Roles For The Dna Damage Checkpoint Gene Hus1 In Responding To Endogenous And Exogenous Stresses In Vivo
The DNA damage response (DDR) represents the primary line of defense against exogenous and endogenous genotoxic agents that threaten the stability of our genomes. The ATM and ATR pathways are central to the response to DNA-damage and their understanding can bring important information in the fight against inborn disease and cancer. While the roles for the ATM pathway in DDR are well understood, the lack of a model organism for the ATR pathway has impeded its understanding. Here I show how we use Hus1, a component of the RAD9-RAD1-HUS1 heterotrimeric clamp and a vital member of the ATR pathway, to deregulate the ATR pathway and dissect its importance in development and disease. This was done by engineering in mice a Hus1 allelic series by combining the wild-type (Hus1+) with a hypomorphic (Hus1neo) or null (Hus1[DELTA]1) alleles. As opposed to the germline constitutive deletion that leads to embryonic lethality, Hus1 hypomorphic mice are born at expected frequencies and show no overt phenotype but have increased levels of genomic instability. Thus they have enough Hus1 to deal with physiologic stress and could be used for our research. iii To investigate the in vivo physiologic relationship between the ATM and ATR pathways I crossed the Hus1 allelic series on an Atm null background and showed that the interaction between the two pathways is critical for normal development. When the two pathways are deregulated simultaneously a synthetic lethal interaction is created. While part of the Hus1/Atm double mutant mice die during development due an apparent incapability to deal with replication stress, the survivors suffer from developmental defects including dwarfism and skeletal defects. To further understand the interplay between these two pathways in response to genotoxins Hus1 single mutant and Hus1/Atm double mutant mice were subjected to specific genotoxin treatments. This analysis showed that in adult tissues there is a clear separation of function between the ATM and ATR pathways, with ATM being the main responde r to DNA breaks while the ATR pathway responds mainly to replication stress such as that caused by mitomycin C. In summary this thesis brings important new information about the relationship between the two main DDR response pathways leaded by ATM and ATR and substantiates their joint role in development and disease. Moreover we now have a better understanding of how these interactions can be used for creating better therapies. iv
DNA damage; ATM; ATR; HUS1; cell cycle checkpoints; cancer; development; genomic instability
Weiss, Robert S.
Nikitin, Alexander; Schimenti, John C.; Alani, Eric
Ph.D. of Physiology
Doctor of Philosophy
dissertation or thesis