guanosine-monophosphate and 8-oxodeoxyguanosine-triphosphate

8-Oxo-7,8,-dihydro-2'-deoxyguanosine triphosphate (8-oxo-dGTP) is a major product of oxidative damage in the nucleotide pool. It is capable of mispairing with adenosine (dA), resulting in futile, mutagenic cycles of base excision repair. Therefore, it is critical that DNA polymerases discriminate against 8-oxo-dGTP at the insertion step. Because of its roles in oxidative DNA damage repair and non-homologous end joining, DNA polymerase lambda (Pol λ) may frequently encounter 8-oxo-dGTP. Here, we have studied the mechanisms of 8-oxo-dGMP incorporation and discrimination by Pol λ. We have solved high resolution crystal structures showing how Pol λ accommodates 8-oxo-dGTP in its active site. The structures indicate that when mispaired with dA, the oxidized nucleotide assumes the mutagenic syn-conformation, and is stabilized by multiple interactions. Steady-state kinetics reveal that two residues lining the dNTP binding pocket, Ala(510) and Asn(513), play differential roles in dNTP selectivity. Specifically, Ala(510) and Asn(513) facilitate incorporation of 8-oxo-dGMP opposite dA and dC, respectively. These residues also modulate the balance between purine and pyrimidine incorporation. Our results shed light on the mechanisms controlling 8-oxo-dGMP incorporation in Pol λ and on the importance of interactions with the incoming dNTP to determine selectivity in family X DNA polymerases.

Topics: Alanine; Asparagine; Catalytic Domain; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA Polymerase beta; Guanosine Monophosphate; Humans; Kinetics; Protein Binding

Reactive oxygen species are produced as side products of oxygen utilization and can lead to the oxidation of nucleic acids and their precursor nucleotides. Among the various oxidized bases, 8-oxo-7,8-dihydroguanine seems to be the most critical during the transfer of genetic information because it can pair with both cytosine and adenine. During the de novo synthesis of guanine nucleotides, GMP is formed first, and it is converted to GDP by guanylate kinase. This enzyme hardly acts on an oxidized form of GMP (8-oxo-GMP) formed by the oxidation of GMP or by the cleavage of 8-oxo-GDP and 8-oxo-GTP by MutT protein. Although the formation of 8-oxo-GDP from 8-oxo-GMP is thus prevented, 8-oxo-GDP itself may be produced by the oxidation of GDP by reactive oxygen species. The 8-oxo-GDP thus formed can be converted to 8-oxo-GTP because nucleoside-diphosphate kinase and adenylate kinase, both of which catalyze the conversion of GDP to GTP, do not discriminate 8-oxo-GDP from normal GDP. The 8-oxo-GTP produced in this way and by the oxidation of GTP can be used for RNA synthesis. This misincorporation is prevented by MutT protein, which has the potential to cleave 8-oxo-GTP as well as 8-oxo-GDP to 8-oxo-GMP. When (14)C-labeled 8-oxo-GTP was applied to CaCl2-permeabilized cells of a mutT(-) mutant strain, it could be incorporated into RNA at 4% of the rate for GTP. Escherichia coli cells appear to possess mechanisms to prevent misincorporation of 8-oxo-7,8-dihydroguanine into RNA.

Topics: Adenosine Triphosphate; Adenylate Kinase; Cytidine Triphosphate; Deoxyguanine Nucleotides; Escherichia coli; Escherichia coli Proteins; Guanosine Monophosphate; Guanylate Kinases; Kinetics; Nucleoside-Diphosphate Kinase; Oxidation-Reduction; Pyrophosphatases; RNA, Bacterial; Uridine Triphosphate

MTH1 hydrolyzes oxidized nucleotide triphosphates, thereby preventing them from being incorporated into DNA. We here present the structures of human MTH1 (1.9Å) and its complex with the product 8-oxo-dGMP (1.8Å). Unexpectedly MTH1 binds the nucleotide in the anti conformation with no direct interaction between the 8-oxo group and the protein. We suggest that the specificity depends on the stabilization of an enol tautomer of the 8-oxo form of dGTP. The binding of the product induces no major structural changes. The structures reveal the mode of nucleotide binding in MTH1 and provide the structural basis for inhibitor design.

Topics: Animals; Crystallography, X-Ray; Deoxyguanine Nucleotides; DNA Repair Enzymes; Drug Design; Guanosine Monophosphate; Humans; Hydrogen Bonding; Mice; Models, Molecular; Oxidation-Reduction; Phosphoric Monoester Hydrolases; Protein Binding; Protein Conformation; Substrate Specificity

DNA 8-oxoguanine (8-oxoG) causes transversions and is also implicated in frameshifts. We previously identified the dNTP pool as a likely source of mutagenic DNA 8-oxoG and demonstrated that DNA mismatch repair prevented oxidation-related frameshifts in mononucleotide repeats. Here, we show that both Klenow fragment and DNA polymerase alpha can utilize 8-oxodGTP and incorporate the oxidized purine into model frameshift targets. Both polymerases incorporated 8-oxodGMP opposite C and A in repetitive DNA sequences and efficiently extended a terminal 8-oxoG. The human MutSalpha mismatch repair factor recognized DNA 8-oxoG efficiently in some contexts that resembled frameshift intermediates in the same C or A repeats. DNA 8-oxoG in other slipped/mispaired structures in the same repeats adopted configurations that prevented recognition by MutSalpha and by the OGG1 DNA glycosylase thereby rendering it invisible to DNA repair. These findings are consistent with a contribution of oxidative DNA damage to frameshifts. They also suggest how mismatch repair might reduce the burden of DNA 8-oxoG and prevent frameshift formation.

Topics: Adenosine; Base Pair Mismatch; Cytosine; Deoxyguanine Nucleotides; DNA; DNA Glycosylases; DNA Repair; DNA-Binding Proteins; Guanine; Guanosine Monophosphate; MutS Homolog 2 Protein; Proto-Oncogene Proteins; Repetitive Sequences, Nucleic Acid

The MutT enzyme from E. coli, in the presence of a divalent cation, catalyzes the hydrolysis of nucleoside- and deoxynucleoside-triphosphate (NTP) substrates by nucleophilic substitution at Pbeta, to yield a nucleotide (NMP) and PPi. The best substrate of MutT is believed to be the mutagenic nucleotide 8-oxo-dGTP, on the basis of its 10(3.4)-fold lower K(m) than that of dGTP (Maki, H., and Sekiguchi, M. (1992) Nature 355, 273-275). To determine the true affinity of MutT for an 8-oxo-nucleotide and to elucidate the kinetic scheme, product inhibition by 8-oxo-dGMP and dGMP and direct binding of these nucleotides to MutT were studied. With Mg(2+)-activated dGTP hydrolysis, 8-oxo-dGMP is a noncompetitive inhibitor with K(I)(sl)(o)(pe) = 49 nM, which is 10(4.6)-fold lower than the K(I)(sl)(o)(pe)of dGMP (1.7 mM). Similarly, the K(I)(intercept) of 8-oxo-dGMP is 10(4.0)-fold lower than that of dGMP. PPi is a linear uncompetitive inhibitor, suggesting that it dissociates first from the product complex, followed by the nucleotide. Noncompetitive inhibition by dGMP and 8-oxo-dGMP indicates an "iso" mechanism in which the nucleotide product leaves an altered form of the enzyme which slowly reverts to the form which binds substrate. Consistent with this kinetic scheme, (1)H-(15)N HSQC titration of MutT with dGMP reveals weak binding and fast exchange from one site with a K(D) = 1.8 mM, in agreement with its K(I)(sl)(o)(pe). With 8-oxo-dGMP, tight binding and slow exchange (n = 1.0 +/- 0.1, K(D) < 0.25 mM) are found. Isothermal calorimetric titration of MutT with 8-oxo-dGMP yields a K(D) of 52 nM, in agreement with its K(I)(sl)(o)(pe). Changing the metal activator from Mg(2+) to Mn(2+) had little effect on the K(I)(sl)(o)(pe) of dGMP or of 8-oxo-dGMP, consistent with the second-sphere enzyme-M(2+)-H(2)O-NTP-M(2+) complex found by NMR (Lin, J., Abeygunawardana, C., Frick, D. N., Bessman, M. J., and Mildvan, A. S. (1997) Biochemistry 36, 1199-1211), but it decreased the K(I) of PPi 12-fold, suggesting direct coordination of the PPi product by the enzyme-bound divalent cation. The tight binding of 8-oxo-dGMP to MutT (DeltaG degrees = -9.8 kcal/mol) is driven by a highly favorable enthalpy ( = -32 +/- 7 kcal/mol), with an unfavorable entropy (<-TDeltaS(o)(binding)> = +22 +/- 7 kcal/mol), as determined by van't Hoff analysis of the effect of temperature on the K(I)(sl)(o)(pe) and by isothermal titration calorimetry in two buffer systems. The binding of 8-o

Topics: Calorimetry; Cations, Divalent; Deoxyguanine Nucleotides; Diphosphates; Enzyme Activation; Enzyme Activators; Enzyme Inhibitors; Escherichia coli Proteins; Guanosine Monophosphate; Kinetics; Macromolecular Substances; Magnesium; Manganese; Models, Chemical; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular; Phosphoric Monoester Hydrolases; Protons; Pyrophosphatases; Temperature; Thermodynamics

Mismatch repair (MMR) corrects replication errors. It requires the MSH2, MSH6, MLH1, and PMS2 proteins which comprise the MutSalpha and MutLalpha heterodimers. Inactivation of MSH2 or MLH1 in human tumors greatly increases spontaneous mutation rates. Oxidation produces many detrimental DNA alterations against which cells deploy multiple protective strategies. The Ogg-1 DNA glycosylase initiates base excision repair (BER) of 8-oxoguanine (8-oxoG) from 8-oxoG:C pairs. The Myh DNA glycosylase removes mismatched adenines incorporated opposite 8-oxoG during replication. Subsequent BER generates 8-oxoG:C pairs, a substrate for excision by Ogg-1. MTH1-an 8-oxodGTPase which eliminates 8-oxodGTP from the dNTP pool-affords additional protection by minimizing 8-oxodGMP incorporation during replication. Here we show that the dNTP pool is, nevertheless, an important source of DNA 8-oxoG and that MMR provides supplementary protection by excising incorporated 8-oxodGMP. Incorporated 8-oxodGMP contributes significantly to the mutator phenotype of MMR-deficient cells. Thus, although BER of 8-oxoG is independent of Msh2, both steady-state and H(2)O(2)-induced DNA 8-oxoG levels are higher in Msh2-defective cells than in their repair-proficient counterparts. Increased expression of MTH1 in MMR-defective cells significantly reduces steady-state and H(2)O(2)-induced DNA 8-oxoG levels. This reduction dramatically diminishes the spontaneous mutation rate of Msh2(-/-) MEFs.

Topics: Animals; Base Pair Mismatch; Deoxyguanine Nucleotides; DNA Repair; Guanosine Monophosphate; Mammals; Oxidation-Reduction