Investigation of DNA damage response at interstitial telomeric sites in Chinese hamster cell lines

Abstract

Telomeres are highly specialised structures that protect the ends of linear chromosomes from damage. Telomeric DNA consists of tandem repeats of a specific sequence (TTAGGG)n that varies in length depending on the species. Two telomere maintenance mechanisms are known to exist. Telomerase, an enzyme that synthesises new tandem repeats onto the end of linear chromosome and ALT (Alternative Lengthening of Chromosomes), which uses recombination to lengthen telomeric sequences. In some mammalian species, both mechanisms are known to co-exist. Telomeric DNA that is found at non-telomere sites are referred to as interstitial telomeric sequences (ITS). Several studies have shown that ITS sites in the Chinese hamster are preferred sites for radiation and spontaneous chromosomal damage. Once damage occurs, repair mechanisms such as HR (homologous recombination) and NHEJ (non-homologous end joined) are activated. However, the contribution to this repair by telomerase and ALT is unknown. The aim of this work therefore is to investigate the contribution that above mention mechanisms (HR, NHEJ, telomerase and ALT) make to ITS repair in Chinese hamster cells. Classical cytogenetic techniques such as Giemsa staining, chromosomal aberration analysis, FISH (Fluorescence in situ hybridazation, IHC (Immunohistochemistry), TIF (Telomere dysfunction induced foci), were used to quantify chromosomal damage after IR (ionising radiation) treatment of normal and repair deficient (HR and NHEJ) Chinese hamster cell lines. Molecular analysis employed the TRAP assay, and C-circle analysis to look at telomerase expression and ALT activity. Finally, siRNA was used to look at two genes which are important in the telomere maintenance pathways, ATRX and HDAC9, were knocked down using siRNA and cells were analysed using Western blotting and qPCR for the effects this had on telomerase and ALT. Our results show that chromatid breaks within ITSs sites were higher than expected in repair deficient cell lines. In addition, repair deficient cells recover more slowly to IR chromosomal damage than control cells, suggesting that HR and NHEJ play a part in the repair of ITS in Chinese hamster cells. siRNA knockdown revealed that ATRX and HDAC9 affected the expression of ALT and telomerase respectively in our cell lines. Taken together, our work provides evidence that show for the first time the co-existence of both telomerase and ALT activity in Chinese hamster cells.