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.

FIG. 1.

Structures of the canonical DNA bases, their deaminated derivatives, and HAP.

FIG. 2.

Confirmation of the ability to detect increased mutagenesis in some of the CC strains after treatment with nitrous acid (in acetate buffer). The data are medians of six to nine experiments ± first and third quartiles.

FIG. 3.

Confirmation of the ability to detect increased mutagenesis in selected CC strains with MNNG. The data are averages of two experiments run on different days.

FIG. 4.

Accumulation of Endo V-recognized modifications in the DNA of rdgB mutants. A 22.5-kbp plasmid (pK96) was isolated from the 11 CC strains, as well as from their 11 ΔrdgB61, 11 nfi-1, and 11 ΔrdgB61 nfi-1 derivatives. The DNA was treated in vitro with Endo V, run on an agarose gel to separate supercoiled from relaxed circular species, transferred to a membrane, and hybridized with a pK96-specific probe. The fraction of relaxed circular DNA was then determined in the total plasmid preparation for every sample, and the averages for the 11 samples of the same genotype ± standard errors were plotted.

FIG. 5.

Spontaneous mutagenesis in the 11 CC strains, as well as in their rdgB, nfi, and rdgB nfi mutant derivatives. The data are medians of eight or nine experiments ± first and third quartiles.

FIG. 6.

Nitrous acid-induced mutagenesis. The data are medians of six experiments ± first and third quartiles. (A) Wild-type (WT) cells versus nfi mutants. (B) rdgB single mutants versus rdgB nfi double mutants.

FIG. 7.

HAP-induced mutagenesis. The data points are averages of four independent measurements ± standard errors.

FIG. 8.

Overall scheme of deaminated purine contamination and decontamination in DNA metabolism. dITP, dITP in DNA precursor pools; H, hypoxanthine in DNA; ???, lack of knowledge about excision repair of hypoxanthines downstream of Endo V nicking. Hypoxanthine in DNA may produce double-strand breaks in three ways: (i) by replication forks running into Endo V-induced nicks (shown), (ii) by direct cleavage by Endo V in single-stranded regions of the replication fork (because its hypoxanthine-specific endonuclease activity attacks both single- and double-stranded DNA [53]), and (iii) by two simultaneous Endo V nicks in the opposite strands of the same DNA duplex. We believe that the two latter mechanisms are less likely than the first one because of the very low density of Endo V-recognized DNA modifications, even in rdgB mutants (5).