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Host-cell reactivation

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The term host cell reactivation or HCR was first used to describe the survival of UV-irradiated bacteriophages, that were transfected to UV-pretreated cells.[1] This phenomenon was first thought to be the result of homologous recombination between both bacteria and phage, but later recognized as enzymatic repair.[2][3][4] Modifications of the assay were later developed, using transient expression plasmid DNA vectors on immortalized fibroblasts,[5] and lately on human lymphocytes.[6]

The HCR assay known also as plasmid reactivation assay, indirectly monitors cellular transcriptional repair system, that is activated by the transcriptional-inhibited damage inflicted by UV-Radiation into the plasmid. Given that UV-induced DNA damage is used as mutagen, the cell uses nucleotide excision repair NER pathway, that is activated by distortion in the DNA helix.[1]

The Host-Cell Reactivation Assay or HCR is a technique used to measure the DNA repair capacity of cell of a particular DNA alteration. In the HCR assay the ability of an intact cell to repair exogenous DNA is measured[7] The host cell is transfected with a damaged plasmid containing a reporter gene, usually luciferase, which has been deactivated due to the damage. The ability of the cell to repair the damage in the plasmid, after it has been introduced to the cell, allows the reporter gene to be reactivated. Earlier versions of this assay were based on the chloramphenicol acetyltransferase (CAT) gene,[5] but the version of the assay using luciferase as reporter gene is as much as 100-fold more sensitive.[1]

See also

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References

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  1. ^ a b c Johnson JM, Latimer JJ (2005). "Analysis of DNA repair using transfection-based host cell reactivation". Molecular Toxicology Protocols. Methods Mol. Biol. Vol. 291. pp. 321–35. doi:10.1385/1-59259-840-4:321. ISBN 1-59259-840-4. PMC 4860737. PMID 15502233.
  2. ^ Rupert CS, Harm W (1966). "Reactivation After Photobiological Damage". Adv. Radiat. Biol. Advances in Radiation Biology. 2: 1–81. doi:10.1016/B978-1-4832-3121-1.50006-2. ISBN 9781483231211. ISSN 0065-3292.
  3. ^ Smith KC, Martignoni KD (December 1976). "Protection of Escherichia coli cells against the lethal effects of ultraviolet and x irradiation by prior x irradiation: a genetic and physiological study". Photochem. Photobiol. 24 (6): 515–23. doi:10.1111/j.1751-1097.1976.tb06868.x. PMID 798210. S2CID 37612164.
  4. ^ Jones DT, Robb FT, Woods DR (December 1980). "Effect of oxygen on Bacteroides fragilis survival after far-ultraviolet irradiation". J. Bacteriol. 144 (3): 1179–81. doi:10.1128/JB.144.3.1179-1181.1980. PMC 294787. PMID 7440505.
  5. ^ a b Protić-Sabljić M, Kraemer KH (October 1985). "One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells". Proc. Natl. Acad. Sci. U.S.A. 82 (19): 6622–6. Bibcode:1985PNAS...82.6622P. doi:10.1073/pnas.82.19.6622. PMC 391262. PMID 2995975.
  6. ^ Athas WF, Hedayati MA, Matanoski GM, Farmer ER, Grossman L (November 1991). "Development and field-test validation of an assay for DNA repair in circulating human lymphocytes". Cancer Res. 51 (21): 5786–93. PMID 1933849.
  7. ^ McCready, S. (2014). An Immunoassay for Measuring Repair of DNA. In P. Keohavong & S. G. Grant (Eds.), Molecular Toxicology Protocols (Vol. 1105, pp. 551–564). Humana Press. doi:10.1007/978-1-62703-739-6_38
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