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

Katherine A Mcallister,C S Downes,Valerie J Mckelvey-Martin,George Mckerr,Akeel A Yasseen

doi:10.3389/fgene.2014.00233

Katherine A Mcallister, C S Downes + Show 3 more

Open Access

https://doi.org/10.3389/fgene.2014.00233

Copy DOI

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.

Highlights

Human thymidine kinase 1 (TK1) is a salvage enzyme that phosphorylates thymidine to create dTMP, which is later converted by thymidylate kinase and nucleoside diphosphate kinase to dTTP, a precursor for DNA metabolism (Segura-Pena et al, 2007)
TK1 deficiency is known to cause a clear increase in DNA damage sensitivity and mutagenicity, and the likelihood of increased carcinogenesis, the DNA repair process on which the salvage enzyme exerts its protective effects remains to be elucidated
While our results in human cells show that TK1 deficiency can reduce the rate of bulk DNA repair, the most prominent finding is the clear role for TK1 in repair of damage occurring in specific gene regions

Summary

Introduction

Human thymidine kinase 1 (TK1) is a salvage enzyme that phosphorylates thymidine to create dTMP, which is later converted by thymidylate kinase and nucleoside diphosphate kinase to dTTP, a precursor for DNA metabolism (Segura-Pena et al, 2007). It has been shown that TK1 is upregulated in different tumor types in response to DNA damage, and that the cellular response to genotoxins causes nuclear localization of TK1; an interesting finding given that the salvage enzyme has previously been regarded as solely cytoplasmic (Chen et al, 2010). These studies suggest that TK1 may somehow affect DNA metabolism in a way not obviously explained by its salvage role

Methods

Results

Conclusion