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    A common mechanism of ATR activation by TOPBP1 and ETAA1

    Thada, Vaughn
    0000-0002-9245-4354
    : http://hdl.handle.net/1803/16455
    : 2021-02-12

    Abstract

    Human cells are constantly exposed to endogenous and exogenous sources of DNA damage that cause thousands of DNA lesions in each cell every day. Accurate repair of these lesions ensures maintenance of genome stability and acts as a barrier to tumorigenesis. The cellular signaling pathways that recognize and repair DNA damage are collectively known as the DNA damage response (DDR). The ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase activates the DDR in response to DNA replication stress, and coordinates the processes of DNA replication, DNA repair, and cell cycle progression. ATR activation is stimulated by an ATR activating protein, and in human cells, there are currently two proteins known to activate ATR: DNA topoisomerase II binding protein 1 (TOPBP1) and ETAA1 activator of ATR kinase (ETAA1). Previous data indicated TOPBP1 and ETAA1 bind the same surface of ATR to trigger kinase activation, but the specific mechanism of ATR activation by TOPBP1 and ETAA1 was not well understood. In this thesis, I further elucidate how TOPBP1 and ETAA1 activate ATR. I found that TOPBP1 and ETAA1 contain predicted coiled-coil motifs within their ATR activation domains (AADs) that are required for ATR binding and activation because these motifs directly contact the ATR-ATR interacting protein (ATRIP) complex. Additionally, I found that TOPBP1, and ETAA1, AAD dimerization enhances ATR activation and that ETAA1 forms oligomeric complexes in cells. Furthermore, I found that forced dimerization of an oligomerization-defective ETAA1 mutant restores ATR signaling, genome stability, and viability in response to DNA damaging agents in ETAA1-deficient cells. Finally, by using a selective DNA polymerase α (Pol α) inhibitor, I found that lagging strand stalling during DNA replication activates TOPBP1-dependent ATR signaling. Together, these results reveal that TOPBP1 and ETAA1 activate ATR via the same biochemical mechanism and identify determinants of ATR checkpoint signaling at stalled replication forks.
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