Overexpression and rapid purification of Escherichia coli formamidopyrimidine–DNA glycosylase

Journal of Biological Chemistry

Doğan

et al. 2009

Self Cite

100

Cockayne syndrome (CS) 6 is a segmental premature aging syndrome with progressive neurological degeneration (1). CS is caused by mutations in CS complementation groups A (CSA) or B (CSB) genes (2, 3). Approximately 80% of CS patients have mutations in the CSB gene, which encodes a 168-kDa protein belonging to the SWI/SNF2 family of chromatin remodeling proteins (4). Cells from CS patients are hypersensitive to UV radiation-induced DNA damage, and the CSB protein is required for the transcription-coupled nucleotide excision repair of UV radiation-induced DNA lesions (cyclobutane pyrimidine dimers and 6-pyrimidine-4-pyrimidone products) (5). CSB is also believed to play a role in transcription elongation and interacts with the RNA polymerase II elongation complex (6). The molecular basis of the progressive neurological defects in CS patients, however, remains unknown; it has been proposed that neurological symptoms in CS may be due to defective repair and/or processing of oxidative DNA damage in CSB-deficient cells (7).Oxidative DNA damage can be caused by endogenous and exogenous agents. Reactive oxygen species, including highly reactive hydroxyl radicals, are formed as byproducts of normal metabolism, mostly during the process of mitochondrial respiration. It has been estimated that up to 2% of all the O 2 consumed by respiration may be released as reactive oxygen species (8, 9). The central nervous system relies exclusively on mitochondria to generate ATP through oxidative metabolism. As a result, neurons are susceptible to increased levels of oxidative stress, and elevated levels of reactive oxygen species have been implicated in the etiology of neurodegenerative diseases including Alzheimer, Parkinson, and Huntington diseases and amyotrophic lateral sclerosis (for a review, see Ref. 10).Hydroxyl radicals attack DNA bases and the sugar-phosphate DNA backbone, generating modified bases and singlestranded DNA (ssDNA) breaks, respectively (11). Many oxida-

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Cockayne Syndrome Group B Protein Stimulates Repair of Formamidopyrimidines by NEIL1 DNA Glycosylase

Müftüoğlu

Journal of Biological Chemistry

Doğan

et al. 2009

Self Cite

100

“…22 Following ethanol precipitation, DNA pellets and supernatant fractions were separated by centrifugation. Supernatant fractions were lyophilized, trimethylsilylated, and analyzed by GC/MS as described (24). For identification and quantification, selected-ion monitoring was used to monitor the characteristic ions of trimethylsilylated FapyA and FapyG and their stable isotopelabeled analogues (25).…”

Section: Analysis By Liquid Chromatography/mass Spectrometry (Lc/ms) mentioning

confidence: 99%

Repair of Formamidopyrimidines in DNA Involves Different Glycosylases

Journal of Biological Chemistry

Haraguchi

et al. 2005

Self Cite

184

148

The oxidatively induced DNA lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino-5-formamidopyrimidine (FapyA) are formed abundantly in DNA of cultured cells or tissues exposed to ionizing radiation or to other free radical-generating systems. In vitro studies indicate that these lesions are miscoding, can block the progression of DNA polymerases, and are substrates for base excision repair. However, no study has yet addressed how these lesions are metabolized in cellular extracts. The synthesis of oligonucleotides containing FapyG and FapyA at defined positions was recently reported. These constructs allowed us to investigate the repair of Fapy lesions in nuclear and mitochondrial extracts from wild type and knock-out mice lacking the two major DNA glycosylases for repair of oxidative DNA damage, OGG1 and NTH1. The background level of FapyG/FapyA in DNA from these mice was also determined. Endogenous FapyG levels in liver DNA from wild type mice were significantly higher than 8-hydroxyguanine levels. FapyG and FapyA were efficiently repaired in nuclear and mitochondrial extracts from wild type animals but not in the glycosylasedeficient mice. Our results indicated that OGG1 and NTH1 are the major DNA glycosylases for the removal of FapyG and FapyA, respectively. Tissue-specific analysis suggested that other DNA glycosylases may contribute to FapyA repair when NTH1 is poorly expressed. We identified NEIL1 in liver mitochondria, which could account for the residual incision activity in the absence of OGG1 and NTH1. FapyG and FapyA levels were significantly elevated in DNA from the knock-out mice, underscoring the biological role of OGG1 and NTH1 in the repair of these lesions.A large number of DNA base modifications are formed by oxidative damage to DNA (for review, see Ref. 1). Some of these lesions are generated at high rates, even in the absence of exogenous DNA-damaging agents. For instance, it was estimated that 100 -500 8-hydroxyguanines 3 are formed per day in a human cell (2). 8-oxoG is one of the most studied DNA lesions, and it is often used as a biomarker of oxidative DNA damage. However, ring-opened formamidopyrimidine lesions, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino-5-formamidopyrimidine (FapyA), are formed at equal or higher levels than 8-oxoG after oxidative stress (3-5). These lesions result from hydroxyl radical attack on guanine and adenine, respectively, followed by one-electron reduction of the hydroxyl adduct radicals (1), which are also intermediates in the formation of 8-oxoG and 8-oxoA (6). Formation of Fapy lesions in DNA upon UV radiation has also been reported (7). FapyA (8) and FapyG (9) are miscoding in vitro, both directing the preferential misincorporation of adenine opposite the lesions by a bacterial DNA polymerase (Klenow exo Ϫ ). Experiments using the methylated analogue of FapyG, i.e. 2,6-diamino-4-hydroxy-5-Nmethylformamidopyrimidine (Me-FapyG), have suggested that formamidopyrimidines might also constitute blocks to DNA poly...

“…After ethanol precipitation, DNA pellets and supernatant fractions were separated by centrifugation. Supernatant fractions were lyophilized, trimethylsilylated, and analyzed by GC-MS (29). For identification and quantification, selected-ion monitoring was used to monitor the characteristic ions of trimethylsilylated FapyGua and its stable isotope-labeled analogues (9).…”

mentioning

confidence: 99%

The Recombination Protein RAD52 Cooperates with the Excision Repair Protein OGG1 for the Repair of Oxidative Lesions in Mammalian Cells

Molecular and Cellular Biology

Maynard

Hashiguchi

et al. 2009

Oxidized bases are common types of DNA modifications. Their accumulation in the genome is linked to aging and degenerative diseases. These modifications are commonly repaired by the base excision repair (BER) pathway. Oxoguanine DNA glycosylase (OGG1) initiates BER of oxidized purine bases. A small number of protein interactions have been identified for OGG1, while very few appear to have functional consequences. We report here that OGG1 interacts with the recombination protein RAD52 in vitro and in vivo. This interaction has reciprocal functional consequences as OGG1 inhibits RAD52 catalytic activities and RAD52 stimulates OGG1 incision activity, likely increasing its turnover rate. RAD52 colocalizes with OGG1 after oxidative stress to cultured cells, but not after the direct induction of double-strand breaks by ionizing radiation. Human cells depleted of RAD52 via small interfering RNA knockdown, and mouse cells lacking the protein via gene knockout showed increased sensitivity to oxidative stress. Moreover, cells depleted of RAD52 show higher accumulation of oxidized bases in their genome than cells with normal levels of RAD52. Our results indicate that RAD52 cooperates with OGG1 to repair oxidative DNA damage and enhances the cellular resistance to oxidative stress. Our observations suggest a coordinated action between these proteins that may be relevant when oxidative lesions positioned close to strand breaks impose a hindrance to RAD52 catalytic activities.Oxidative DNA damage is generated at high levels in mammalian cells, even in cells not exposed to exogenous sources of reactive oxygen species. Several kinds of DNA modifications are formed upon oxidative stress (8). The most prevalent modifications, quantitatively, are single-strand breaks and oxidized bases. Clustered DNA damage, when two or more modifications are closely positioned in opposite strands, is detectable after gamma irradiation and has recently been shown to be generated by normal oxidative metabolism (3,35). One unique aspect of such clustered lesions is that they can be converted into double-strand breaks (DSB) if a DNA glycosylase removes the two opposite bases and an apurinic/apyrimidinic (AP)-endonuclease cleaves the resulting abasic sites. Thus, although quantitatively minor, DSB are possible outcomes of oxidative DNA damage.