The ATR-dependent DNA damage response (DDR) is essential for genome integrity and constitutes an important tumor suppressive system. DNA replication stress has been linked to cellular transformation, and represents some of the early events leading to tumorigenesis in mammals. Our work has centered on proteins that are part of the `Zyxin' LIM family, characterized by the presence of tandem LIM domains at their C terminus, which we found were important for the repression of the DNA damage response in human cells. Specifically, we have discovered that Ajuba is essential for efficient repression of the ATR pathway in unperturbed cells. Depletion of Ajuba leads to activation of ATR, followed by a potent apoptotic response. We have discovered that Ajuba associates with the RPA complex, itself central to the ATR activation pathway, and have detected a direct interaction in vitro between Ajuba and the RPA70 subunit. RPA70 is composed of OB oligonucleotide/oligosaccharide-binding (OB) folds essential for single strand DNA binding and assembly of the ATR activation complex. Based on these observations, we hypothesize that Ajuba is an important repressor of inappropriate ATR activation in S phase through a direct interaction with RPA70. We are proposing to study in detail the direct contacts between Ajuba and RPA, and to analyze the significance of this interaction in human cells. Our results are expected to improve our understanding of the regulation of the DNA damage response in human cells, itself implicated in early events in cellular transformation and cancer.
Cells possess molecular mechanisms that sense DNA damage, leading to DNA repair or cell death. This proposal focuses on the early events leading to the induction of the ATR repair pathway, and possibly other DNA repair systems. Because cancer cells are often defective in DNA repair, and exhibit high genomic instability, understanding these events is important to discover the early events that can lead to the cancer phenotype at the cellular level.
