The ATR protein kinase sits atop a complex signaling cascade that is activated by DNA replication and hyper- activated by replication stress or DNA double-strand breaks. Activation of ATR by genotoxic stress is essential for the ability of cells to survive stress and represents a barrier to transformation of normal cells to a pathological condition that promotes tumorigenesis. The downstream effectors and consequences of ATR signaling are now being understood, however early events in signaling, such as the initial activation of ATR kinase at sites of gentoxic stress, are still poorly understood. This proposal focuses on the biochemical mechanism for ATR activation at sites of DNA damage. This proposal features the TOPBP1 protein, which physically interacts with ATR at sites of DNA damage and is required for ATR activation during the replication stress and DNA damage responses. TOPBP1 is a BRCT repeat containing protein that likely acts as a scaffold to link checkpoint proteins together into active signaling centers. In this proposal we combine biochemical studies using purified factors, functional studies in Xenopus egg extracts, and in vivo studies in cultured cells to mount an in-depth exploration of how TOPBP1 activates ATR at sites of DNA damage. Recent studies on the ETAA1 protein have shown that TOPBP1 is not alone in its ability to activate ATR, and our preliminary studies suggest that a third and possibly more ATR activators are present in human cells. In this proposal we will also study these new activators, with the long-term goal of building a comprehensive, systems-level view of how ATR signaling is initiated. The work is divided into three Aims.
In Aim 1, we examine how active ATR signaling centers form at sites of damage. The recruitment mechanism is complex, and likely involves multiple protein-protein interactions between TOPBP1 and its binding partners. Recent work from our group has shown that soluble TOPBP1 is held in an auto-inhibitory conformation and thus an additional goal of Aim 1 is to determine how this auto- inhibition is resolved at sites of damage.
In Aim 2 we examine how ATR may use a negative feedback loop to regulate assembly of additional signaling centers at sites of damage. We will also explore the possibility that an ATR signaling center is mobile on DNA. In the final Aim we will define a rule-book for how different ATR activators are utilized, how their functions are related (or not), and how their activities are integrated during an ATR response.
Understanding how normal cells are transformed into cancer cells is one of the long-term goals of cancer biology. The ATR protein kinase protects cells from the cancer-promoting effects of DNA damage, and thus represents an important barrier to tumorigenesis. This proposal seeks a biochemical understanding of how the ATR kinase is activated by DNA damage, and successful completion of the proposed experiments will be important to the development of ATR-based anti-cancer therapeutics, which have already shown promise in pre-clinical testing.
| Wong, Matthew M; Belew, Mezmur D; Kwieraga, Amanda et al. (2018) Programmed DNA Breaks Activate the Germline Genome in Caenorhabditis elegans. Dev Cell 46:302-315.e5 |
