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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

Effect of thymidine salvage depletion in Raji TK1+ cells. (A) depicts the repair of bulk DNA, while graphs (B,C) show the % TP53 and hTERT tail signals over the repair time period after 5 Gy-irradiation. The rapid gene repair observed clearly demonstrates that culturing of TK1+ cells in thymidine free media has no effect on the level of preferential repair. Fifty cells were analyzed at each time-point for each experimental replicate. Each data point represents the mean ± SEM of three independent replicate experiments.
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Figure 5: Effect of thymidine salvage depletion in Raji TK1+ cells. (A) depicts the repair of bulk DNA, while graphs (B,C) show the % TP53 and hTERT tail signals over the repair time period after 5 Gy-irradiation. The rapid gene repair observed clearly demonstrates that culturing of TK1+ cells in thymidine free media has no effect on the level of preferential repair. Fifty cells were analyzed at each time-point for each experimental replicate. Each data point represents the mean ± SEM of three independent replicate experiments.

Mentions: The growth of TK1+ cells in thymidine free media (Raji TK+ Thy-) had no impact on the proficiency of single strand repair of bulk DNA in Raji TK1+ cells (Figure 5A) or gene region-specific repair of TP53 (Figure 5B) or hTERT (Figure 5C). Figure 5B shows TP53 repair in Raji TK1+ Thy+ and Raji TK1+ Thy- cells. Statistical analysis found no significant difference in the % of TP53 tail signals between TK1+ Thy+ and Raji TK1+ Thy- cells (p > 0.05). Statistical analysis likewise found no significant difference in the % hTERT tail signals at any given time points. These experiments show that culturing of TK1+ cells in thymidine free media has no effect on the level of preferential repair.


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)

Effect of thymidine salvage depletion in Raji TK1+ cells. (A) depicts the repair of bulk DNA, while graphs (B,C) show the % TP53 and hTERT tail signals over the repair time period after 5 Gy-irradiation. The rapid gene repair observed clearly demonstrates that culturing of TK1+ cells in thymidine free media has no effect on the level of preferential repair. Fifty cells were analyzed at each time-point for each experimental replicate. Each data point represents the mean ± SEM of three independent replicate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Effect of thymidine salvage depletion in Raji TK1+ cells. (A) depicts the repair of bulk DNA, while graphs (B,C) show the % TP53 and hTERT tail signals over the repair time period after 5 Gy-irradiation. The rapid gene repair observed clearly demonstrates that culturing of TK1+ cells in thymidine free media has no effect on the level of preferential repair. Fifty cells were analyzed at each time-point for each experimental replicate. Each data point represents the mean ± SEM of three independent replicate experiments.
Mentions: The growth of TK1+ cells in thymidine free media (Raji TK+ Thy-) had no impact on the proficiency of single strand repair of bulk DNA in Raji TK1+ cells (Figure 5A) or gene region-specific repair of TP53 (Figure 5B) or hTERT (Figure 5C). Figure 5B shows TP53 repair in Raji TK1+ Thy+ and Raji TK1+ Thy- cells. Statistical analysis found no significant difference in the % of TP53 tail signals between TK1+ Thy+ and Raji TK1+ Thy- cells (p > 0.05). Statistical analysis likewise found no significant difference in the % hTERT tail signals at any given time points. These experiments show that culturing of TK1+ cells in thymidine free media has no effect on the level of preferential repair.

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