Analysis of DNA Repair Using Transfection-Based Host Cell Reactivation

Johnson, Jennifer M.; Latimer, Jean Johanna

doi:10.1385/1-59259-840-4:321

Cited by 15 publications

(15 citation statements)

References 28 publications

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“…The foundation of the assay lies in the ability of transcription blocking DNA damage to impede expression of a transiently transfected reporter gene; repair restores transcription of the reporter gene, which may encode enzymes such as chloramphenicol acetyltransferase (CAT) and luciferase, or a fluorescent protein (105). A major strength of HCR assays, stemming from the in vitro generation of damaged reporter plasmid DNA, is the ability to measure the in vivo repair of specific DNA lesions in intact cells.…”

Section: Evidence Of Inter-individual Drc Differences From Direct mentioning

confidence: 99%

Inter-individual variation in DNA repair capacity: A need for multi-pathway functional assays to promote translational DNA repair research

2014

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Why does a constant barrage of DNA damage lead to disease in some individuals, while others remain healthy? This article surveys current work addressing the implications of inter-individual variation in DNA repair capacity for human health, and discusses the status of DNA repair assays as potential clinical tools for personalized prevention or treatment of disease. In particular, we highlight research showing that there are significant inter-individual variations in DNA Repair Capacity (DRC), and that measuring these differences provides important biological insight regarding disease susceptibility and cancer treatment efficacy. We emphasize work showing that it is important to measure repair capacity in multiple pathways, and that functional assays are required to fill a gap left by genome wide association studies, global gene expression and proteomics. Finally, we discuss research that will be needed to overcome barriers that currently limit the use of DNA repair assays in the clinic.

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Section: Evidence Of Inter-individual Drc Differences From Direct mentioning

confidence: 99%

Inter-individual variation in DNA repair capacity: A need for multi-pathway functional assays to promote translational DNA repair research

2014

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“…Some of these methods are based on the detection of chromosome breakage (reviewed in [70]) including the Comet assay (see [71][72][73] for review). Other approaches measure the activity of nDNA repair enzymes [74,75] or the amount of damage localized to a plasmid (i.e., to exogenous DNA) that a cell can "reactivate" or repair (socalled host cell reactivation; reviewed in [76]). The common limitation of these methods is that they cannot be used to investigate nDNA repair processes ongoing in the brain in a celltype-specific manner.…”

Section: Nuclear Dna Repairmentioning

confidence: 99%

Accumulation of nuclear DNA damage or neuron loss: Molecular basis for a new approach to understanding selective neuronal vulnerability in neurodegenerative diseases

et al. 2008

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According to a long-standing hypothesis, aging is mainly caused by accumulation of nuclear (n) DNA damage in differentiated cells such as neurons due to insufficient nDNA repair during lifetime. In line with this hypothesis it was until recently widely accepted that neuron loss is a general consequence of normal aging, explaining some degree of decline in brain function during aging. However, with the advent of more accurate procedures for counting neurons, it is currently widely accepted that there is widespread preservation of neuron numbers in the aging brain, and the changes that do occur are relatively specific to certain brain regions and types of neurons. Whether accumulation of nDNA damage and decline in nDNA repair is a general phenomenon in the aging brain or also shows cell-type specificity is, however, not known. It has not been possible to address this issue with the biochemical and molecular-biological methods available to study nDNA damage and nDNA repair. Rather, it was the introduction of autoradiographic methods to study quantitatively the relative amounts of nDNA damage (measured as nDNA single-strand breaks) and nDNA repair (measured as unscheduled DNA synthesis) on tissue sections that made it possible to address this question in a cell-type-specific manner under physiological conditions. The results of these studies revealed a formerly unknown inverse relationship between age-related accumulation of nDNA damage and age-related impairment in nDNA repair on the one hand, and the age-related, selective, loss of neurons on the other hand. This inverse relation may not only reflect a fundamental process of aging in the central nervous system but also provide the molecular basis for a new approach to understand the selective neuronal vulnerability in neurodegenerative diseases, particularly Alzheimer's disease.

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“…We asked next whether EBV DNase has the potential to affect DNA repair. We carried out HCR assay to investigate the effect of DNase on DNA repair (63). The HCR assay can easily measure the capacity for DNA repair quantitatively for various types of DNA damage (64,65).…”

Section: Resultsmentioning

confidence: 99%

“…In the HCR assay, either the empty vector or plasmid expressing DNase, control plasmid pRL-CMV, and either a damaged or untreated luciferase reporter plasmid (pCMV-Luc) were cotransfected into TW01 epithelial cells. Because DNA-damaging chemicals and UV are the most common causes of damage used experimentally (63), first, we assayed the effect of DNase on repair of chemical-induced damage. Reporter plasmids were treated with N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG) or cisplatin for 3 h, purified on columns and cotransfected with DNase or vector plasmid and the internal control pRL-CMV.…”

Section: Resultsmentioning

confidence: 99%

Epstein–Barr Virus DNase (BGLF5) induces genomic instability in human epithelial cells

Liu

Chang

et al. 2009

Nucleic Acids Research

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Epstein–Barr Virus (EBV) DNase (BGLF5) is an alkaline nuclease and has been suggested to be important in the viral life cycle. However, its effect on host cells remains unknown. Serological and histopathological studies implied that EBV DNase seems to be correlated with carcinogenesis. Therefore, we investigate the effect of EBV DNase on epithelial cells. Here, we report that expression of EBV DNase induces increased formation of micronucleus, an indicator of genomic instability, in human epithelial cells. We also demonstrate, using γH2AX formation and comet assay, that EBV DNase induces DNA damage. Furthermore, using host cell reactivation assay, we find that EBV DNase expression repressed damaged DNA repair in various epithelial cells. Western blot and quantitative PCR analyses reveal that expression of repair-related genes is reduced significantly in cells expressing EBV DNase. Host shut-off mutants eliminate shut-off expression of repair genes and repress damaged DNA repair, suggesting that shut-off function of BGLF5 contributes to repression of DNA repair. In addition, EBV DNase caused chromosomal aberrations and increased the microsatellite instability (MSI) and frequency of genetic mutation in human epithelial cells. Together, we propose that EBV DNase induces genomic instability in epithelial cells, which may be through induction of DNA damage and also repression of DNA repair, subsequently increases MSI and genetic mutations, and may contribute consequently to the carcinogenesis of human epithelial cells.

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Analysis of DNA Repair Using Transfection-Based Host Cell Reactivation

Cited by 15 publications

References 28 publications

Inter-individual variation in DNA repair capacity: A need for multi-pathway functional assays to promote translational DNA repair research

Inter-individual variation in DNA repair capacity: A need for multi-pathway functional assays to promote translational DNA repair research

Accumulation of nuclear DNA damage or neuron loss: Molecular basis for a new approach to understanding selective neuronal vulnerability in neurodegenerative diseases

Epstein–Barr Virus DNase (BGLF5) induces genomic instability in human epithelial cells

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