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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

Investigation of TP53 and hTERT status in Raji cells using RTPCR. (A) RNA extraction. Lane 1 Werner Syndrome (WS), lane 2 TK1+, lane 3 TK1-, lane 4 GM38, M is a 1 kb ladder. (B) β-actin gene expression internal control check as detected by a 435 amplicon in all cell-lines. Lane 1 WS, lane 2 TK1+, lane 3 TK1-, lane 4 GM38 and lane 5 PCR water control. M is a 100 bp ladder. (C) HTERT gene expression detected by a 200 bp amplicon. HTERT gene expression was found in the WS cell-line, TK1+ and TK1- cells, but not GM38. Lane 1 WS, lane 2 TK1+, lane 3 Raji TK1-, lane 4 GM38 lane 5 –RTPCR control (-RT) WS, lane 6 –RT TK1+, lane 7 –RT TK1-, lane 8 –RT GM38, lane 9 PCR water control. M is a 100 bp ladder (D) TP53 gene expression as detected by a 1220 bp band present in all cells. Lane 1 GM38, lane 2 GM38 –RT, lane 3 Raji TK1+, lane 4 Raji TK1+ –RT, lane 5 Raji TK1-, lane 6 Raji TK1- –RT, lane 7 PCR water control. M is a 100 bp ladder.
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Figure 2: Investigation of TP53 and hTERT status in Raji cells using RTPCR. (A) RNA extraction. Lane 1 Werner Syndrome (WS), lane 2 TK1+, lane 3 TK1-, lane 4 GM38, M is a 1 kb ladder. (B) β-actin gene expression internal control check as detected by a 435 amplicon in all cell-lines. Lane 1 WS, lane 2 TK1+, lane 3 TK1-, lane 4 GM38 and lane 5 PCR water control. M is a 100 bp ladder. (C) HTERT gene expression detected by a 200 bp amplicon. HTERT gene expression was found in the WS cell-line, TK1+ and TK1- cells, but not GM38. Lane 1 WS, lane 2 TK1+, lane 3 Raji TK1-, lane 4 GM38 lane 5 –RTPCR control (-RT) WS, lane 6 –RT TK1+, lane 7 –RT TK1-, lane 8 –RT GM38, lane 9 PCR water control. M is a 100 bp ladder (D) TP53 gene expression as detected by a 1220 bp band present in all cells. Lane 1 GM38, lane 2 GM38 –RT, lane 3 Raji TK1+, lane 4 Raji TK1+ –RT, lane 5 Raji TK1-, lane 6 Raji TK1- –RT, lane 7 PCR water control. M is a 100 bp ladder.

Mentions: We have used TP53 and hTERT as markers for transcribed gene repair: it was therefore necessary to check they are in fact transcribed in both cell lines. They are, as shown in (Figure 2). A sample was considered positive for the hTERT gene expression by the presence of a 200 base pair amplicon (Figure 2C), as was the case for every cell-line except the fibroblast cell-line GM38. TP53 gene expression was also detected in both Raji clones and the normal fibroblast, GM38, as shown in Figure 2D by the presence of a 1220-base pair amplicon. Sequencing data of the TP53 gene in the Raji cell-lines also confirmed that the alleles were identical in both, and therefore any difference in DNA repair could not be accounted for by mutations in this key DNA repair gene (sequencing data not shown, supplementary).


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)

Investigation of TP53 and hTERT status in Raji cells using RTPCR. (A) RNA extraction. Lane 1 Werner Syndrome (WS), lane 2 TK1+, lane 3 TK1-, lane 4 GM38, M is a 1 kb ladder. (B) β-actin gene expression internal control check as detected by a 435 amplicon in all cell-lines. Lane 1 WS, lane 2 TK1+, lane 3 TK1-, lane 4 GM38 and lane 5 PCR water control. M is a 100 bp ladder. (C) HTERT gene expression detected by a 200 bp amplicon. HTERT gene expression was found in the WS cell-line, TK1+ and TK1- cells, but not GM38. Lane 1 WS, lane 2 TK1+, lane 3 Raji TK1-, lane 4 GM38 lane 5 –RTPCR control (-RT) WS, lane 6 –RT TK1+, lane 7 –RT TK1-, lane 8 –RT GM38, lane 9 PCR water control. M is a 100 bp ladder (D) TP53 gene expression as detected by a 1220 bp band present in all cells. Lane 1 GM38, lane 2 GM38 –RT, lane 3 Raji TK1+, lane 4 Raji TK1+ –RT, lane 5 Raji TK1-, lane 6 Raji TK1- –RT, lane 7 PCR water control. M is a 100 bp ladder.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Investigation of TP53 and hTERT status in Raji cells using RTPCR. (A) RNA extraction. Lane 1 Werner Syndrome (WS), lane 2 TK1+, lane 3 TK1-, lane 4 GM38, M is a 1 kb ladder. (B) β-actin gene expression internal control check as detected by a 435 amplicon in all cell-lines. Lane 1 WS, lane 2 TK1+, lane 3 TK1-, lane 4 GM38 and lane 5 PCR water control. M is a 100 bp ladder. (C) HTERT gene expression detected by a 200 bp amplicon. HTERT gene expression was found in the WS cell-line, TK1+ and TK1- cells, but not GM38. Lane 1 WS, lane 2 TK1+, lane 3 Raji TK1-, lane 4 GM38 lane 5 –RTPCR control (-RT) WS, lane 6 –RT TK1+, lane 7 –RT TK1-, lane 8 –RT GM38, lane 9 PCR water control. M is a 100 bp ladder (D) TP53 gene expression as detected by a 1220 bp band present in all cells. Lane 1 GM38, lane 2 GM38 –RT, lane 3 Raji TK1+, lane 4 Raji TK1+ –RT, lane 5 Raji TK1-, lane 6 Raji TK1- –RT, lane 7 PCR water control. M is a 100 bp ladder.
Mentions: We have used TP53 and hTERT as markers for transcribed gene repair: it was therefore necessary to check they are in fact transcribed in both cell lines. They are, as shown in (Figure 2). A sample was considered positive for the hTERT gene expression by the presence of a 200 base pair amplicon (Figure 2C), as was the case for every cell-line except the fibroblast cell-line GM38. TP53 gene expression was also detected in both Raji clones and the normal fibroblast, GM38, as shown in Figure 2D by the presence of a 1220-base pair amplicon. Sequencing data of the TP53 gene in the Raji cell-lines also confirmed that the alleles were identical in both, and therefore any difference in DNA repair could not be accounted for by mutations in this key DNA repair gene (sequencing data not shown, supplementary).

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