Genetic analysis of ATR, a central regulator of genome stability

To protect the DNA against the constant assaults of endogenous and environmental agents, cells have developed conserved signaling pathways, known as the DNA damage response. Such responses are important to maintain genome stability and prevent human disease. The ATR kinase plays a critical role during the DNA Damage Response, as well as during unperturbed DNA replication. Despite the vital importance of ATR, little is known about its structure and the precise regulation of its activity. In this study we investigate the structure-function relationship of ATR using DT40 chicken cells. In particular, we examine the role of tandem helical motifs, called HEAT repeats, that comprise the non-kinase portion of ATR. We found that Atr mutants, expressing an Atr protein version where single/multiple HEATs were removed, behaved similarly to the null cell line. In contrast, hybrid Atr mutants expressing equivalent regions of human ATR were fully functional. Our observations suggest that all ATR functions are tightly dependent on the integrity of HEAT repeats. Although we cannot exclude that these motifs mediate specific protein-protein interactions, it is likely that their main role is to contribute to an overall structural function, holding ATR configuration in place. ATR is an essential gene and mutations in this gene have been implicated in different human diseases, such as cancer and Seckel syndrome. In this thesis, we have also modeled mutations implicated in such disorders using DT40 and human cells. In particular, we found that two novel missense ATR Seckel mutations (M1159I and K1665N) do not affect protein function, but instead impact ATR splicing, potentially by affecting binding of splice factors to sequence-specific regulators of splicing. Additionally, we have also investigated the effect of a non-Seckel mutation (Q2144R) linked to cancer predisposition in patients. Our results indicate that this mutation has a profound effect on ATR activity. Checkpoint signaling is abrogated in the presence of this substitution and, as a consequence, cells seem to accumulate chromosomal defects resulting in cell death. These findings indicate that Q2144 residue, which is part of a potential SQ site, could be key in the ATR– mediated response to DNA damage. 2020-08-09