Biochemical characterization of a novel hypoxanthine/xanthine dNTP pyrophosphatase from Methanococcus jannaschii

Abstract

A novel dNTP pyrophosphatase, Mj0226 from Methanococcus jannaschii, which catalyzes the hydrolysis of nucleoside triphosphates to the monophosphate and PPi, has been characterized. Mj0226 protein catalyzes hydrolysis of two major substrates, dITP and XTP, suggesting that the 6-keto group of hypoxanthine and xanthine is critical for interaction with the protein. Under optimal reaction conditions the k(ca)(t) /K(m) value for these substrates was approximately 10 000 times that with dATP. Neither endonuclease nor 3'-exonuclease activities were detected in this protein. Interestingly, dITP was efficiently inserted opposite a dC residue in a DNA template and four dNTPs were also incorporated opposite a hypoxanthine residue in template DNA by DNA polymerase I. Two protein homologs of Mj0226 from Escherichia coli and Archaeoglobus fulgidus were also cloned and purified. These have catalytic activities similar to Mj0226 protein under optimal conditions. The implications of these results have significance in understanding how homologous proteins, including Mj0226, act biologically in many organisms. It seems likely that Mj0226 and its homologs have a major role in preventing mutations caused by incorporation of dITP and XTP formed spontaneously in the nucleotide pool into DNA. This report is the first identification and functional characterization of an enzyme hydrolyzing non-canonical nucleotides, dITP and XTP.

Repair System for Noncanonical Purines in Escherichia coli

“…Our data suggest that RdgB is capable of efficiently hydrolyzing dHAPTP. Surprisingly, Chung et al have reported that Mj0226 displays a substrate specificity for dHAPTP that is at least 1,500-fold less than that for xanthine triphosphate (XTP), at least 1,000-fold less than that for dITP, and actually lower than that for dGTP (4). However, in our hands, the pyrophosphatase activity of RdgB against GTP can be significantly inhibited by addition of HAP to the reaction (unpublished results).…”

“…The RecA protein plays a central role in homologous recombination and, in conjunction with the LexA protein, induces the SOS regulon in response to DNA damage (14,36,45). The RdgB protein, along with the human and Methanococcus jannaschii homologs, have been shown to have deoxyribonucleoside triphosphate pyrophosphatase activity on several purine base analog nucleotide triphosphates (4,34). Additionally, Clyman and Cunningham have shown that an rdgB mutant displays a hyperrecombinogenic phenotype and shows elevated levels of SOS expression.…”

ITPase-deficient mice show growth retardation and die before weaning

“…[1][2][3] These modifications often cause a nucleotide to pair with an alternate partner nucleotide in DNA. 4,5 For example, 8-oxo-dGTP, an oxidized product of dGTP, can pair with adenine as well as cytosine during replication, resulting in a transversion mutation. 4 8-Oxoguanine in DNA appears through two independent pathways, either by an incorporation of the oxidized precursor, 8-oxo-dGTP during DNA synthesis, or by the direct oxidation of a guanine base in DNA.…”

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

“…Similarly, RdgB is the E. coli dITPase (10,14,15), whose function is to intercept the noncanonical DNA precursor dITP before hypoxanthine incorporates into DNA in place of guanine. If hypoxanthine forms within DNA by adenine deamination (36), endonuclease V initiates its removal (84,85).…”