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FISH comets show that the salvage enzyme TK1 contributes to gene-specific DNA repair.

McAllister KA, Yasseen AA, McKerr G, Downes CS, McKelvey-Martin VJ - Front Genet (2014)

Bottom Line: Thymidine kinase 1 (TK1) is a salvage enzyme that phosphorylates thymidine, imported from surrounding fluids, to create dTMP, which is further phosphorylated to the DNA precursor dTTP.We have examined preferential strand break repair of DNA domains in TK1(+) and TK1(-) clones of the Raji cell line, by the Comet-FISH technique, in bulk DNA and in the actively transcribed tumor suppressor (TP53) and human telomerase reverse transcriptase (hTERT) gene regions, over 1 h after 5Gy γ-irradiation.TK1 therefore does not exert its protective effects through dTTP pools, but through another unidentified mechanism, which affects sensitivity to and mutagenicity by DNA damaging agents.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, University of Ulster Coleraine, UK.

ABSTRACT
Thymidine kinase 1 (TK1) is a salvage enzyme that phosphorylates thymidine, imported from surrounding fluids, to create dTMP, which is further phosphorylated to the DNA precursor dTTP. TK1 deficiency has for a long time been known to cause increased cellular sensitivity to DNA damage. We have examined preferential strand break repair of DNA domains in TK1(+) and TK1(-) clones of the Raji cell line, by the Comet-FISH technique, in bulk DNA and in the actively transcribed tumor suppressor (TP53) and human telomerase reverse transcriptase (hTERT) gene regions, over 1 h after 5Gy γ-irradiation. Results showed that repair of the TP53 and hTERT gene regions was more efficient in TK1(+) compared to TK1(-) cells, a trend also reflected to a lesser degree in genomic DNA repair between the cell-lines. The targeted gene-specific repair in TK(+) cells occurred rapidly, mainly over the first 15 min repair-period. Therefore, TK1 is needed for preferential repair of actively transcribed regions, through a previously unsuspected mechanism. In principle, TK1 could exert its protective effects through supply of a supplementary dTTP pool for accurate repair of damaged genes; but Raji TK1(+) cells in thymidine free media still show preferential repair of transcribed regions. TK1 therefore does not exert its protective effects through dTTP pools, but through another unidentified mechanism, which affects sensitivity to and mutagenicity by DNA damaging agents.

No MeSH data available.


Related in: MedlinePlus

Confirmation of cellular phenotype in Raji cells. (A) Effect of culturing TK1- cells and (B) TK1+ cells in 5 μg/ml TFT compared to untreated control cells. Culture of Raji cells in TFT proved toxic to the TK1+ clones whereas TK1- cells were resistant. Only cells containing functional TK1 can incorporate TFT and uptake of the chemical results in cell death. Therefore Raji TK1- cells lack TK1 functional protein. (C) Tritium counts obtained from Raji TK1+ and TK1- clones during the TK assay expressed in counts per minute (CPM). The graph shows that the incorporation of 3H-thymidine is greatly reduced in Raji TK1- cells compared to TK1+. The ability of cells to incorporate 3H-thymidine into nucleic acids is an approximate measure of cellular thymidine kinase activity; therefore Raji TK1- cells lack functional TK1 protein. The results in each graph data-point represent the mean ± SEM of three independent experiments.
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Figure 1: Confirmation of cellular phenotype in Raji cells. (A) Effect of culturing TK1- cells and (B) TK1+ cells in 5 μg/ml TFT compared to untreated control cells. Culture of Raji cells in TFT proved toxic to the TK1+ clones whereas TK1- cells were resistant. Only cells containing functional TK1 can incorporate TFT and uptake of the chemical results in cell death. Therefore Raji TK1- cells lack TK1 functional protein. (C) Tritium counts obtained from Raji TK1+ and TK1- clones during the TK assay expressed in counts per minute (CPM). The graph shows that the incorporation of 3H-thymidine is greatly reduced in Raji TK1- cells compared to TK1+. The ability of cells to incorporate 3H-thymidine into nucleic acids is an approximate measure of cellular thymidine kinase activity; therefore Raji TK1- cells lack functional TK1 protein. The results in each graph data-point represent the mean ± SEM of three independent experiments.

Mentions: The Raji cells used are from lines that were originally created in the 1990s: we have therefore checked that they have retained their original phenotypes. The TK1+ line retain sensitivity to the toxic thymidine analog trifluorothymidine (Karran et al., 1990), while TK1- cells which cannot metabolize it remain resistant. Correspondingly, incorporation of tritiated thymidine is linear in TK1+ cells, negligible in TK1- (Figure 1). The TK1- nature of the Raji clones, first established several years ago (Hampar et al., 1971), has therefore been maintained.


FISH comets show that the salvage enzyme TK1 contributes to gene-specific DNA repair.

McAllister KA, Yasseen AA, McKerr G, Downes CS, McKelvey-Martin VJ - Front Genet (2014)

Confirmation of cellular phenotype in Raji cells. (A) Effect of culturing TK1- cells and (B) TK1+ cells in 5 μg/ml TFT compared to untreated control cells. Culture of Raji cells in TFT proved toxic to the TK1+ clones whereas TK1- cells were resistant. Only cells containing functional TK1 can incorporate TFT and uptake of the chemical results in cell death. Therefore Raji TK1- cells lack TK1 functional protein. (C) Tritium counts obtained from Raji TK1+ and TK1- clones during the TK assay expressed in counts per minute (CPM). The graph shows that the incorporation of 3H-thymidine is greatly reduced in Raji TK1- cells compared to TK1+. The ability of cells to incorporate 3H-thymidine into nucleic acids is an approximate measure of cellular thymidine kinase activity; therefore Raji TK1- cells lack functional TK1 protein. The results in each graph data-point represent the mean ± SEM of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126492&req=5

Figure 1: Confirmation of cellular phenotype in Raji cells. (A) Effect of culturing TK1- cells and (B) TK1+ cells in 5 μg/ml TFT compared to untreated control cells. Culture of Raji cells in TFT proved toxic to the TK1+ clones whereas TK1- cells were resistant. Only cells containing functional TK1 can incorporate TFT and uptake of the chemical results in cell death. Therefore Raji TK1- cells lack TK1 functional protein. (C) Tritium counts obtained from Raji TK1+ and TK1- clones during the TK assay expressed in counts per minute (CPM). The graph shows that the incorporation of 3H-thymidine is greatly reduced in Raji TK1- cells compared to TK1+. The ability of cells to incorporate 3H-thymidine into nucleic acids is an approximate measure of cellular thymidine kinase activity; therefore Raji TK1- cells lack functional TK1 protein. The results in each graph data-point represent the mean ± SEM of three independent experiments.
Mentions: The Raji cells used are from lines that were originally created in the 1990s: we have therefore checked that they have retained their original phenotypes. The TK1+ line retain sensitivity to the toxic thymidine analog trifluorothymidine (Karran et al., 1990), while TK1- cells which cannot metabolize it remain resistant. Correspondingly, incorporation of tritiated thymidine is linear in TK1+ cells, negligible in TK1- (Figure 1). The TK1- nature of the Raji clones, first established several years ago (Hampar et al., 1971), has therefore been maintained.

Bottom Line: Thymidine kinase 1 (TK1) is a salvage enzyme that phosphorylates thymidine, imported from surrounding fluids, to create dTMP, which is further phosphorylated to the DNA precursor dTTP.We have examined preferential strand break repair of DNA domains in TK1(+) and TK1(-) clones of the Raji cell line, by the Comet-FISH technique, in bulk DNA and in the actively transcribed tumor suppressor (TP53) and human telomerase reverse transcriptase (hTERT) gene regions, over 1 h after 5Gy γ-irradiation.TK1 therefore does not exert its protective effects through dTTP pools, but through another unidentified mechanism, which affects sensitivity to and mutagenicity by DNA damaging agents.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, University of Ulster Coleraine, UK.

ABSTRACT
Thymidine kinase 1 (TK1) is a salvage enzyme that phosphorylates thymidine, imported from surrounding fluids, to create dTMP, which is further phosphorylated to the DNA precursor dTTP. TK1 deficiency has for a long time been known to cause increased cellular sensitivity to DNA damage. We have examined preferential strand break repair of DNA domains in TK1(+) and TK1(-) clones of the Raji cell line, by the Comet-FISH technique, in bulk DNA and in the actively transcribed tumor suppressor (TP53) and human telomerase reverse transcriptase (hTERT) gene regions, over 1 h after 5Gy γ-irradiation. Results showed that repair of the TP53 and hTERT gene regions was more efficient in TK1(+) compared to TK1(-) cells, a trend also reflected to a lesser degree in genomic DNA repair between the cell-lines. The targeted gene-specific repair in TK(+) cells occurred rapidly, mainly over the first 15 min repair-period. Therefore, TK1 is needed for preferential repair of actively transcribed regions, through a previously unsuspected mechanism. In principle, TK1 could exert its protective effects through supply of a supplementary dTTP pool for accurate repair of damaged genes; but Raji TK1(+) cells in thymidine free media still show preferential repair of transcribed regions. TK1 therefore does not exert its protective effects through dTTP pools, but through another unidentified mechanism, which affects sensitivity to and mutagenicity by DNA damaging agents.

No MeSH data available.


Related in: MedlinePlus