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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

Digital imaging of comet-FISH cells. The position of TP53 and hTERT hybridization spots in the comet head or tail specifies whether it lies in, or close to, a region of intact or damaged DNA. Observations for imaging were made at a final magnification of ×600 (Nikon ×60 Fluor lens). (A) An untreated Raji cells displays minimal DNA damage as evidenced by the intact comet head and minimal comet tail. Two TP53 (red arrows) and two hTERT (green arrows) hybridization spots are visible in the intact comet head. (B,C) Immediately following 5Gy γ-irradaition a large comet tail is visualized, indicating a large amount of overall DNA damage, with red TP53 and green hTERT hybridization spots in the comet tail indicating that radiation-induced strand breaks have occurred within or close to the vivinity of both genes of (B) TK+ and (C) TK- cells. (D). At 15 min of repair the TK+ cells have repaired most gene specific damage as depicted here for the TP53 gene region, as evidenced here by the FISH spots being located in the head. (E) Raji TK- cells at 60 min post-irradiation typically feature TP53 and/or HTERT FISH spots located in the comet-head as gene repair has begun to recover. However, FISH tail spots still remain in higher numbers than TK+, this picture showing an example of damage remaining in the TP53 locci of TK- cells at 60 min.
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Figure 3: Digital imaging of comet-FISH cells. The position of TP53 and hTERT hybridization spots in the comet head or tail specifies whether it lies in, or close to, a region of intact or damaged DNA. Observations for imaging were made at a final magnification of ×600 (Nikon ×60 Fluor lens). (A) An untreated Raji cells displays minimal DNA damage as evidenced by the intact comet head and minimal comet tail. Two TP53 (red arrows) and two hTERT (green arrows) hybridization spots are visible in the intact comet head. (B,C) Immediately following 5Gy γ-irradaition a large comet tail is visualized, indicating a large amount of overall DNA damage, with red TP53 and green hTERT hybridization spots in the comet tail indicating that radiation-induced strand breaks have occurred within or close to the vivinity of both genes of (B) TK+ and (C) TK- cells. (D). At 15 min of repair the TK+ cells have repaired most gene specific damage as depicted here for the TP53 gene region, as evidenced here by the FISH spots being located in the head. (E) Raji TK- cells at 60 min post-irradiation typically feature TP53 and/or HTERT FISH spots located in the comet-head as gene repair has begun to recover. However, FISH tail spots still remain in higher numbers than TK+, this picture showing an example of damage remaining in the TP53 locci of TK- cells at 60 min.

Mentions: Comet-FISH experiments were evaluated by quantifying the number of TP53 and hTERT hybridization spots located in the comet head or tail of TK1+ and TK1- at each repair incubation time as shown both in representative images in Figure 3 and the data in Table 1. FISH comet tail spots were almost entirely absent in both un-irradiated controls; and on account of the aneuploidy and the post-replicative elements in the population, the average spot number in the comet head was above two (Figure 3A). Post-irradiation, both cell-lines showed an increase in TP53 and hTERT probe signal in the comet tail, which is indicative of damage to both gene regions (Figures 3B,C). A notable finding in Raji TK1+ cells (Table 1) was a rapid rate of gene-specific repair that featured significant decreases in the number of TP53 and hTERT tail spots at 15 min compared to TK1- (p < 0.0001) and also at 30–60 min (p < 0.0001). Repair continued at a slower rate for the remainder of the repair period in TK+ cells so that by 60 min there was no signal left of either probe in the tail (Figure 3D). In comparison, gene-specific repair was found to be stalled in TK1- cells and the comet-tail FISH signals for each gene probe began to diminish only after 15 min (Figure 3E).


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)

Digital imaging of comet-FISH cells. The position of TP53 and hTERT hybridization spots in the comet head or tail specifies whether it lies in, or close to, a region of intact or damaged DNA. Observations for imaging were made at a final magnification of ×600 (Nikon ×60 Fluor lens). (A) An untreated Raji cells displays minimal DNA damage as evidenced by the intact comet head and minimal comet tail. Two TP53 (red arrows) and two hTERT (green arrows) hybridization spots are visible in the intact comet head. (B,C) Immediately following 5Gy γ-irradaition a large comet tail is visualized, indicating a large amount of overall DNA damage, with red TP53 and green hTERT hybridization spots in the comet tail indicating that radiation-induced strand breaks have occurred within or close to the vivinity of both genes of (B) TK+ and (C) TK- cells. (D). At 15 min of repair the TK+ cells have repaired most gene specific damage as depicted here for the TP53 gene region, as evidenced here by the FISH spots being located in the head. (E) Raji TK- cells at 60 min post-irradiation typically feature TP53 and/or HTERT FISH spots located in the comet-head as gene repair has begun to recover. However, FISH tail spots still remain in higher numbers than TK+, this picture showing an example of damage remaining in the TP53 locci of TK- cells at 60 min.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Digital imaging of comet-FISH cells. The position of TP53 and hTERT hybridization spots in the comet head or tail specifies whether it lies in, or close to, a region of intact or damaged DNA. Observations for imaging were made at a final magnification of ×600 (Nikon ×60 Fluor lens). (A) An untreated Raji cells displays minimal DNA damage as evidenced by the intact comet head and minimal comet tail. Two TP53 (red arrows) and two hTERT (green arrows) hybridization spots are visible in the intact comet head. (B,C) Immediately following 5Gy γ-irradaition a large comet tail is visualized, indicating a large amount of overall DNA damage, with red TP53 and green hTERT hybridization spots in the comet tail indicating that radiation-induced strand breaks have occurred within or close to the vivinity of both genes of (B) TK+ and (C) TK- cells. (D). At 15 min of repair the TK+ cells have repaired most gene specific damage as depicted here for the TP53 gene region, as evidenced here by the FISH spots being located in the head. (E) Raji TK- cells at 60 min post-irradiation typically feature TP53 and/or HTERT FISH spots located in the comet-head as gene repair has begun to recover. However, FISH tail spots still remain in higher numbers than TK+, this picture showing an example of damage remaining in the TP53 locci of TK- cells at 60 min.
Mentions: Comet-FISH experiments were evaluated by quantifying the number of TP53 and hTERT hybridization spots located in the comet head or tail of TK1+ and TK1- at each repair incubation time as shown both in representative images in Figure 3 and the data in Table 1. FISH comet tail spots were almost entirely absent in both un-irradiated controls; and on account of the aneuploidy and the post-replicative elements in the population, the average spot number in the comet head was above two (Figure 3A). Post-irradiation, both cell-lines showed an increase in TP53 and hTERT probe signal in the comet tail, which is indicative of damage to both gene regions (Figures 3B,C). A notable finding in Raji TK1+ cells (Table 1) was a rapid rate of gene-specific repair that featured significant decreases in the number of TP53 and hTERT tail spots at 15 min compared to TK1- (p < 0.0001) and also at 30–60 min (p < 0.0001). Repair continued at a slower rate for the remainder of the repair period in TK+ cells so that by 60 min there was no signal left of either probe in the tail (Figure 3D). In comparison, gene-specific repair was found to be stalled in TK1- cells and the comet-tail FISH signals for each gene probe began to diminish only after 15 min (Figure 3E).

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