Studio del ruolo delle protein-chinasi ATR e ATM nell'omeostasi dei telomeri
Author(s)
Ingegnere, Tiziano
Date Issued
May 16, 2014
Type
Doctoral Thesis
Abstract
Telomere integrity is essential to maintain chromosome stability and to avoid chromosome end fusion. Telomeres comprise tandem DNA repeats bound by a multiprotein complex known as shelterin. Sheltering contributes to maintenance of telomere integrity and prevents activation of DNA damage response (DDR) at telomeres. In particular it has been shown that TRF1 is important to prevent telomere breakage following replication fork stalling at telomeres. More recently, combined deletion of all the shelterin components revealed that this complex act to protect telomeres from a number of DNA-rearranging process including homologous recombination, NHEJ, ATM/ATR signaling and DNA resection. It has been shown that short dysfunctional telomeres activate DDRs mediated by ATM and ATR checkpoint kinases, which play a crucial role in the cellular response to DNA lesion. Paradoxically, ATR and ATM were found associated to telomeres in a cell cycle manner and in the absence of DNA damage, suggesting a role in the maintenance of telomere homeostasis. Indeed mice in which it was induced ATM loss of function show premature aging and have shorter telomeres than normal cells (Wong K.K., 2003). However, later studies failed to reveal a role of ATM in the elongation of short telomeres by telomerase (wong et al., 2003; Feldser et al 2006). More recently, it was demonstrated that severe reduction of ATR levels in primary murine fibroblasts, does not impair telomere length but induce the appearance of a number of futures typical of fragile sites. Similar finding was reported in murine cells lacking ATM (Qi et al 2003; wong et al., 2003). Collectively these results suggest that ATM and ATR participate to telomere protection in mammalian cells by preventing telomere fragility and recombination. In the effort to better understand the function of ATR and ATM in a telomeric context we respectively inhibited the proteins with UCN-01, an inhibitor of the Chk1 pathway responsive under ATR activation, and KU55933 a direct inhibitor of ATM. We tested this two inhibitor in Hela cells and human fibroblast. Furthermore, to induce a stalled DNA replication fork we added fluorodesossiuridine (FdU) in combination with the inhibitors. Here, we report by using telomere-fluorescence in situ hybridization (Telo-FISH) on metaphase chromosomes that in Hela cells and in human fibroblast both the inhibitors not seems to increase the telomere fusions. Via chromosome orientation-FISH we investigate if there are different involvement of the two protein in the type of chromosome fusions during the replication of leading and lagging strand. No differences are reported. With the Telomeric oligonucleotide ligation assay (T-OLA) we analyze the length of the 3’-overhang, the single strand region at the end of the telomeres. Our results show no difference of 3’-overhang length between control and the inhibitors treatment. Altogether this results seems to indicate that the inhibition by Chk1 and ATM pathway don’t affects the protective structure of telomeres. On the other hands we report a substantial increase of Multiple telomeric signal (MTS) in Hela cells but not in human fibroblast. We also show that the inhibition of Chk1 and ATM, mainly in Human fibroblast, lead to Telomere deletion (TD) and Sister Chromatid loss (SCL). Together these findings leads us to think that in absence of the ATM and ATR signaling telomeres resemble common fragile sites. In the effort to better understand our data we test our inhibitor on a common fragile site: FRAXA. The data from a linfoblastoid line with FRAXA, 32B, partially fit with the telomer’s one. Especially with Hela results. We suppose that the difference between Hela and 32B on one site, and Human Fibroblast on the other may be attributed to the lack of an active telomerase in Fibroblast.
The attention for the interaction between DNA Repair Machinery and Telomer are growing in the scientific community. Particularly the mechanism that brings some protein, as ATM or ATR as we show, to remodelling their response in a telomeric context.
Additional information
Dottorato di ricerca in Evoluzione biologica e biochimica
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