Résumé
The Tel1 protein in Saccharomyces cerevisiae and its mammalian orthologue, ATM, are the members of the evolutionary conserved PI3K-related kinase family, crucial for maintaining genome stability. Historically described for its role in preserving telomere length, Tel1 is at present well-established as a key regulator of Double-Stranded DNA Breaks (DSB) metabolism, orchestrating DSB detection, DNA Damage Response activation and completion (including ef- ficient end resection), as well as DSB-induced modulation of chromatin state and cell cycle progression.More recently, Tel1 has been studied in the context of replicative stress, a condition charac- terized by impaired replication fork progression. In S. cerevisiae and human cell lines suffering from camptothecin (CTP)-induced replicative stress, Tel1 is essential for stabilization of reversed replication forks. These DNA intermediates, generated by reannealing of the newly synthesized DNA strands, resemble one-ended DSB and are susceptible to nucleolytic degradation, which, if not properly regulated, can result in severe genome instability. By binding the reversed fork tip, Tel1 regulates the accurate processing and resolution of reversed replication forks.We used a functionally validated Tel1 construct, tagged with yEGFP (yeast enhanced-GFP), to monitor Tel1’s behaviour by epifluorescence microscopy in two scenarios: 1) during chemically or enzymatically induced DSB, or 2) in response to pharmacologically or endogenously triggered replicative stress that both culminate with fork reversal. We detected that Tel1 forms nuclear foci in all these conditions, but the kinetics of foci formation, their number per nucleus and their subnuclear localization were different, strongly depending on the underlying DNA intermediate. From these features, we show that yEGFP-Tel1 is a powerful tool to gain new knowledge on DSB and DSB-like signalling. Moreover, we introduce this construct as a marker to track reversed replication forks, thus filling a technical gap in the replication stress field.