8-hydroxyguanine and Cockayne-Syndrome

8-Oxoguanine (8-oxoG) is a major oxidized base found in DNA due to endogenous or exogenous pro-oxidant agents. In the absence of repair, this lesion has a high mutation potency giving rise mainly to G:C to A:T transversions. 8-oxoG can be removed by the classical base excision repair pathway but can also be eliminated by a transcription-coupled repair (TCR) process that needs the wild type activities of CSB, XPG, XPB, XPD, BRCA1, BRCA2 and MSH2 proteins. The lack of TCR of oxidative lesions may lead to dramatic hereditary diseases like Cockayne syndrome. Accumulation of unrepaired oxidized bases in brain cells may explain the progressive neurological deterioration found in some DNA repair-deficient patients.

Topics: Cockayne Syndrome; DNA Repair; Guanine; Humans; Nervous System Diseases; Transcription, Genetic

Multiple DNA repair processes are required to maintain the integrity of the cellular genome. Recent advances, including elucidation of three-dimensional structures of DNA repair enzymes, and the cloning and characterization of DNA repair genes implicated in human inherited disease, have given new insights into the surprising complexity of cellular responses to DNA damage.

Topics: Animals; Cockayne Syndrome; DNA; DNA Repair; Guanine; Humans; Methylation; Xeroderma Pigmentosum

Reduced host cell reactivation (HCR) of a reporter gene containing 8-oxoguanine (8-oxoG) lesions in Cockayne syndrome (CS) fibroblasts has previously been attributed to increased 8-oxoG-mediated inhibition of transcription resulting from a deficiency in repair. This interpretation has been challenged by a report suggesting reduced expression from an 8-oxoG containing reporter gene occurs in all cells by a mechanism involving gene inactivation by 8-oxoG DNA glycosylase and this inactivation is strongly enhanced in the absence of the CS group B (CSB) protein. The observation of reduced gene expression in the absence of CSB protein led to speculation that decreased HCR in CS cells results from enhanced gene inactivation rather than reduced gene reactivation. Using an adenovirus-based β-galactosidase (β-gal) reporter gene assay, we have examined the effect of methylene blue plus visible light (MB + VL)-induced 8-oxoG lesions on the time course of gene expression in normal and CSA and CSB mutant human SV40-transformed fibroblasts, repair proficient and CSB mutant Chinese hamster ovary (CHO) cells and normal mouse embryo fibroblasts. We demonstrate that MB + VL treatment of the reporter leads to reduced expression of the damaged β-gal reporter relative to control at early time points following infection in all cells, consistent with in vivo inhibition of RNA polII-mediated transcription. In addition, we have demonstrated HCR of reporter gene expression occurs in all cell types examined. A significant reduction in the rate of gene reactivation in human SV40-transformed cells lacking functional CSA or CSB compared to normal cells was found. Similarly, a significant reduction in the rate of reactivation in CHO cells lacking functional CSB (CHO-UV61) was observed compared to the wild-type parental counterpart (CHO-AA8). The data presented demonstrate that expression of an oxidatively damaged reporter gene is reactivated over time and that CSA and CSB are required for normal reactivation.

Topics: Adenoviridae; Animals; beta-Galactosidase; Cell Line, Transformed; CHO Cells; Cockayne Syndrome; Cricetinae; Cricetulus; DNA Damage; DNA Helicases; DNA Repair Enzymes; Gene Expression Regulation, Viral; Genes, Reporter; Guanine; Humans; Poly-ADP-Ribose Binding Proteins; Transcription Factors; Ultraviolet Rays

Cells from Cockayne syndrome patients are characterized by a deficiency in transcription-coupled repair (TCR) of UV-induced lesions. These cells have also been shown to be sensitive to oxidative stress and defective in TCR of some oxidative lesions. Because some discrepancies about this pathway have been recently reported in the literature, we describe here a system that allows us to analyze the effect of a unique 8-oxoguanine (8-oxoG) lesion on gene transcription in vivo. We have constructed nonreplicative shuttle vectors containing a single 8-oxoG in the transcribed strand of the luciferase reporter gene. We have positioned this unique lesion in different sequence contexts and we have tested the effect of two promoters with different transcriptional strength on the level of transcriptional bypass/pause due to the presence of the lesion. When we transfected DNA repair-deficient mouse cell lines with these shuttle vectors, we found a approximately 50% decrease in relative luciferase activity in Ogg1(-/-) and Csb(-/-) embryonic mouse cell lines. In Csb(-/-)/Ogg1(-/-) cells, this decrease was even more important achieving eventually up to 90% inhibition of luciferase expression depending upon the promoter strength and the position of the lesion. These results show clearly that a unique 8-oxoG exhibits different effect on gene expression depending upon the nucleotidic sequence around it and needs the wild-type activities of Csb and Ogg1 proteins to be fully repaired.

Topics: Animals; Bacteria; Base Sequence; Cell Line; Cockayne Syndrome; DNA Glycosylases; DNA Repair; DNA Repair Enzymes; Embryo, Mammalian; Fibroblasts; Guanine; Luciferases; Mice; Mice, Knockout; Mutagenesis; Mutagenicity Tests; Poly-ADP-Ribose Binding Proteins; Promoter Regions, Genetic; Transcription, Genetic

It has been previously reported that the elevated accumulation of repair incision intermediates in cells from patients with combined characteristics of xeroderma pigmentosum complementation group D (XP-D) and Cockayne syndrome (CS) XP-D/CS fibroblasts following UV irradiation is caused by an "uncontrolled" incision of undamaged genomic DNA induced by UV-DNA-lesions which apparently are not removed. This could be an explanation for the extreme sensitivity of these cells to UV light. In the present study, we confirm the immediate DNA breakage following UV irradiation also for CS group B (CS-B) fibroblasts by DNA migration in the "comet assay" and extend these findings to other lesions such as 8-oxodeoxyguanosine (8-oxodG), selectively induced by KBrO3 treatment. In contrast, X-ray exposure does not induce differential DNA breakage. This indicates that additional lesions other than the UV-induced photoproducts (cyclobutane pyrimidine dimers, CPD, and 6-pyrimidine-4-pyrimidone products, 6-4 PP), such as 8-oxodG, specifically induced by KBrO3, are likely to trigger "uncontrolled" DNA breakage in the undamaged genomic DNA in the CS-B fibroblasts, thus accounting for some of the clinical features of these patients.

Topics: Bromates; Chromosomal Instability; Cockayne Syndrome; Comet Assay; DNA; DNA Damage; DNA Repair; Fibroblasts; Guanine; Humans; Oxidation-Reduction; Photochemistry; Pyrimidine Dimers; Radiation Tolerance; Transcription, Genetic; Ultraviolet Rays; Xeroderma Pigmentosum

Cockayne syndrome (CS) is a genetic human disease with clinical symptoms that include neurodegeneration and premature aging. The disease is caused by the disruption of CSA, CSB, or some types of xeroderma pigmentosum genes. It is known that the CSB protein coded by the CS group B gene plays a role in the repair of 8-hydroxyguanine (8-OH-Gua) in transcription-coupled and non-strand discriminating modes. Recently we reported a defect of CSB mutant cells in the repair of another oxidatively modified lesion 8-hydroxyadenine (8-OH-Ade). We show here that primary fibroblasts from CS patients lack the ability to efficiently repair these particular types of oxidatively induced DNA damages. Primary fibroblasts of 11 CS patients and 6 control individuals were exposed to 2 Gy of ionizing radiation to induce oxidative DNA damage and allowed to repair the damage. DNA from cells was analyzed using liquid chromatography/isotope dilution mass spectrometry to measure the biologically important lesions 8-OH-Gua and 8-OH-Ade. After irradiation, no significant change in background levels of 8-OH-Gua and 8-OH-Ade was observed in control human cells, indicating their complete cellular repair. In contrast, cells from CS patients accumulated significant amounts of these lesions, providing evidence for a lack of DNA repair. This was supported by the observation that incision of 8-OH-Gua- or 8-OH-Ade-containing oligodeoxynucleotides by whole cell extracts of fibroblasts from CS patients was deficient compared to control individuals. This study suggests that the cells from CS patients accumulate oxidatively induced specific DNA base lesions, especially after oxidative stress. A deficiency in cellular repair of oxidative DNA damage might contribute to developmental defects in CS patients.

Topics: Adenine; Adult; Cell Line; Child; Child, Preschool; Cockayne Syndrome; DNA; DNA Damage; DNA Repair; Endonucleases; Exonucleases; Female; Guanine; Humans; Infant; Male; Oxidative Stress

Reactive oxygen species, which are prevalent in mitochondria, cause oxidative DNA damage including the mutagenic DNA lesion 7,8-dihydroxyguanine (8-oxoG). Oxidative damage to mitochondrial DNA has been implicated as a causative factor in a wide variety of degenerative diseases, and in cancer and aging. 8-oxoG is repaired efficiently in mammalian mitochondrial DNA by enzymes in the base excision repair pathway, including the 8-oxoguanine glycosylase (OGG1), which incizes the lesion in the first step of repair. Cockayne syndrome (CS) is a segmental premature aging syndrome in humans that has two complementation groups, CSA and CSB. Previous studies showed that CSB-deficient cells have reduced capacity to repair 8-oxoG. This study examines the role of the CSB gene in regulating repair of 8-oxoG in mitochondrial DNA in human and mouse cells. 8-oxoG repair was measured in liver cells from CSB deficient mice and in human CS-B cells carrying expression vectors for wild type or mutant forms of the human CSB gene. For the first time we report that CSB stimulates repair of 8-oxoG in mammalian mitochondrial DNA. Furthermore, evidence is presented to support the hypothesis that wild type CSB regulates expression of OGG1.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Line, Transformed; Cockayne Syndrome; DNA Helicases; DNA Primers; DNA Repair; DNA Repair Enzymes; DNA-Formamidopyrimidine Glycosylase; DNA, Mitochondrial; Guanine; Humans; Mice; Mice, Inbred C57BL; N-Glycosyl Hydrolases; Poly-ADP-Ribose Binding Proteins

We have previously reported that the Cockayne syndrome group B gene product (CSB) contributes to base excision repair (BER) of 8-hydroxyguanine (8-OH-Gua) and the importance of motifs V and VI of the putative helicase domains of CSB in BER of 8-OH-Gua. To further elucidate the function of CSB in BER, we investigated its role in the pathway involving human 8-OH-Gua glycosylase/apurinic lyase (hOgg1). Depletion of CSB protein with anti-CSB antibody reduced the 8-OH-Gua incision rate of wild type cell extracts but not of CSB null and motif VI mutant cell extracts, suggesting a direct contribution of CSB to the catalytic process of 8-OH-Gua incision and the importance of its motif VI in this pathway. Introduction of recombinant purified CSB partially complemented the depletion of CSB as shown by the recovery of the incision activity. This complementation could not fully recover the deficiency of the incision activity in WCE from CS-B null and mutant cell lines, suggesting that some additional factor(s) are necessary for the full activity. Electrophoretic mobility shift assays (EMSAs) showed a defect in binding of CSB null and motif VI mutant cell extracts to 8-OH-Gua-containing oligonucleotides. We detected less hOgg1 transcript and protein in the cell extracts from CS-B null and mutant cells, suggesting hOgg1 may be the missing component. Pull-down of hOgg1 by histidine-tagged CSB and co-localization of those two proteins after gamma-radiation indicated their co-existence in vivo, particularly under cellular stress. However, we did not detect any functional and physical interaction between purified CSB and hOgg1 by incision, gel shift and yeast two-hybrid assays, suggesting that even though hOgg1 and CSB might be in a common protein complex, they may not interact directly. We conclude that CSB functions in the catalysis of 8-OH-Gua BER and in the maintenance of efficient hOgg1 expression, and that motif VI of the putative helicase domain of CSB is crucial in these functions.

Topics: Amino Acid Motifs; Blotting, Western; Cell Extracts; Cell Line, Transformed; Cells, Cultured; Cockayne Syndrome; DNA; DNA Helicases; DNA Primers; DNA Repair; DNA Repair Enzymes; DNA-Formamidopyrimidine Glycosylase; Electrophoretic Mobility Shift Assay; Fluorescent Antibody Technique; Guanine; Humans; Mutagenesis, Site-Directed; Mutation; N-Glycosyl Hydrolases; Poly-ADP-Ribose Binding Proteins; Polymerase Chain Reaction; Promoter Regions, Genetic; Recombinant Proteins; Saccharomyces cerevisiae; Transfection; Two-Hybrid System Techniques

Cockayne syndrome (CS) is a rare inherited human genetic disorder characterized by UV sensitivity, developmental abnormalities and premature aging. The cellular and molecular phenotypes of CS include increased sensitivity to oxidative and UV-induced DNA lesions. The CSB protein is thought to play a pivotal role in transcription-coupled repair and CS-B cells are defective in the repair of the transcribed strand of active genes, both after exposure to UV and in the presence of oxidative DNA lesions. A previous study has indicated that a conserved helicase ATPase motif II residue is essential for the function of the CSB protein in responding to UV-induced DNA damage in a hamster cell line. Due to the limitations in studying a complex human disorder in another species, this study introduced the site-directed mutation of the ATPase motif II in the human CSB gene in an isogenic human cell line. The CSB mutant allele was tested for genetic complementation of UV-sensitive phenotypes in the human CS-B cell line CS1AN.S3.G2. In addition, the incision of an 8-oxoguanine lesion by extracts of the CS-B cell lines stably transfected with the wild-type or ATPase mutant CSB gene has been investigated. The ATPase motif II point mutation (E646Q) abolished the function of the CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery and apoptosis. Interestingly, whole-cell extract prepared from these mutant cells retained wild-type incision activity on an oligonucleotide containing a single 8-oxoguanine lesion, whereas the absence of the CSB gene altogether resulted in reduced incision activity relative to wild-type. These results suggest damage-specific functional requirements for CSB in the repair of UV-induced and oxidative lesions in human cells. The transfection of the mutant or wild-type CSB gene into the CS1AN.S3.G2 cells did not alter the expression of the subset of genes examined by cDNA array analysis.

Topics: Adenosine Triphosphatases; Amino Acid Motifs; Amino Acid Sequence; Apoptosis; Cell Extracts; Cell Line; Cell Survival; Cockayne Syndrome; Cytosine; DNA Damage; DNA Helicases; DNA Repair; DNA Repair Enzymes; Fibroblasts; Gene Expression Profiling; Genetic Complementation Test; Guanine; Humans; Hydrogen Peroxide; Mutation; Oligonucleotide Array Sequence Analysis; Poly-ADP-Ribose Binding Proteins; Protein Structure, Tertiary; Radiation Tolerance; RNA; Thymine; Ultraviolet Rays

Cockayne Syndrome (CS) is a human genetic disorder with two complementation groups, CS-A and CS-B. The CSB gene product is involved in transcription-coupled repair of DNA damage but may participate in other pathways of DNA metabolism. The present study investigated the role of different conserved helicase motifs of CSB in base excision repair. Stably transformed human cell lines with site-directed CSB mutations in different motifs within its putative helicase domain were established. We find that CSB null and helicase motif V and VI mutants had greater sensitivity than wild type cells to gamma-radiation. Whole cell extracts from CSB null and motif V/VI mutants had lower activity of 8-hydroxyguanine incision in DNA than wild type cells. Also, 8-hydroxyguanine accumulated more in CSB null and motif VI mutant cells than in wild type cells after exposure to gamma-radiation. We conclude that a deficiency in general genome base excision repair of selective modified DNA base(s) might contribute to CS pathogenesis. Furthermore, whereas the disruption of helicase motifs V or VI results in a CSB phenotype, mutations in other helicase motifs do not cause this effect. The biological functions of CSB in different DNA repair pathways may be mediated by distinct functional motifs of the protein.

Topics: Amino Acid Sequence; Cell Line, Transformed; Cockayne Syndrome; DNA; DNA Helicases; DNA Repair; DNA Repair Enzymes; Genome; Guanine; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Oxidative Stress; Poly-ADP-Ribose Binding Proteins

Analysis of transcription-coupled repair (TCR) of oxidative lesions here reveals strand-specific removal of 8-oxo-guanine (8-oxoG) and thymine glycol both in normal human cells and xeroderma pigmentosum (XP) cells defective in nucleotide excision repair. In contrast, Cockayne syndrome (CS) cells including CS-B, XP-B/CS, XP-D/CS, and XP-G/CS not only lack TCR but cannot remove 8-oxoG in a transcribed sequence, despite its proficient repair when not transcribed. The XP-G/CS defect uniquely slows lesion removal in nontranscribed sequences. Defective TCR leads to a mutation frequency at 8-oxoG of 30%-40% compared to the normal 1%-4%. Surprisingly, unrepaired 8-oxoG blocks transcription by RNA polymerase II. These data imply that TCR is required for polymerase release to allow repair and that CS results from defects in TCR of oxidative lesions.

Topics: Cell Line; Cockayne Syndrome; DNA Helicases; DNA Repair; DNA Repair Enzymes; DNA-Binding Proteins; Endonucleases; Fibroblasts; Guanine; Humans; Mutagenesis; Nuclear Proteins; Oxidation-Reduction; Oxidative Stress; Plasmids; Poly-ADP-Ribose Binding Proteins; RNA Polymerase II; Transcription Factor TFIIH; Transcription Factors; Transcription Factors, TFII; Transcription, Genetic; Transfection; Xeroderma Pigmentosum

The incision of the 8-oxoguanine in DNA by normal and Cockayne Syndrome (CS) cell extracts has been investigated. The incision in extracts derived from CS cells was approximately 50% of the incision level compared with extracts prepared from normal cells. In contrast, the incision rate of uracil and thymine glycol was not defective in CS cells. The deficiency in 8-oxoguanine incision was also demonstrated in a CS family. Whereas the proband had markedly less incision compared with the normal siblings, the parents had intermediate levels. The low level of 8-oxoguanine-DNA glycosylase in CS extracts correlates with the reduced expression of the 8-oxoguanine-DNA glycosylase gene (hOGG1) in CS cells. Both the levels of expression of the hOGG1 gene and the incision of 8-oxoguanine in DNAincreased markedly after transfection of CS-B cells with the CSB gene. We suggest that the CSB mutation leads to deficient transcription of the hOGG1 gene and thus to deficient repair of 8-oxoguanine in DNA.

Topics: Base Sequence; Cell Line; Cockayne Syndrome; DNA; DNA Helicases; DNA Primers; DNA Repair; DNA Repair Enzymes; DNA-Formamidopyrimidine Glycosylase; Down-Regulation; Guanine; Humans; N-Glycosyl Hydrolases; Poly-ADP-Ribose Binding Proteins; Transfection

Living organisms are constantly exposed to oxidative stress from environmental agents and from endogenous metabolic processes. The resulting oxidative modifications occur in proteins, lipids and DNA. Since proteins and lipids are readily degraded and resynthesized, the most significant consequence of the oxidative stress is thought to be the DNA modifications, which can become permanent via the formation of mutations and other types of genomic instability. Many different DNA base changes have been seen following some form of oxidative stress, and these lesions are widely considered as instigators for the development of cancer and are also implicated in the process of aging. Several studies have documented that oxidative DNA lesions accumulate with aging, and it appears that the major site of this accumulation is mitochondrial DNA rather than nuclear DNA. The DNA repair mechanisms involved in the removal of oxidative DNA lesions are much more complex than previously considered. They involve base excision repair (BER) pathways and nucleotide excision repair (NER) pathways, and there is currently a great deal of interest in clarification of the pathways and their interactions. We have used a number of different approaches to explore the mechanism of the repair processes, and we are able to examine the repair of different types of lesions and to measure different steps of the repair processes. Furthermore, we can measure the DNA damage processing in the nuclear DNA and separately, in the mitochondrial DNA. Contrary to widely held notions, mitochondria have efficient DNA repair of oxidative DNA damage and we are exploring the mechanisms. In a human disorder, Cockayne syndrome (CS), characterized by premature aging, there appear to be deficiencies in the repair of oxidative DNA damage in the nuclear DNA, and this may be the major underlying cause of the disease.

Topics: Aging; Cockayne Syndrome; DNA Damage; DNA Repair; DNA, Mitochondrial; Guanine; Humans; Oxidation-Reduction