Abstract
DNA damage response pathways are essential for genome stability and cell survival. Specifically, the ATR kinase is activated by DNA replication stress. An early event in this activation is the recruitment and phosphorylation of RPA, a single stranded DNA binding complex composed of three subunits, RPA70, RPA32 and RPA14. We have previously shown that the LIM protein Ajuba associates with RPA, and that depletion of Ajuba leads to potent activation of ATR. In this study, we provide evidence that the Ajuba-RPA interaction occurs through direct protein contact with RPA70, and that their association is cell cycle-regulated and is reduced upon DNA replication stress. We propose a model in which Ajuba negatively regulates the ATR pathway by directly interacting with RPA70, thereby preventing inappropriate ATR activation. Our results provide a framework to further our understanding of the mechanism of ATR regulation in human cells in the context of cellular transformation.
Highlights
Cells possess conserved mechanisms that have evolved to sense, respond to and repair specific types of DNA damage[1]
In order to determine whether Ajuba exhibits association with the whole RPA complex, or to a particular subunit of RPA, co-immunoprecipitations were performed with HTC75 cell extracts using antibodies to each subunit separately
We found no perceptible effect on the amount of Ajuba associated with RPA in S phase cells, and concluded that the dissociation seen upon hydroxyurea treatment was not merely due to delay in S phase, and was the consequence of DNA replication stress
Summary
Cells possess conserved mechanisms that have evolved to sense, respond to and repair specific types of DNA damage[1]. Role given its ability to bind single stranded DNA, thereby preventing secondary structures incompatible with replication or repair, and a signaling role related to the assembly of the ATR activation complex on RPA70N. Our current model is that Ajuba exerts an inhibitory activity to full-blown activation of the ATR kinase, and is essential to prevent cell death by apoptosis in unperturbed cells This inhibitory effect correlates with our observation that Ajuba associates with the RPA complex in the cell[14], and associates with a core component of early ATR activation. We show that the interaction between Ajuba and the RPA complex is direct and occurs through the RPA70 subunit We found that this association occurs in the nucleus, and that the amounts of Ajuba along with the degree of co-localization between Ajuba and RPA are increased in S phase, suggesting cell cycle-dependent regulation of the localization of Ajuba, and perhaps of its interaction with RPA as well. We propose a model for Ajuba in preventing inappropriate ATR induction during S phase
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