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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

TP53 and hTERT gene-repair in TK1- and TK1+ cells and comparison with the overall genome. (A) shows the % TP53 tail signals and (B) the % hTERT tail signals over the repair time period after 5 Gy-irradiation. (C,D) compares DNA repair between the overall genome and specific gene-regions. These findings demonstrate that preferential repair is occurring in the TP53 and hTERT domains of Raji TK1+ but not in TK1- cells. In each graph, 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 4: TP53 and hTERT gene-repair in TK1- and TK1+ cells and comparison with the overall genome. (A) shows the % TP53 tail signals and (B) the % hTERT tail signals over the repair time period after 5 Gy-irradiation. (C,D) compares DNA repair between the overall genome and specific gene-regions. These findings demonstrate that preferential repair is occurring in the TP53 and hTERT domains of Raji TK1+ but not in TK1- cells. In each graph, 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 level of strand break repair in the specific gene-regions was compared between TK1+ and TK1- cells using the parameter “% TP53/hTERT signals in the comet tail” (Figures 4A,B). There was found to be a significant reduction (p < 0.0001) in the % of TP53 and hTERT tail signals at each time-point in Raji TK1+ cells compared to TK1- (Figures 4A,B). The most prominent occurred at 15 min, with a difference between the Raji clones of 35.18 ± 3.56 for TP53 tail spots and 29.82 ± 4.34 for hTERT tail spots. The delayed reduction of % FISH signals in the comet tail for each gene over the hourly incubation period demonstrates that TK1- are severely compromised in gene region repair kinetics compared to TK1+.


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)

TP53 and hTERT gene-repair in TK1- and TK1+ cells and comparison with the overall genome. (A) shows the % TP53 tail signals and (B) the % hTERT tail signals over the repair time period after 5 Gy-irradiation. (C,D) compares DNA repair between the overall genome and specific gene-regions. These findings demonstrate that preferential repair is occurring in the TP53 and hTERT domains of Raji TK1+ but not in TK1- cells. In each graph, 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 4: TP53 and hTERT gene-repair in TK1- and TK1+ cells and comparison with the overall genome. (A) shows the % TP53 tail signals and (B) the % hTERT tail signals over the repair time period after 5 Gy-irradiation. (C,D) compares DNA repair between the overall genome and specific gene-regions. These findings demonstrate that preferential repair is occurring in the TP53 and hTERT domains of Raji TK1+ but not in TK1- cells. In each graph, 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 level of strand break repair in the specific gene-regions was compared between TK1+ and TK1- cells using the parameter “% TP53/hTERT signals in the comet tail” (Figures 4A,B). There was found to be a significant reduction (p < 0.0001) in the % of TP53 and hTERT tail signals at each time-point in Raji TK1+ cells compared to TK1- (Figures 4A,B). The most prominent occurred at 15 min, with a difference between the Raji clones of 35.18 ± 3.56 for TP53 tail spots and 29.82 ± 4.34 for hTERT tail spots. The delayed reduction of % FISH signals in the comet tail for each gene over the hourly incubation period demonstrates that TK1- are severely compromised in gene region repair kinetics compared to TK1+.

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