Human erythrocyte “ITPase”: An ITP pyrophosphohydrolase

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...

Section: From the Department Of Biological Sciences University At Alsupporting

confidence: 54%

Substrate Specificity of RdgB Protein, a Deoxyribonucleoside Triphosphate Pyrophosphohydrolase

Burgis

Cunningham

2007

“…Although mammalian ITPase activities have been identified in human erythrocytes (9), rabbit liver (10), and several rat tissues (17), no gene has been cloned and characterized. Recently a novel bacterial nucleoside triphosphate pyrophosphatase Mj0226, from Methanococcus jannaschii, was revealed by structure-based identification and subsequent biochemical analysis (18).…”

mentioning

confidence: 99%

Cloning, Expression, and Characterization of a Human Inosine Triphosphate Pyrophosphatase Encoded by the ITPAGene

Lin

McLennan

Kong

et al. 2001

129

156

ITP and dITP exist in all cells. dITP is potentially mutagenic, and the levels of these nucleotides are controlled by inosine triphosphate pyrophosphatase (EC 3.6.1.19). Here we report the cloning, expression, and characterization of a 21.5-kDa human inosine triphosphate pyrophosphatase (hITPase), an enzyme whose activity has been reported in many animal tissues and studied in populations but whose protein sequence has not been determined before. At the optimal pH of 10.0, recombinant hITPase hydrolyzed ITP, dITP, and xanthosine 5-triphosphate to their respective monophosphates whereas activity with other nucleoside triphosphates was low. K m values for ITP, dITP, and xanthosine 5-triphosphate were 0.51, 0.31, and 0.57 mM, respectively, and k cat values were 580, 360, and 640 s ؊1 , respectively. A divalent cation was absolutely required for activity. The gene encoding the hITPase cDNA sequence was localized by radiation hybrid mapping to chromosome 20p in the interval D20S113-D20S97, the same interval in which the ITPA inosine triphosphatase gene was previously localized. A BLAST search revealed the existence of many similar sequences in organisms ranging from bacteria to mammals. The function of this ubiquitous protein family is proposed to be the elimination of minor potentially mutagenic or clastogenic purine nucleoside triphosphates from the cell.

“…The inosine triphosphate pyrophosphohydrolase activity has also been identified in human erythrocytes (19). The human inosine triphosphate pyrophosphohydrolase gene has been cloned and named as hITPase, which encodes a protein homologous to the M. jannaschii Mj0226 protein (20).…”

mentioning

confidence: 99%

Thermotoga maritima MazG Protein Has Both Nucleoside Triphosphate Pyrophosphohydrolase and Pyrophosphatase Activities

Zhang

Inouye

2003

MazG proteins form a widely conserved family among bacteria, but their cellular function is still unknown. Here we report that Thermotoga maritima MazG protein (Tm-MazG), the product of the TM0913 gene, has both nucleoside triphosphate pyrophosphohydrolase (NTPase) and pyrophosphatase activities. Tm-MazG catalyzes the hydrolysis of all eight canonical ribo-and deoxyribonucleoside triphosphates to their corresponding nucleoside monophosphates and PP i and subsequently hydrolyzes the resultant PP i to P i . The NTPase activity with deoxyribonucleoside triphosphates as substrate is higher than corresponding ribonucleoside triphosphates. dGTP is the best substrate among the deoxyribonucleoside triphosphates, and GTP is the best among the ribonucleoside triphosphates. Both NTPase and pyrophosphatase activities were enhanced at higher temperatures and blocked by the ␣,␤-methyleneadenosine triphosphate, which cannot be hydrolyzed by Tm-MazG. Furthermore, PP i is an inhibitor for the Tm-MazG NTPase activity. Significant decreases in the NTPase activity and concomitant increases in the pyrophosphatase activity were observed when mutations were introduced at the highly conserved amino acid residues in Tm-MazG N-terminal region (E41Q/E42Q, E45Q, E61Q, R97A/R98A, and K118A). These results demonstrated that Tm-MazG has dual enzymatic functions, NTPase and pyrophosphatase, and that these two enzymatic activities are coordinated.