Substrate specificity of Deinococcus radiodurans Fpg protein.

A DNA repair enzyme has recently been isolated from the ionizing radiation-resistant bacterium Deinococcus radiodurans [Bauche, C., and Laval, J. (1999) J. Bacteriol. 181, 262-269]. This enzyme is a homologue of the Fpg protein of Escherichia coli. We investigated the substrate specificity of this enzyme for products of oxidative DNA base damage using gas chromatography/isotope-dilution mass spectrometry and DNA substrates, which were either gamma-irradiated or treated with H(2)O(2)/Fe(III)-EDTA/ascorbic acid. Excision of purine lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), 4,6-diamino-5-formamidopyrimidine (FapyAde), and 8-hydroxyguanine (8-OH-Gua) was observed among 17 lesions detected in damaged DNA substrates. The extent of excision was determined as a function of enzyme concentration, time, and substrate concentration. FapyGua and FapyAde were excised with similar specificities from three DNA substrates, whereas 8-OH-Gua was the least preferred lesion. The results show that D. radiodurans Fpg protein and its homologue E. coli Fpg protein excise the same modified DNA bases, but the excision rates of these enzymes are significantly different. Formamidopyrimidines are preferred substrates of D. radiodurans Fpg protein over 8-OH-Gua, whereas E. coli Fpg protein excises these three lesions with similar efficiencies from various DNA substrates. Substrate specificities of these enzymes were also compared with that of Saccharomyces cerevisiae Ogg1 protein, which excises FapyGua and 8-OH-Gua, but not FapyAde.     

Substrate specificity of the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase): excision of purine lesions in DNA produced by ionizing radiation or photosensitization.

We have investigated the excision of a variety of modified bases from DNA by the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase) [Boiteux, S., O'Connor, T. R., Lederer, F., Gouyette, A., & Laval, J. (1990) J. Biol. Chem. 265, 3916-3922]. DNA used as a substrate was modified either by exposure to ionizing radiation or by photosensitization using visible light in the presence of methylene blue (MB). The technique of gas chromatography/mass spectrometry, which can unambiguously identify and quantitate pyrimidine- and purine-derived lesions in DNA, was used for analysis of hydrolyzed and derivatized DNA samples. Thirteen products resulting from pyrimidines and purines were detected in gamma-irradiated DNA, whereas only the formation of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 8-hydroxyguanine (8-OH-Gua) was observed in visible light/MB-treated DNA. Analysis of gamma-irradiated DNA after incubation with the Fpg protein followed by precipitation revealed that the Fpg protein significantly excised 4,6-diamino-5-formamidopyrimidine (FapyAde), FapyGua, and 8-OH-Gua. The excision of a small but detectable amount of 8-hydroxyadenine was also observed. The detection of these products in the supernatant fractions of the same samples confirmed their excision by the enzyme. Nine pyrimidine-derived lesions were not excised. The Fpg protein also excised FapyGua and 8-OH-Gua from visible light/MB-treated DNA. The presence of these products in the supernatant fractions confirmed their excision.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of single mutations in the OGG1 gene found in human tumors on the substrate specificity of the Ogg1 protein

We have investigated the effect of single amino acid substitutions of conserved arginines on the catalytic activities of the human Ogg1 protein (α-hOgg1-Ser³²⁶) (wild-type α-hOgg1). Mutant forms of hOgg1 with mutations Arg⁴⁶→Gln (α-hOgg1-Gln⁴⁶) and Arg¹⁵⁴→His (α-hOgg1-His¹⁵⁴) have previously been identified in human tumors. The mutant proteins α-hOgg1-Gln⁴⁶ and α-hOgg1-His¹⁵⁴ were expressed in Escherichia coli and purified to homogeneity. The substrate specificities of these proteins and wild-type α-hOgg1 were investigated using γ-irradiated DNA and the technique of gas chromatography/isotope-dilution mass spectrometry. All three enzymes excised 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 8-hydroxyguanine (8-OH-Gua) from γ-irradiated DNA containing a multiplicity of base lesions. Michaelis–Menten kinetics of excision were measured. Significant differences between excision kinetics of these three enzymes were observed. Excision of FapyGua and 8-OH-Gua by wild-type α-hOgg1 was greater than that by α-hOgg1-Gln⁴⁶ and α-hOgg1-His¹⁵⁴. The latter mutant protein was less active than the former. The diminished activity of the mutant proteins was more pronounced for 8-OH-Gua than for FapyGua. Cleavage assays were also performed using ³²P-labeled 34mer oligonucleotide duplexes containing a single 8-OH-Gua paired to each of the four DNA bases. The results obtained with the oligonucleotide containing the 8-OH-Gua/Cyt pair were in good agreement with those observed with γ-irradiated DNA. Wild-type α-hOgg1 and its mutants repaired the three mismatches less efficiently than the 8-OH-Gua/Cyt pair. The substitution of Arg¹⁵⁴, in addition to diminishing the activity on 8-OH-Gua, relaxes the selectivity found in the wild-type α-hOgg1 for the base opposite 8-OH-Gua. Taken together the results show that the mutant forms α-hOgg1-Gln⁴⁶ and α-hOgg1-His¹⁵⁴ found in human tumors are defective in their catalytic capacities.     

Substrate specificity of the Ogg1 protein of Saccharomyces cerevisiae: excision of guanine lesions produced in DNA by ionizing radiation- or hydrogen peroxide/metal ion-generated free radicals.

We have investigated the substrate specificity of the Ogg1 protein of Saccharomyces cerevisiae (yOgg1 protein) for excision of modified DNA bases from oxidatively damaged DNA substrates using gas chromatography/isotope dilution mass spectrometry. Four DNA substrates prepared by treatment with H2O2/Fe(III)-EDTA/ascorbic acid, H2O2/Cu(II) and gamma-irradiation under N2O or air were used. The results showed that 8-hydroxyguanine (8-OH-Gua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) were efficiently excised from DNA exposed to ionizing radiation in the presence of N2O or air. On the other hand, 8-OH-Gua and FapyGua were not excised from H2O2/Fe(III)-EDTA/ascorbic acid-treated and H2O2/Cu(II)-treated DNA respectively. Fourteen other lesions, including the adenine lesions 8-hydroxyadenine and 4,6-diamino-5-formamidopyrimidine, were not excised from any of the DNA substrates. Kinetics of excision significantly depended on the nature of the damaged DNA substrates. The findings suggest that, in addition to 8-OH-Gua, FapyGua may also be a primary substrate of yOgg1 in cells. The results also show significant differences between the substrate specificities of yOgg1 protein and its functional analog Fpg protein in Escherichia coli.     

Kinetics of excision of purine lesions from DNA by Escherichia coli Fpg protein.

The kinetics of excision of damaged purine bases from oxidatively damaged DNA by Escherichia coli Fpg protein were investigated. DNA substrates, prepared by treatment with H2O2/Fe(III)-EDTA or by gamma-irradiation under N2O or air, were incubated with Fpg protein, followed by precipitation of DNA. Precipitated DNA and supernatant fractions were analyzed by gas chromatography/isotope-dilution mass spectrometry. Kinetic studies revealed efficient excision of 8-hydroxyguanine (8-OH-Gua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4, 6-diamino-5-formamidopyrimidine (FapyAde). Thirteen other modified bases in the oxidized DNA substrates, including 5-hydroxycytosine and 5-hydroxyuracil, were not excised. Excision was measured as a function of enzyme concentration, substrate concentration, time and temperature. The rate of release of modified purine bases from the three damaged DNA substrates varied significantly even though each DNA substrate contained similar levels of oxidative damage. Specificity constants (kcat/KM) for the excision reaction indicated similar preferences of Fpg protein for excision of 8-OH-Gua, FapyGua and FapyAde from each DNA substrate. These findings suggest that, in addition to 8-OH-Gua, FapyGua and FapyAde may be primary substrates for this enzyme in cells.     

Repair of oxidative DNA damage in Drosophila melanogaster: identification and characterization of dOgg1, a second DNA glycosylase activity for 8-hydroxyguanine and formamidopyrimidines

In Drosophila, the S3 ribosomal protein has been shown to act as a DNA glycosylase/AP lyase capable of releasing 8-hydroxyguanine (8-OH-Gua) in damaged DNA. Here we describe a second Drosophila protein (dOgg1) with 8-OH-Gua and abasic (AP) site DNA repair activities. The Drosophila OGG1 gene codes for a protein of 327 amino acids, which shows 33 and 37% identity with the yeast and human Ogg1 proteins, respectively. The DNA glycosylase activity of purified dOgg1 was investigated using γ-irradiated DNA and gas chromatography/isotope dilution mass spectrometry (GC/IDMS). The dOgg1 protein excises 8-OH-Gua and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from γ-irradiated DNA. with k_cat/K_M values of 21.0 × 10^–5 and 11.2 × 10^–5 (min^–1 nM^–1), respectively. Enzymatic assays using oligodeoxyribonucleotides containing a single lesion show that dOgg1 displays a marked preference for DNA duplexes containing 8-OH-Gua, 8-OH-Ade or an AP site placed opposite a cytosine. The cleavage of the 8-OH-Gua-containing strand results from the excision of the damaged base followed by a β-elimination reaction at the 3′-side of the resulting AP site. Cleavage of 8-OH-Gua.C duplex involves the formation of a reaction intermediate that is converted into a stable covalent adduct in the presence of sodium borohydre. dOgg1 complements the mutator phenotype of fpg mutY mutants of Escherichia coli. Whole-mount in situ hybridizations on tissues at different stages of Drosophila development reveal that the dOGG1 messenger is expressed uniformly at a low level in cells in which mitotic division occurs. Therefore, Drosophila possesses two DNA glycosylase activities that can excise 8-OH-Gua and formamidopyrimidines from DNA, dOgg1 and the ribosomal protein S3.     

Effect of single mutations on the specificity of Escherichia coli FPG protein for excision of purine lesions from DNA damaged by free radicals

The formamidopyrimidine N-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that is specific for the removal of purine-derived lesions from DNA damaged by free radicals and other oxidative processes. We investigated the effect of single mutations on the specificity of this enzyme for three purine-derived lesions in DNA damaged by free radicals. These damaging agents generate a multiplicity of base products in DNA, with the yields depending on the damaging agent. Wild type Fpg protein (wt-Fpg) removes 8-hydroxyguanine (8-OH-Gua), 4,6-diamino-5-formamidopyrimidine (FapyAde), and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from damaged DNA with similar specificities. We generated five mutant forms of this enzyme with mutations involving Lys-57-->Gly (FpgK57G), Lys-57-->Arg (FpgK57R), Lys-155-->Ala (FpgK155A), Pro-2-->Gly (FpgP2G), and Pro-2-->Glu (FpgP2E), and purified them to homogeneity. FpgK57G and FpgK57R were functional for removal of FapyAde and FapyGua with a reduced activity when compared with wt-Fpg. The removal of 8-OH-Gua was different in that the specificity of FpgK57G was significantly lower for its removal from irradiated DNA, whereas wt-Fpg, FpgK57G, and FpgK57R excised 8-OH-Gua from H2O2/Fe(III)-EDTA/ascorbic acid-treated DNA with almost the same specificity. FpgK155A and FpgP2G had very low activity and FpgP2E exhibited no activity at all. Michaelis-Menten kinetics of excision was measured and kinetic constants were obtained. The results indicate an important role of Lys-57 residue in the activity of Fpg protein for 8-OH-Gua, but a lesser significant role for formamidopyrimidines. Mutations involving Lys-155 and Pro-2 had a dramatic effect with Pro-2-->Glu leading to complete loss of activity, indicating a significant role of these residues. The results show that point mutations significantly change the specificity of Fpg protein and suggest that point mutations are also expected to change specificities of other DNA repair enzymes.     

Suppression of chemically induced and spontaneously occurring oxidative mutagenesis by three alleles of human OGG1 gene encoding 8-hydroxyguanine DNA glycosylase

8-Hydroxyguanine (8-OH-G) is an oxidatively damaged guanine base that causes G:C to T:A transversion mutations. To counteract the mutagenicity of 8-OH-G in DNA, humans possess the hOGG1 gene, which encodes 8-OH-G DNA glycosylase. Interestingly, genetic polymorphisms at codon 326 (hOGG1-Ser326 versus hOGG1-Cys326) and at codon 46 (hOGG1-Arg46 versus hOGG1-Gln46) exist in human populations. hOGG1-Ser326 and -Cys326 have Arg at codon 46, and hOGG1-Gln46 has Ser at codon 326. In this study, we examined the abilities of three forms of GST-hOGG1 (hOGG1-Ser326, -Cys326 and -Gln46) to suppress chemically induced oxidative mutagenesis using Salmonella typhimurium strains YG3001 and YG3002. These strains are the mutMST derivatives of Ames tester strains TA1535 (uvrB-) and TA1975 (uvrB+), respectively. The mutMST gene encodes a functional counterpart of the OGG1 gene. Mutations induced by 4-nitroquinoline 1-oxide were by more than 95% suppressed by the expression of any of three forms of GST-hOGG1 in strain YG3002. Expression of GST-hOGG1 also reduced by 40 and 60%, respectively, the numbers of His+ revertants induced by methylene blue plus visible light and benzo[a]pyrene plus visible light in strain YG3001. hOGG1-Gln46 displayed a slightly weaker ability to suppress the mutations induced by methylene blue plus visible light than did other two forms although the differences were not statistically significant. About 85 and 95% of spontaneous mutagenesis in strain YG3021 and YG3022, the mutMST mutYST double mutants of strain TA1535 and TA1975, respectively, were suppressed by the expression of any of hOGG1 alleles. hOGG1-Gln46 displayed a weaker suppression than did other two forms in strain YG3022 and the difference was statistically significant. These results suggest that three alleles of the hOGG1 gene efficiently suppress chemically induced and spontaneously occurring oxidative mutagenesis, and that hOGG1-Gln46 may have a weaker ability to suppress the mutations.     

The human OGG1 gene: structure, functions, and its implication in the process of carcinogenesis

A particularly important stress for all cells is the one produced by reactive oxygen species (ROS) that are formed as byproducts of cell metabolism. Among DNA damages induced by ROS, 8-hydroxyguanine (8-OH-G) is certainly the product that has retained most of the attention in the past few years. The biological relevance of 8-OH-G in DNA has been unveiled by the study of Escherichia coli and Saccharomyces cerevisiae genes involved in the neutralization of the mutagenic effects of 8-OH-G. These genes, fpg and mutY for E. coli and OGG1 for yeast, code for DNA glycosylases. Inactivation of any of those genes leads to a spontaneous mutator phenotype, characterized by the increase in GC to TA transversions. In yeast, the OGG1 gene encodes a DNA glycosylase/AP lyase that excises 8-OH-G from DNA. In human cells, the OGG1 gene is localized on chromosome 3p25 and encodes two forms of hOgg1 protein which result from an alternative splicing of a single messenger RNA. The alpha-hOgg1 protein has a nuclear localization whereas the beta-hOgg1 is targeted to the mitochondrion. Biochemical studies on the alpha-hOgg1 protein show that it is a DNA glycosylase/AP lyase that excises 8-OH-G and Fapy-G from gamma-irradiated DNA. Several approaches have been used to study the biological role of OGG1 in mammalian cells, ranging from its overexpression in cell lines to the generation of homozygous ogg1-/- null mice. Furthermore, to explore a possible role in the prevention of cancer, the cDNA coding for alpha-hOgg1 has been sequenced in human tumors. All these results point to 8-OH-G as an endogenous source of mutations in eukaryotes and to its likely involvement in the process of carcinogenesis. A review of the recent literature on the mammalian Ogg1 proteins, the main repair system involved in the elimination of this mutagenic lesion, is presented.     

Comparison of the levels of 8-hydroxyguanine in DNA as measured by gas chromatography mass spectrometry following hydrolysis of DNA by Escherichia coli Fpg protein or formic acid

8-hydroxyguanine (8-OH-Gua) is one of many lesions generated in DNA by oxidative processes including free radicals. It is the most extensively investigated lesion, due to its miscoding properties and its potential role in mutagenesis, carcinogenesis and aging, and also to the existence of analytical methods using HPLC and gas chromatography mass spectrometry (GC/MS). Some studies raised the possibility of artifacts generated during sample preparation. We investigated several experimental conditions in order to eliminate possible artifacts during the measurement of 8-OH-Gua by GC/MS. Derivatization has been reported to produce artifacts by oxidation of guanine to 8-OH-Gua in acid-hydrolysates of DNA, although the extent of artifacts seems to depend on experimental conditions. For removal of 8-OH-Gua from DNA, we used either formic acid hydrolysis or specific enzymatic hydrolysis with Escherichia coli Fpg protein. Derivatization of enzyme-hydrolysates should not generate additional 8-OH-Gua because of the absence of guanine, which is not released by the enzyme, whereas guanine released by acid may be oxidized to yield 8-OH-Gua. The measurement of 8-OH-Gua in calf thymus DNA by GC/isotope-dilution MS (GC/IDMS) using these two different hydrolyses yielded similar levels of 8-OH-Gua. This indicated that no artifacts occurred during derivatization of acid-hydrolysates of DNA. Pyridine instead of acetonitrile and room temperature were used during derivatization. Pyridine reduced the level of 8-OH-Gua, when compared with acetonitrile, indicating its potential to prevent oxidation. Two different stable-isotope labeled analogs of 8-OH-Gua used as internal standards for GC/IDMS analysis yielded similar results. A comparison of the present results with the results of recent trials by the European Standards Committee for Oxidative DNA Damage (ESCODD) is also presented.     

Substrate specificities of the ntg1 and ntg2 proteins of Saccharomyces cerevisiae for oxidized DNA bases are not identical.

Two genes of Saccharomyces cerevisiae, NTG1 and NTG2, encode proteins with a significant sequence homology to the endonuclease III of Escherichia coli. The Ntg1 and Ntg2 proteins were overexpressed in E.coli and purified to apparent homogeneity. The substrate specificity of Ntg1 and Ntg2 proteins for modified bases in oxidatively damaged DNA was investigated using gas chromatography/isotope-dilution mass spectrometry. The substrate used was calf-thymus DNA exposed to gamma-radiation in N2O-saturated aqueous solution. The results reveal excision by Ntg1 and Ntg2 proteins of six pyrimidine-derived lesions, 5-hydroxy-6-hydrothymine, 5-hydroxy-6-hydrouracil, 5-hydroxy-5-methylhydantoin, 5-hydroxyuracil, 5-hydroxycytosine and thymine glycol, and two purine-derived lesions, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine from gamma-irradiated DNA. In contrast, Ntg1 and Ntg2 proteins do not release 8-hydroxyguanine or 8-hydroxyadenine from gamma-irradiated DNA. The Ntg1 and Ntg2 proteins also release 2, 6-diamino-4-hydroxy-5-N-methylformamido-pyrimidine from damaged poly(dG-dC).poly(dG-dC). Excision was measured as a function of enzyme concentration and time. Furthermore, kinetic parameters were determined for each lesion. The results show that kinetic constants varied among the different lesions for the same enzyme. We also investigated the capacity of the Ntg1 and Ntg2 proteins to cleave 34mer DNA duplexes containing a single 8-OH-Gua residue mispaired with each of the four DNA bases. The results show that the Ntg1 protein preferentially cleaves a DNA duplex containing 8-OH-Gua mispaired with a guanine. Moreover, the Ntg1 protein releases free 8-OH-Gua from 8-OH-Gua/Gua duplex but not from duplexes containing 8-OH-Gua mispaired with adenine, thymine or cytosine. In contrast, the Ntg2 protein does not incise duplexes containing 8-OH-Gua mispaired with any of the four DNA bases. These results demonstrate that substrate specificities of the Ntg1 and Ntg2 proteins are similar but not identical and clearly different from that of the endonuclease III of E.coli and its homologues in Schizosaccharomyces pombe or human cells.     

Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase

Human angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a zinc metalloprotease whose closest homolog is angiotensin I-converting enzyme. To begin to elucidate the physiological role of ACE2, ACE2 was purified, and its catalytic activity was characterized. ACE2 proteolytic activity has a pH optimum of 6.5 and is enhanced by monovalent anions, which is consistent with the activity of ACE. ACE2 activity is increased approximately 10-fold by Cl(-) and F(-) but is unaffected by Br(-). ACE2 was screened for hydrolytic activity against a panel of 126 biological peptides, using liquid chromatography-mass spectrometry detection. Eleven of the peptides were hydrolyzed by ACE2, and in each case, the proteolytic activity resulted in removal of the C-terminal residue only. ACE2 hydrolyzes three of the peptides with high catalytic efficiency: angiotensin II () (k(cat)/K(m) = 1.9 x 10(6) m(-1) s(-1)), apelin-13 (k(cat)/K(m) = 2.1 x 10(6) m(-1) s(-1)), and dynorphin A 1-13 (k(cat)/K(m) = 3.1 x 10(6) m(-1) s(-1)). The ACE2 catalytic efficiency is 400-fold higher with angiotensin II () as a substrate than with angiotensin I (). ACE2 also efficiently hydrolyzes des-Arg(9)-bradykinin (k(cat)/K(m) = 1.3 x 10(5) m(-1) s(-1)), but it does not hydrolyze bradykinin. An alignment of the ACE2 peptide substrates reveals a consensus sequence of: Pro-X((1-3 residues))-Pro-Hydrophobic, where hydrolysis occurs between proline and the hydrophobic amino acid.     

A kinetically trapped intermediate of FK506 binding protein forms in vitro: chaperone machinery dominates protein folding in vivo

We have characterised the stability, binding and enzymatic properties of three human FK506 binding proteins (FKBP-12) differing only by the length and sequence of their N-terminus. One construct has a short hexa-his tag (6H-FKBP12); the second longer fusion protein (6HL-FKBP12) contains an additional thrombin protease cleavage site; the third has the long fusion tag removed and is essentially native FKBP-12 (cFKBP12). The proteins were purified both under native conditions and also using a refolding protocol. All three natively purified proteins have, within experimental error, the same peptidyl-prolyl isomerase (PPIase) activity (k(cat)/K(m) approximately 1 x 10(6)M(-1)s(-1)), and bind a natural inhibitor, rapamycin, with the same high affinity (K(d) approximately 6 nM). However, refolding of the protein containing the longer tag in vitro results in reduced PPIase activity (the k(cat)/K(m) was reduced from 1 x 10(6)M(-1)s(-1) to 0.81 x 10(6)M(-1)s(-1)) and a 6-fold affinity loss for rapamycin. Addition of both the long and short N-terminal his-tags slows the refolding kinetics of FKBP-12. However, the shorter his-tagged fusion protein regains fully native activity (> or =95%) while the longer regains only approximately 80-85% of native activity. Equilibrium urea denaturation titrations, isothermal titration calorimetry (ITC), analytical gel-filtration, and fluorescence binding data show that this loss of activity is not due to gross misfolding events, but is rather caused by the formation of a stable but subtly misfolded protein that has reduced peptidyl-prolyl isomerase (PPIase) activity and reduced affinity for rapamycin. The difference in behaviour between the in vitro refolded and native forms is due to the dominant role of the cellular chaperone/folding machinery.     

Rapid phosphotransfer to CheY from a CheA protein lacking the CheY-binding domain

The histidine protein kinase CheA plays a central role in the bacterial chemotaxis signal transduction pathway. Autophosphorylated CheA passes its phosphoryl group to CheY very rapidly (k(cat) approximately 750 s(-)(1)). Phospho-CheY in turn influences the direction of flagellar rotation. The autophosphorylation site of CheA (His(48)) resides in its N-terminal P1 domain. The adjacent P2 domain provides a high-affinity binding site for CheY, which might facilitate the phosphotransfer reaction by tethering CheY in close proximity to the phosphodonor located in P1. To explore the contribution of P2 to the CheA --> CheY phosphotransfer reaction in the Escherichia coli chemotaxis system, we examined the transfer kinetics of a mutant CheA protein (CheADeltaP2) in which the 98 amino acid P2 domain had been replaced with an 11 amino acid linker. We used rapid-quench and stopped-flow fluorescence experiments to monitor phosphotransfer to CheY from phosphorylated wild-type CheA and from phosphorylated CheADeltaP2. The CheADeltaP2 reaction rates were significantly slower and the K(m) value was markedly higher than the corresponding values for wild-type CheA. These results indicate that binding of CheY to the P2 domain of CheA indeed contributes to the rapid kinetics of phosphotransfer. Although phosphotransfer was slower with CheADeltaP2 (k(cat)/K(m) approximately 1.5 x 10(6) M(-)(1) s(-)(1)) than with wild-type CheA (k(cat)/K(m) approximately 10(8) M(-)(1) s(-)(1)), it was still orders of magnitude faster than the kinetics of CheY phosphorylation by phosphoimidazole and other small molecule phosphodonors (k(cat)/K(m) approximately 5-50 M(-)(1) s(-)(1)). We conclude that the P1 domain of CheA also makes significant contributions to phosphotransfer rates in chemotactic signaling.     

Serine 19 of human 6-pyruvoyltetrahydropterin synthase is phosphorylated by cGMP protein kinase II.

6-Pyruvoyltetrahydropterin synthase (PTPS) participates in tetrahydrobiopterin cofactor biosynthesis. We previously identified in a PTPS-deficient patient an inactive PTPS allele with an Arg(16) to Cys codon mutation. Arg(16) is located in the protein surface exposed phosphorylation motif Arg(16)-Arg-Ile-Ser, with Ser(19) as the putative phosphorylation site for serine-threonine protein kinases. Purification of recombinant PTPS-S19A from bacterial cells resulted in an active enzyme (k(cat)/K(m) = 6.4 x 10(3) M(-1) s(-1)), which was similar to wild-type PTPS (k(cat)/K(m) = 4.1 x 10(3) M(-1) s(-1)). In assays with purified enzymes, wild-type but not PTPS-S19A was a specific substrate for the cGMP-dependent protein kinase (cGK) type I and II. Upon expression in COS-1 cells, PTPS-S19A was stable but not phosphorylated and had a reduced activity of approximately 33% in comparison to wild-type PTPS. Extracts from several human cell lines, including brain, contained a kinase that bound to and phosphorylated immobilized wild-type, but not mutant PTPS. Addition of cGMP stimulated phosphotransferase activity 2-fold. Extracts from transfected COS-1 cells overexpressing cGKII stimulated Ser(19) phosphorylation more than 100-fold, but only 4-fold from cGKI overexpressing cells. Moreover, fibroblast extracts from mice lacking cGKII exhibited significantly reduced phosphorylation of PTPS. These results suggest that Ser(19) of human PTPS may be a substrate for cGKII phosphorylation also in vivo, a modification that is essential for normal activity.     

Acute arsenite-induced 8-hydroxyguanine is associated with inhibition of repair activity in cultured human cells

8-Hydroxyguanine (8-OH-Gua) is one of the major modified bases in DNA produced by oxidative damage. Human lung carcinoma cells (A549) were treated with 0.5-2mM sodium arsenite for 4h. By an immunohistochemical type procedure, 8-OH-Gua was clearly detected in A549 cells using a fluorescence microscope and an increase in the percentage of A549 cells with oxidative DNA damage was observed using flow cytometry. The formation of 8-OH-Gua in DNA was also detected by a HPLC-ECD. A dose-dependent increase in oxidative DNA damage in A549 cells with increasing arsenite concentrations was obtained. Therefore, oxidative stress is induced after arsenite treatment. Furthermore, we also found that arsenite decreased the activity of the 8-OH-Gua repair enzyme, hOGG1 (8-oxoguanine-DNA glycosylase 1) as well as its gene and protein expression. We conclude that the 8-OH-Gua level in cultured human cells increases partly by the generation of reactive oxygen species (ROS) and partly by the influence on hOGG1 expression, followed by the inhibition of the repair activity for 8-OH-Gua.     

Singlet oxygen induced DNA damage.

Singlet oxygen generated by photoexcitation and by chemiexcitation selectively reacts with the guanine moiety in nucleosides (kq + kr about 5 x 10(6) M-1s-1) and in DNA. The oxidation products include 8-oxo-7-hydro-deoxyguanosine (8-oxodG; also called 8-hydroxydeoxyguanosine) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). Singlet oxygen also causes alkali-labile sites and single-strand breaks in DNA. The biological consequences include a loss of transforming activity as studied with plasmids and bacteriophage DNA, and mutagenicity and genotoxicity. Employing shuttle vectors, it was shown that double-stranded vectors carrying singlet oxygen induced lesions seem to be processed in mammalian cells by DNA repair mechanisms efficient in preserving the biological activity of the plasmid but highly mutagenic in mammalian cells. Biological protection against singlet oxygen is afforded by quenchers, notably carotenoids and tocopherols. Major repair occurs by excision of the oxidized deoxyguanosine moieties by the Fpg protein, preventing mismatch of 8-oxodG with dA, which would generate G:C to T:A transversions.     

Contribution of the second OB fold of ribosomal protein S1 from Escherichia coli to the recognition of TmRNA

Escherichia coli ribosomal protein S1 is composed of six repeating homologous oligonucleotide/oligosaccharide-binding fold (OB folds). In trans-translation, S1 plays a role in delivering transfer-messenger RNA (tmRNA) to stalled ribosomes. The second OB fold of S1 was found to be protected from tryptic digestion in the presence of tmRNA. Truncated S1 mutant Delta2, in which the first and second OB folds were deleted, showed significantly decreased tmRNA-binding activity. Furthermore, the E. coli S1 homolog (BS1) from Bacillus subtilis, which corresponds to the four C-terminal OB folds of E. coli S1, showed no interaction with E. coli tmRNA, as judged by the results of a gel shift assay. Surface plasmon resonance analysis revealed that mutant Delta2 and BS1 had decreased association rate constants (ka, 0.59 x 10(3) M(-1).S(-1); and ka, 1.89 x 10(3) M(-1).S(-1)), while they retained the respective dissociation rate constants (kd, 0.67 x 10(-3) S(-1); and kd, 0.53 x 10(-3) S(-1)), in comparison with wild-type protein S1 (ka, 3.32 x 10(3) M(-1).S(-1); and kd, 0.56 x 10(-3) S(-1)). These results suggest that the second OB fold in protein S1 is essential for the recognition of tmRNA, while the four C-terminal OB folds play a role in stabilizing the S1-tmRNA complex.