Two-Wavelength Fluorescence Assay for DNA Repair

Roguev, Assen; Russev, George

doi:10.1006/abio.2000.4865

Cited by 22 publications

(16 citation statements)

References 30 publications

(29 reference statements)

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“…The primers were 5′‐CCATTGACGTCAATGGGA and 5′‐GGGTGCTCAGGTAGTGGTT and the thermal cycle parameters were: denaturation at 95°C for 60 s; annealing at 59°C for 45 s; extension at 72°C for 60 s; 18 PCR cycles. The ratio between the PCR products obtained from the Trioxsalen treated samples and the control were used to calculate the average number of lesions/kbp using the Poisson formulae [Roguev and Russev, 2000].…”

Section: Methodsmentioning

confidence: 99%

Repair of DNA interstrand crosslinks may take place at the nuclear matrix

Atanassov

Gospodinov

Stoimenov

et al. 2005

J of Cellular Biochemistry

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Host cell reactivation assay using Trioxsalen-crosslinked plasmid pEGFP-N1 showed that human cells were able to repair Trioxsalen interstrand crosslinks (ICL). To study the mechanism of this repair pathway, cells were transfected with the plasmids pEGFP-1, which did not contain the promoter of the egfp gene, and with pEGFP-G-, which did not contain the egfp gene. Neither of these plasmids alone was able to express the green fluorescent protein. After cotransfection with the two plasmids, 1%-2% of the cells developed fluorescent signal, which showed that recombination events had taken place in these cells to create DNA constructs containing the promoter and the gene properly aligned. When one or both of the plasmids were crosslinked with Trioxsalen, the recombination rate increased several fold. To identify the nuclear compartment where recombination takes place, cells were transfected with crosslinked pEGFP-N1 and the amount of plasmid DNA in the different nuclear fractions was determined. The results showed that Trioxsalen crosslinking increased the percentage of matrix attached plasmid DNA in a dose-dependent way. Immunoblotting experiments showed that after transfection with Trioxsalen crosslinked plasmids the homologous recombination protein Rad51 also associated with the nuclear matrix fraction. These studies provide a model system for investigating the precise molecular mechanisms that appear to couple repair of DNA ICL with nuclear matrix attachment.

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Section: Methodsmentioning

confidence: 99%

Repair of DNA interstrand crosslinks may take place at the nuclear matrix

Atanassov

Gospodinov

Stoimenov

et al. 2005

J of Cellular Biochemistry

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“…Mutagenesis of the wild type gene yielded improved variants as well as colour variants such as Cyan and Yellow Fluorescent Proteins [24]. Unfortunately, the emission maxima of YFP and GFP excited by 488 nm laser light are in close proximity [3]. Thereby, the fluorescence spectra of both proteins overlap in wide areas, so that they cannot be clearly distinguished using FACS-technology.…”

Section: Resultsmentioning

confidence: 99%

A modified fluorimetric host cell reactivation assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts

et al. 2010

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BackgroundThe Host Cell Reactivation Assay (HCRA) is widely used to identify circumstances and substances affecting the repair capacity of cells, however, it is restricted by the transfection procedure used and the sensitivity of the detection method. Primary skin cells are particularly difficult to transfect, and therefore sensitive methods are needed to detect any variations due to the cell-type or inter-individual differences or changes induced by diverse substances.A sensitive and repeatable method to detect the repair capacity of skin cells would be useful in two different aspects: On the one hand, to identify substances influencing the repair capacity in a positive manner (these substances could be promising ingredients for cosmetic products) and on the other hand, to exclude the negative effects of substances on the repair capacity (this could serve as one step further towards replacing or at least reducing animal testing).ResultsIn this paper, we present a rapid and sensitive assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts based on two wave-length Green Fluorescent Protein (GFP) and DsRed reporter technology in order to test different substances and their potential to influence the DNA repair capacity. For the detection of plasmid restoration, we used FACS technology, which, in comparison to luminometer technology, is highly sensitive and allows single cell based analysis.The usefulness of this assay and studying the repair capacity is demonstrated by the evidence that DNA repair is repressed by Cyclosporin A in fibroblasts.ConclusionsThe methodology described in this paper determines the DNA repair capacity in different types of human skin cells. The described transfection protocol is suitable for the transfection of melanocytes, keratinocytes and fibroblasts, reaching efficacies suitable for the detection of the restored plasmids by FACS technology. Therefore the repair capacity of different cell types can be compared with each other. The described assay is also highly flexible, and the activity of other repair mechanisms can be determined using modifications of this method.

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“…2), which showed that G1 phase cells repair trioxsalen ICLs very poorly. Using the Poisson formula which links the percentage of plasmids without ICLs to the average number of ICLs [21], it was calculated that they repaired less than 0.1 ICL/ kbp, while the S phase HeLa cells were able to repair about 0.2-0.3 ICL/kbp over the 12 h period.…”

Section: Icl Repairmentioning

confidence: 99%

Enhanced repair of DNA interstrand crosslinks in S phase

Mladenova¹,

Russev²

2006

FEBS Letters

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Hela cells synchronized in G1 and S phases of the cell cycle were transfected with pEGFP crosslinked with trioxsalen. Twelve hours later the number of fluorescent cells was determined by fluorescent microscopy. Cells in S phase have repaired 0.2-0.3 ICL/kb over the 12 h period, while cells in G1 phase repaired interstrand crosslinks much more poorly. The crosslinked plasmids were efficiently recruited to the nuclear matrix both in G1 phase and S-phase, which showed that the poor repair of G1 cells was a result of a lack of DNA replication rather than of a lack of matrix attachment.

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Two-Wavelength Fluorescence Assay for DNA Repair

Cited by 22 publications

References 30 publications

Repair of DNA interstrand crosslinks may take place at the nuclear matrix

Repair of DNA interstrand crosslinks may take place at the nuclear matrix

A modified fluorimetric host cell reactivation assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts

Enhanced repair of DNA interstrand crosslinks in S phase

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