation Is Nonmutagenic in Escherichia coli

Abstract

Endonuclease V, encoded by the nfi gene, initiates removal of the base analogs hypoxanthine and xanthine from DNA, acting to prevent mutagenesis from purine base deamination within the DNA. On the other hand, the RdgB nucleotide hydrolase in Escherichia coli is proposed to prevent hypoxanthine and xanthine incorporation into DNA by intercepting the noncanonical DNA precursors dITP and dXTP. Because many base analogs are mutagenic when incorporated into DNA, it is intuitive to think of RdgB as acting to prevent similar mutagenesis from deaminated purines in the DNA precursor pools. To test this idea, we used a set of Claire Cupples' strains to detect changes in spontaneous mutagenesis spectra, as well as in nitrous acid-induced mutagenesis spectra, in wild-type cells and in rdgB single, nfi single, and rdgB nfi double mutants. We found neither a significant increase in spontaneous mutagenesis in rdgB and nfi single mutants or the double mutant nor any changes in nitrous acid-induced mutagenesis for rdgB mutant strains. We conclude that incorporation of deaminated purines into DNA is nonmutagenic.Three of the four DNA bases-cytosine, adenine, and guanine-have amino groups. Deamination of these three bases ( Fig. 1) generates the base analogs uracil, hypoxanthine, and xanthine, which have pairing specificities different from the original bases. Therefore, the cell employs excision repair to prevent mutagenesis due to spontaneous DNA base deamination. For example, in Escherichia coli, uracil-DNA glycosylase encoded by the ung gene initiates excision of uracils from DNA (12, 30). The ung mutants experience a 30-fold increase in GC3AT transitions (13), owing to the deamination of cytosine to uracil in the DNA (31). Deamination of adenine to hypoxanthine in the DNA (24) is also known to cause mutations: hypoxanthine preferentially base pairs with cytosine (3,18,35,39,51), and its formation in DNA, for example, as a result of nitrous acid exposure, leads to AT3GC transitions (45). Hypoxanthine removal from DNA in E. coli is initiated by the product of the nfi gene, endonuclease V (Endo V) (53, 54), and, to a much lesser extent, by the product of the alkA gene, 3-methyladenine-DNA glycosylase (43,44). Although nfi mutants do not show increased spontaneous mutagenesis, they are more mutable by nitrous acid (45), an agent known to promote deamination of DNA bases (55).Bases can deaminate not only in DNA but in nucleotides as well, both chemically (32, 46) and enzymatically (56). Deamination of DNA precursors should lead to accumulation of noncanonical nucleoside triphosphates in the cell's DNA precursor pool. These noncanonical nucleotides, such as dITP, dXTP, and dUTP, can be incorporated into the newly synthesized DNA, albeit less efficiently than regular DNA precursors (3,38,42,51). Noncanonical DNA precursors are actively intercepted and removed from the DNA precursor pool. For example, dUTP is hydrolyzed to dUMP by deoxyuridine triphosphatase (2, 16), encoded by the dut gene (19). Indirect evidence (5; B. Budke and A....

ThemutTDefect Does Not Elevate Chromosomal Fragmentation inEscherichia coliBecause of the Surprisingly Low Levels of MutM/MutY-Recognized DNA Modifications

“…At this point we measured the rate of AT3CG transversions in our wild-type and mutT mutant strains to compare them with the reported data. We found that, whereas the increase in mutagenesis due to the mutT defect matches the values reported in the previous studies, the absolute level of AT3CG transversions is up to two orders of magnitude lower because of the much lower basal level in our wild-type strain (which is confirmed in an independent study [12]). We have noticed an adaptive-like mutagenesis (26) in the CC101 strain, about a twofold increase in the number of Lac ϩ colonies between day 2 and day 8 ( Fig.…”

“…The main reason is that while the mutagenicity of our mutT allele is the same as the normalized mutation rates used in the previous studies (Table 3), the mutation rate in the CC101 strain is 22 to 125 times lower. It should be noted that a similarly low level of mutagenesis was already reported for this CC101 strain in an independent study from this laboratory (12). The calculated level of three to four 8-oxo-G modifications in the DNA of mutT mutants is 10 times lower than the steadystate level of endonuclease V-recognized modifications in the DNA of rdgB mutants (about 42 per genome equivalent) (10) and is even lower than the steady-state level of uracil excision events in dut mutants (16 per genome equivalent) (43) ( Table 4).…”

“…To quantify lac reversion in the CC101 background, strains were propagated on M9-glucose minimal medium to maintain the episome, and 2-to 7-day-old colonies were inoculated in LB broth. For CC101, 10 ml of a saturated culture was concentrated and spread on M9-lactose minimal medium (12), while the ⌬mutT mutants had 10 l of each dilution spotted on the plate. Since the small amount of glucose present in lactose preparations allowed formation of Lac Ϫ microcolonies, cell titer was determined by counting these microcolonies on M9-lactose plates using a stereomicroscope.…”

Defects in purine nucleotide metabolism lead to substantial incorporation of xanthine and hypoxanthine into DNA and RNA

“…With respect to mutagenesis, while there is some controversy about the ability of hypoxanthine to induce mutations in E. coli (4,44), dI, dX, and dU are all established mutagens in higher organisms. So it is reasonable to assume that genetic polymorphisms that raise the steady-state levels of dX and dI in DNA in humans would exacerbate the toxicity associated with the nitrosative stress of chronic inflammation, with further increases in dX and dI exceeding the cell's repair capacity and contributing to mutations and cancer risk.…”

Molybdenum cofactor-dependent resistance to N-hydroxylated base analogs in Escherichia coli is independent of MobA function

“…Ham1p is a specific pyrophosphatase that hydrolyzes HAP deoxyribonucleoside triphosphate to the corresponding mononucleotide, thus preventing incorporation of the analog into DNA [4,8]. While E. coli contains a homolog for the HAM1 gene, named rdgB [9,10], this system apparently plays a back-up role in HAP detoxification [11,12]. Its main function is likely the removal of (d)ITP or (d)XTP from the cellular (d)NTP pools [10][11][12].…”