Fluorometric Analysis of DNA Unwinding (FADU) as a Method for Detecting Repair-induced DNA Strand Breaks in UV-irradiated Mammalian Cells¶

Baumstark‐Khan, Christa; Hentschel, Uwe; Nikandrova, Yelizabeta; Krug, Jens; Horneck, G.

doi:10.1562/0031-8655(2000)072<0477:faoduf>2.0.co;2

Cited by 46 publications

(26 citation statements)

References 43 publications

(51 reference statements)

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“…Before the ligation step, alkali treatment of the nicked DNA produces single-stranded regions at the sites of the initial damage. Thus, the extent of DNA double-strandedness provides a measure of the ongoing but still incomplete repair (25,26). This can be quantified by fluorometric analysis of DNA unwinding (FADU) (27) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Mammalian ribonucleotide reductase subunit p53R2 is required for mitochondrial DNA replication and DNA repair in quiescent cells

Pontarin

Ferraro

Bee

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

114

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In postmitotic mammalian cells, protein p53R2 substitutes for protein R2 as a subunit of ribonucleotide reductase. In human patients with mutations in RRM2B, the gene for p53R2, mitochondrial (mt) DNA synthesis is defective, and skeletal muscle presents severe mtDNA depletion. Skin fibroblasts isolated from a patient with a lethal homozygous missense mutation of p53R2 grow normally in culture with an unchanged complement of mtDNA. During active growth, the four dNTP pools do not differ in size from normal controls, whereas during quiescence, the dCTP and dGTP pools decrease to 50% of the control. We investigate the ability of these mutated fibroblasts to synthesize mtDNA and repair DNA after exposure to UV irradiation. Ethidium bromide depleted both mutant and normal cells of mtDNA. On withdrawal of the drug, mtDNA recovered equally well in cycling mutant and control cells, whereas during quiescence, the mutant fibroblasts remained deficient. Addition of deoxynucleosides to the medium increased intracellular dNTP pools and normalized mtDNA synthesis. Quiescent mutant fibroblasts were also deficient in the repair of UV-induced DNA damage, as indicated by delayed recovery of dsDNA analyzed by fluorometric analysis of DNA unwinding and the more extensive and prolonged phosphorylation of histone H2AX after irradiation. Supplementation by deoxynucleosides improved DNA repair. Our results show that in nontransformed cells only during quiescence, protein p53R2 is required for maintenance of mtDNA and for optimal DNA repair after UV damage.DNA precursors | dNTP de novo synthesis | cell cycle | mitochondrial disease D NA replication and repair require the continued synthesis of the four dNTPs. They are synthesized by evolutionary-related ribonucleotide reductases operating with slightly different mechanisms in aerobic and anaerobic organisms (1). Each ribonucleotide reductase provides the required amounts of all four dNTPs. A similar allosteric mechanism, maintained throughout evolution, regulates both the enzyme's activity and its substrate specificity. Cells contain small dNTP pools of similar sizes, approximately 10-fold larger during DNA replication than during quiescence. Regulation of pool sizes by ribonucleotide reductases is of great importance for correct DNA replication, and changes in the actual sizes or in their balance lead to increased mutation rates (2). For mammalian cells, the induction of mutations by pool imbalances has been described in detail, along with possible mechanisms (3). In yeast, a recent elegant study (4) linked specific amino acid substitutions in the catalytic subunit of ribonucleotide reductase to defined pool imbalances, which result in increased mutation rates.In mammalian cells, the canonical ribonucleotide reductase is a complex between two proteins: the large catalytic protein R1 that contains the allosteric sites and the smaller protein R2 that contributes a stable tyrosyl free radical during the reaction (1). Both proteins are transcriptionally activated during early S-phase (5) a...

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Section: Resultsmentioning

confidence: 99%

Mammalian ribonucleotide reductase subunit p53R2 is required for mitochondrial DNA replication and DNA repair in quiescent cells

Pontarin

Ferraro

Bee

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

114

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“…DNA strand breaks are observed extensively in cells under UV-B irradiation [87, 88]. UV-B-induced ROS [89] as well as DNA lesions (CPDs and 6-4PPs) may cause primary as well as secondary breaks, respectively.…”

Section: Uv-induced Dna Double Strand Breaksmentioning

confidence: 99%

Molecular Mechanisms of Ultraviolet Radiation‐Induced DNA Damage and Repair

Rastogi

Richa

Kumar

et al. 2010

Journal of Nucleic Acids

940

735

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DNA is one of the prime molecules, and its stability is of utmost importance for proper functioning and existence of all living systems. Genotoxic chemicals and radiations exert adverse effects on genome stability. Ultraviolet radiation (UVR) (mainly UV-B: 280–315 nm) is one of the powerful agents that can alter the normal state of life by inducing a variety of mutagenic and cytotoxic DNA lesions such as cyclobutane-pyrimidine dimers (CPDs), 6-4 photoproducts (6-4PPs), and their Dewar valence isomers as well as DNA strand breaks by interfering the genome integrity. To counteract these lesions, organisms have developed a number of highly conserved repair mechanisms such as photoreactivation, base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). Additionally, double-strand break repair (by homologous recombination and nonhomologous end joining), SOS response, cell-cycle checkpoints, and programmed cell death (apoptosis) are also operative in various organisms with the expense of specific gene products. This review deals with UV-induced alterations in DNA and its maintenance by various repair mechanisms.

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“…For ssDNA, the resulting CD spectrum at high dye–DNA ratio showed a 2/1 pattern with increasing wavelength, changing sign at 506 nm (Kubista et al ., ; Baumstark‐Khan et al. , ).…”

Section: Introductionmentioning

confidence: 99%

Cell‐death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques

Atale

Gupta

Yadav³

et al. 2014

Journal of Microscopy

263

171

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Summary Apoptosis, a genetically programmed cellular event leads to biochemical and morphological changes in cells. Alterations in DNA caused by several factors affect nucleus and ultimately the entire cell leading to compromised function of the organ and organism. DNA, a master regulator of the cellular events, is an important biomolecule with regards to cell growth, cell death, cell migration and cell differentiation. It is therefore imperative to develop the staining techniques that may lead to visualize the changes in nucleus where DNA is housed, to comprehend the cellular pathophysiology. Over the years a number of nuclear staining techniques such as propidium iodide, Hoechst‐33342, 4’, 6‐diamidino‐2‐phenylindole (DAPI), Acridine orange–Ethidium bromide staining, among others have been developed to assess the changes in DNA. Some nonnuclear staining techniques such as Annexin‐V staining, which although does not stain DNA, but helps to identify the events that result from DNA alteration and leads to initiation of apoptotic cell death. In this review, we have briefly discussed some of the most commonly used fluorescent and nonfluorescent staining techniques that identify apoptotic changes in cell, DNA and the nucleus. These techniques help in differentiating several cellular and nuclear phenotypes that result from DNA damage and have been identified as specific to necrosis or early and late apoptosis as well as scores of other nuclear deformities occurring inside the cells.

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Fluorometric Analysis of DNA Unwinding (FADU) as a Method for Detecting Repair-induced DNA Strand Breaks in UV-irradiated Mammalian Cells¶

Cited by 46 publications

References 43 publications

Mammalian ribonucleotide reductase subunit p53R2 is required for mitochondrial DNA replication and DNA repair in quiescent cells

Mammalian ribonucleotide reductase subunit p53R2 is required for mitochondrial DNA replication and DNA repair in quiescent cells

Molecular Mechanisms of Ultraviolet Radiation‐Induced DNA Damage and Repair

Cell‐death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques

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