Deamination of nucleobases in DNA and RNA results in the formation of xanthine (X), hypoxanthine (I), oxanine, and uracil, all of which are miscoding and mutagenic in DNA and can interfere with RNA editing and function. Among many forms of nucleic acid damage, deamination arises from several unrelated mechanisms, including hydrolysis, nitrosative chemistry, and deaminase enzymes. Here we present a fourth mechanism contributing to the burden of nucleobase deamination: incorporation of hypoxanthine and xanthine into DNA and RNA caused by defects in purine nucleotide metabolism. Using Escherichia coli and Saccharomyces cerevisiae with defined mutations in purine metabolism in conjunction with analytical methods for quantifying deaminated nucleobases in DNA and RNA, we observed large increases (up to 600-fold) in hypoxanthine in both DNA and RNA in cells unable to convert IMP to XMP or AMP (IMP dehydrogenase, guaB; adenylosuccinate synthetase, purA, and ADE12), and unable to remove dITP/ITP and dXTP/XTP from the nucleotide pool (dITP/XTP pyrophosphohydrolase, rdgB and HAM1). Conversely, modest changes in xanthine levels were observed in RNA (but not DNA) from E. coli lacking purA and rdgB and the enzyme converting XMP to GMP (GMP synthetase, guaA). These observations suggest that disturbances in purine metabolism caused by known genetic polymorphisms could increase the burden of mutagenic deaminated nucleobases in DNA and interfere with gene expression and RNA function, a situation possibly exacerbated by the nitrosative stress of concurrent inflammation. The results also suggest a mechanistic basis for the pathophysiology of human inborn errors of purine nucleotide metabolism.DNA and RNA damage | mass spectrometry | nucleobase deamination | purine metabolism | DNA repair T he chemical modification of nucleobases in DNA and RNA can arise from both physiological and adventitious mechanisms at all stages of nucleic acid metabolism. This is particularly true for deaminated versions of the nucleobases. As shown in Fig. 1 for purines, nucleobase deamination in DNA and RNA leads to the formation of 2′-deoxy-and ribonucleoside forms of hypoxanthine (2′-deoxyinosine, dI; inosine, Ino) from adenine, xanthine (2′-deoxyxanthosine, dX; xanthosine, Xao) and oxanine (2′-deoxyoxanosine, dO; oxanosine, Oxo) from guanine, and uracil (2′-deoxyuridine, dU; uridine, Urd) from cytosine (1). All of these products are miscoding and mutagenic in DNA (2-4) and can interfere with RNA editing (5) and the function of noncoding RNAs (6).There are three recognized mechanisms that contribute to nucleobase deamination in DNA and RNA, the simplest of which is hydrolysis (7). A second source of nucleobase deamination is associated with the nitrosative stress caused by increases in nitric oxide-derived nitrous anhydride during inflammation (1). A third mechanism is associated with deaminase enzymes acting on RNA and DNA, with activation-induced cytidine deaminase converting cytidine to uridine during immunoglobulin diversification in B lymphocytes an...

We have previously reported the identification of a DNA repair system in Escherichia coli for the prevention of the stable incorporation of noncanonical purine dNTPs into DNA. We hypothesized that the RdgB protein is active on 2-deoxy-N-6-hydroxylaminopurine triphosphate (dHAPTP) as well as deoxyinosine triphosphate. Here we show that RdgB protein and RdgB homologs from Saccharomyces cerevisiae, mouse, and human all possess deoxyribonucleoside triphosphate pyrophosphohydrolase activity and that all four RdgB homologs have high specificity for dHAPTP and deoxyinosine triphosphate compared with the four canonical dNTPs and several other noncanonical (d)NTPs. Kinetic analysis reveals that the major source of the substrate specificity lies in changes in K m for the various substrates. The expression of these enzymes in E. coli complements defects that are caused by the incorporation of HAP and an endogenous noncanonical purine into DNA. Our data support a preemptive role for the RdgB homologs in excluding endogenous and exogenous modified purine dNTPs from incorporation into DNA.Our laboratory has shown that an Escherichia coli recA200(Ts) rdgB double mutant is inviable at the nonpermissive temperature and suggested that in the absence of RdgB a lesion develops in DNA that requires repair by a RecA-mediated event (1). Overexpression of the purA gene suppresses the temperature-sensitive phenotype of rdgB recA200(Ts) mutants (2). The purA gene encodes adenylosuccinate synthase, which catalyzes the first step in the conversion of IMP to AMP (3). This result suggests that RdgB may play a role in modulating nucleotide pools. The yeast homolog of RdgB has been named HAM1. Pavlov and co-workers (4, 5) have reported that HAM1 mutants of Saccharomyces cerevisiae display hypersensitivity to N-6-hydroxylaminopurine (HAP) 2 and a hypermutable phenotype upon HAP exposure. They suggested the possibility that HAM1 may be a deoxyribonucleoside triphosphate pyrophosphohydrolase and that it may play a role in detoxifying 2Ј-deoxy-HAP triphosphate. E. coli cells that are deficient in molybdopterin biosynthesis (moa strains) display a hypersensitive and mutagenic phenotype upon exposure to HAP (6). We have recently reported that inactivation of the rdgB gene in a moa background resulted in increased HAP sensitivity, an increase in the level of mutagenesis, and increased recombination and SOS induction upon HAP exposure (7). Our results support a model for the exclusion of HAP residues from DNA that includes a molybdoenzyme and RdgB protein and suggest that the repair system excludes an unknown endogenous noncanonical purine from DNA.Recent work in our laboratory has shown that endonuclease V is an important component of a repair pathway for noncanonical purines that have been incorporated into DNA (7). We have demonstrated that inactivation of the nfi gene in E. coli results in a reversal of HAP sensitivity, as well as the hyperrecombination and SOS-induced phenotypes of moa and moa rdgB strains exposed to HAP. Moreover, transduction o...

Exposure of Escherichia coli strains deficient in molybdopterin biosynthesis (moa) to the purine base N-6-hydroxylaminopurine (HAP) is mutagenic and toxic. We show that moa mutants exposed to HAP also exhibit elevated mutagenesis, a hyperrecombination phenotype, and increased SOS induction. The E. coli rdgB gene encodes a protein homologous to a deoxyribonucleotide triphosphate pyrophosphatase from Methanococcus jannaschii that shows a preference for purine base analogs. moa rdgB mutants are extremely sensitive to killing by HAP and exhibit increased mutagenesis, recombination, and SOS induction upon HAP exposure. Disruption of the endonuclease V gene, nfi, rescues the HAP sensitivity displayed by moa and moa rdgB mutants and reduces the level of recombination and SOS induction, but it increases the level of mutagenesis. Our results suggest that endonuclease V incision of DNA containing HAP leads to increased recombination and SOS induction and even cell death. Double-strand break repair mutants display an increase in HAP sensitivity, which can be reversed by an nfi mutation. This suggests that cell killing may result from an increase in double-strand breaks generated when replication forks encounter endonuclease V-nicked DNA. We propose a pathway for the removal of HAP from purine pools, from deoxynucleotide triphosphate pools, and from DNA, and we suggest a general model for excluding purine base analogs from DNA. The system for HAP removal consists of a molybdoenzyme, thought to detoxify HAP, a deoxyribonucleotide triphosphate pyrophosphatase that removes noncanonical deoxyribonucleotide triphosphates from replication precursor pools, and an endonuclease that initiates the removal of HAP from DNA.Extensive investigation has shown that purine base analogs can be mutagenic to cells. 2-Aminopurine has been shown to promote base-pair transitions in Escherichia coli and is highly mutagenic (47). It has been shown that the Klenow fragment of DNA polymerase I inserts either deoxythymidine triphosphate or deoxycytosine triphosphate opposite N-6-hydroxylaminopurine (HAP) in an oligonucleotide template and that HAP induces both A:T to G:C and G:C to A:T transition mutations in E. coli at a similar frequency (44). Both xanthine and hypoxanthine are readily taken up by cells and are quickly converted to their corresponding nucleotides during purine salvage. Furthermore, hypoxanthine, IMP, and XMP can arise endogenously from purine interconversion and from the deamination of canonical bases (51,52,62). Inosine residues in DNA have been shown to result in transition mutations, and xanthosine residues in DNA are assumed to result in transition mutations as well (22). Therefore, it is important for an organism to possess enzymes that protect it from exogenous and endogenous purine base analogs.Schaaper and colleagues have shown that E. coli strains deficient in molybdopterin biosynthesis are hypersensitive to HAP for both mutagenesis and toxicity (29). They established that HAP sensitivity is conferred by the inactivation of...