Periodic Reporting for period 1 - DNAcheck (Mechanistic analysis of DNA damage signaling and bypass upon replication of damaged DNA template in human cells.) Periodo di rendicontazione: 2018-10-01 al 2020-09-30 Sintesi del contesto e degli obiettivi generali del progetto The genetic information encoded by DNA is constantly threatened by endogenous or exogenous sources of damage. To safeguard the genome against these insults, cells have evolved DNA damage checkpoints that sense the presence of damaged DNA, block cell cycle progression and ensure that DNA is fully repaired before resuming the cell cycle. However, in many cases, unrepaired lesions remain in the DNA when cells enter S-phase. In this scenario, cells employ DNA damage tolerance mechanisms to complete genome replication and prevent fork breakage. Importantly, these pathways are not restricted to the site of stalling but can also function behind the fork at single-stranded(ss)DNA gaps originated by re-priming of DNA synthesis downstream of lesions. While it is well known that the accumulation of single-stranded DNA (ssDNA) is the signal that riggers the checkpoint response, it is less clear how and where ssDNA actually arises. Generally, it is assumed to accumulate at stalled replication forks by an uncoupling between replicative helicase and polymerase movement. However, recent evidence found in budding yeast support a model where, in response to polymerase-blocking lesions, replication forks do not stall but recover efficiently by re-priming downstream of the lesions, thus leaving ssDNA gaps behind the fork. It is currently unknown whether this model also applies to vertebrate cells. Hence, this project aimed to address the fundamental question of how DNA damage is sensed during genome replication in human cells. Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti In the course of this project, we have identified a remarkable mechanism that promote the robust activation of the replication checkpoint in human cell lines, which involves the action of different nucleases at ssDNA gaps generated behind replication forks in the presence of lesions on the DNA template. Remarkably, this mechanism did not apply when lesion-less replication stress was induced. Thus, these results have shown further evidence of a dichotomy between the cellular response to damaged DNA template versus replisome problems. The results of the project have been presented to the scientific community at multiple conferences and workshops and are expected to be published within a year. Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora) The project results have helped to better understand how human cells sense and deal with replicative stress, a process that is highly relevant to human disease such as cancer. Interestingly, deregulation of nuclease levels has been reported in different types of cancer. Hence, these nucleases may offer a target for cancer therapeutic development.