8-hydroxyguanine and isoguanine

Topics: Animals; DNA Damage; DNA Glycosylases; DNA Repair Enzymes; DNA, Mitochondrial; Genome; Guanine; Humans; Mitochondria; Neurodegenerative Diseases; Nucleotides; Oxidative Stress; Phosphoric Monoester Hydrolases

Oligodeoxyribonucleotides containing 8-hydroxyguanine and 2-hydroxyadenine, purine lesions produced in cells by reactive oxygen species, were synthesized and inserted into vector DNAs to introduce each lesion at a predetermined site. The manipulated DNAs were transfected into living cells, and the mutants induced by each DNA lesion were collected and analyzed. In addition, the mutations induced by damaged DNA precursors with the two oxidized purine bases were studied by the use of chemically synthesized nucleoside triphosphates. In this review article, the author summarizes the mutagenic potentials of the two oxidized purine bases, by focusing on experiments examined by the author and his collaborators.

Topics: Animals; DNA; DNA Damage; Genetic Vectors; Guanine; Mutagenesis; Oligodeoxyribonucleotides; Oxidation-Reduction; Purine Nucleotides; Reactive Oxygen Species

In mammalian cells, more than one genome has to be maintained throughout the entire life of the cell, one in the nucleus and the other in mitochondria. It seems likely that the genomes in mitochondria are highly exposed to reactive oxygen species (ROS) as a result of their respiratory function. Human MTH1 (hMTH1) protein hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP, 8-oxo-dATP, and 2-hydroxy (OH)-dATP, thus suggesting that these oxidized nucleotides are deleterious for cells. Here, we report that a single-nucleotide polymorphism (SNP) in the human MTH1 gene alters splicing patterns of hMTH1 transcripts, and that a novel hMTH1 polypeptide with an additional mitochondrial targeting signal is produced from the altered hMTH1 mRNAs; thus, intracellular location of hMTH1 is likely to be affected by a SNP. These observations strongly suggest that errors caused by oxidized nucleotides in mitochondria have to be avoided in order to maintain the mitochondrial genome, as well as the nuclear genome, in human cells. Based on these observations, we further characterized expression and intracellular localization of 8-oxoG DNA glycosylase (hOGG1) and 2-OH-A/adenine DNA glycosylase (hMYH) in human cells. These two enzymes initiate base excision repair reactions for oxidized bases in DNA generated by direct oxidation of DNA or by incorporation of oxidized nucleotides. We describe the detection of the authentic hOGG1 and hMYH proteins in mitochondria, as well as nuclei in human cells, and how their intracellular localization is regulated by alternative splicing of each transcript.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Animals; Cell Compartmentation; Cell Nucleus; DNA Damage; DNA Glycosylases; DNA Repair; DNA-Formamidopyrimidine Glycosylase; DNA, Mitochondrial; Fungal Proteins; Guanine; HeLa Cells; Humans; Membrane Proteins; Mitochondria; Molecular Sequence Data; N-Glycosyl Hydrolases; Oxidants; Oxidation-Reduction; Oxidative Stress; Point Mutation; Polymorphism, Genetic; Protein Sorting Signals; Protein Transport; Purine Nucleosides; RNA Splicing; RNA, Messenger; Saccharomyces cerevisiae Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Species Specificity; Transfection

Oligodeoxyribonucleotides containing a DNA lesion were synthesized and inserted into vector DNAs to introduce the lesion at a predetermined site. The manipulated DNAs were transfected into living mammalian cells and the mutants induced by the DNA lesion were collected and analyzed. This approach was applied to various DNA lesions produced in cells by reactive oxygen species, chemicals, and ultraviolet light. In addition, the mutations induced by damaged DNA precursors were studied by the use of chemically synthesized nucleoside triphosphates. In this review article, the author summarizes the results obtained by these types of experiments, focusing on two oxidatively damaged compounds, 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) and 2-hydroxyadenine (1,2-dihydro-2-oxoadenine).

Topics: 3T3 Cells; Animals; Base Sequence; DNA Damage; Genes, ras; Genetic Vectors; Guanine; Humans; Mice; Molecular Sequence Data; Mutation; Transformation, Genetic

Cytosine deaminase (CDA) from Escherichia coli was shown to catalyze the deamination of isoguanine (2-oxoadenine) to xanthine. Isoguanine is an oxidation product of adenine in DNA that is mutagenic to the cell. The isoguanine deaminase activity in E. coli was partially purified by ammonium sulfate fractionation, gel filtration, and anion exchange chromatography. The active protein was identified by peptide mass fingerprint analysis as cytosine deaminase. The kinetic constants for the deamination of isoguanine at pH 7.7 are as follows: k(cat) = 49 s(-1), K(m) = 72 μM, and k(cat)/K(m) = 6.7 × 10(5) M(-1) s(-1). The kinetic constants for the deamination of cytosine are as follows: k(cat) = 45 s(-1), K(m) = 302 μM, and k(cat)/K(m) = 1.5 × 10(5) M(-1) s(-1). Under these reaction conditions, isoguanine is the better substrate for cytosine deaminase. The three-dimensional structure of CDA was determined with isoguanine in the active site.

Topics: Catalysis; Cytosine; Deamination; Escherichia coli Proteins; Guanine; Guanine Deaminase; Kinetics; Substrate Specificity

The blockage of transcription elongation by RNA polymerase II (RNAPII) at DNA lesions on the transcribed strand is a serious challenge to accurate transcription. Transcription-coupled DNA repair (TCR), which is assumed to be initiated by the blockage of transcription, rapidly removes lesions on the transcribed strand of expressed genes and allows the resumption of transcription. Although helix-distorting bulky damage such as a cyclobutane pyrimidine dimer is known to block transcription elongation and to be repaired by TCR, it is not clear whether oxidative DNA lesions are repaired by TCR. First, we examined whether transcription elongation by RNAPII is stalled at sites of 2-hydroxyadenine (2-OH-A), 8-oxoadenine (8-oxoA), 8-oxoguanine (8-oxoG), or thymine glycol (Tg) on the transcribed strand. Our results indicate that RNAPII incorporated nucleotides opposite the lesions and then stalled. In addition, we found that transcription elongation factor TFIIS (SII) enabled RNAPII to bypass 8-oxoG but not the other types of damage, while transcription initiation and elongation factor TFIIF did not bypass 8-oxoG. These results suggest that SII is important for preventing cellular death due to oxidative DNA damage, assisting RNAPII to bypass 8-oxoG.

Topics: Adenine; Base Sequence; DNA Damage; DNA Repair; Guanine; HeLa Cells; Humans; Models, Chemical; Models, Genetic; Molecular Sequence Data; Oxygen; RNA Polymerase II; Thymine; Transcription, Genetic; Transcriptional Elongation Factors

An enzyme activity introducing an alkali-labile site at 2-hydroxyadenine (2-OH-A) in double-stranded oligonucleotides was detected in nuclear extracts of Jurkat cells. This activity co-eluted with activities toward adenine paired with guanine and 8-oxo-7,8-dihydroguanine (8-oxoG) as a single peak corresponding to a 55 kDa molecular mass on gel filtration chromatography. Further co-purification was then done. Western blotting revealed that these activities also co-purified with a 52 kDa polypeptide which reacted with antibodies against human MYH (anti-hMYH). Recombinant hMYH has essentially similar activities to the partially purified enzyme. Thus, hMYH is likely to possess both adenine and 2-OH-A DNA glycosylase activities. In nuclear extracts from Jurkat cells, a 52 kDa polypeptide was detected with a small amount of 53 kDa polypeptide, while in mitochondrial extracts a 57 kDa polypeptide was detected using anti-hMYH. With amplification of the 5'-regions of the hMYH cDNA, 10 forms of hMYH transcripts were identified and subgrouped into three types, each with a unique 5' sequence. These hMYH transcripts are likely to encode multiple authentic hMYH polypeptides including the 52, 53 and 57 kDa polypeptides detected in Jurkat cells.

Topics: Adenine; Amino Acid Sequence; Blotting, Western; Cell Extracts; Cell Nucleus; DNA; DNA Glycosylases; DNA Repair; Gene Expression Profiling; Guanine; HeLa Cells; Humans; Isoenzymes; Jurkat Cells; Mitochondria; Molecular Sequence Data; Molecular Weight; N-Glycosyl Hydrolases; Peptides; Recombinant Proteins; RNA, Messenger; Sequence Alignment; Substrate Specificity

The induction of SCE by ribo- and deoxyribonucleosides of 8-hydroxyguanine, 5-hydroxycytosine, and 2-hydroxyadenine was tested using human peripheral blood lymphocytes. All of these compounds caused an increase in SCE frequency. The potency of SCE induction was as follows: 5-OH-C, 5-OH-dC > 8.OH-G > 8-OH-dG > 2-OH-A, 2-OH-dA. These results suggest that the oxidized nucleosides are incorporated into DNA with different efficiencies (or are repaired with different efficiencies) and exhibit genotoxicity in human blood cells. Ribo- and deoxyribo-derivatives of 5-OH-Cyt and 2-OH-Ade also showed mutagenic activity in Salmonella typhimurium TA 100.

Topics: Cytosine; Deoxyribonucleosides; DNA Damage; Guanine; Humans; In Vitro Techniques; Lymphocytes; Mutagenicity Tests; Mutagens; Oxidation-Reduction; Point Mutation; Ribonucleosides; Salmonella typhimurium; Sister Chromatid Exchange

Oxygen radicals are known to play a role in causing cellular DNA damage, which is involved in carcinogenesis. 8-Hydroxyguanine (8-OH-Gua) is a major form of oxidative DNA damage and is known as a useful marker of DNA oxidation. Recently, we found another type of oxidative DNA damage, 2-hydroxyadenine (2-OH-Ade), which has a mutation frequency comparable to that of 8-OH-Gua. We compared the repair activities for two types of oxidative DNA damage, 8-OH-Gua and 2-OH-Ade, in 7-week-old male Sprague-Dawley (SD) rat organs. The repair activities were measured by an endonuclease nicking assay using 22 mer [32P]-end-labeled double-stranded DNA substrates, which contained either 8-OH-Gua (opposite C) or 2-OH-Ade (opposite T or C). In all of the SD rat organs we studied, the nicking activity for 2-OH-Ade was not detected, while that for 8-OH-Gua was clearly detected with the same conditions. Moreover, the 2-OH-Ade nicking activity was not induced in Wistar rat kidney extracts prepared after ferric nitrilotriacetate (Fe-NTA) treatment, which is known to increase 8-OH-Gua repair activity. These results suggest that 2-OH-Ade might not be repaired by the glycosylase type mechanism in mammalian cells.

Topics: Animals; DNA Damage; DNA Repair; Endonucleases; Ferric Compounds; Guanine; Kidney; Liver; Lung; Male; Nitrilotriacetic Acid; Rats; Rats, Sprague-Dawley; Rats, Wistar