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DNA replication is essential for cell survival. During DNA replication, cells encounter many challenges, such as dNTP depletion, transcription intermediates, chemical adducts, double strand breaks, and heterochromatin. Replication stress induced by these challenges activates the sensor kinase ATR in mammals (Mec1 in S. cerevisiae), which coordinates a multifaceted cellular response that is essential for maintaining genome integrity. Over the last few decades, ATR’s role in controlling the checkpoint upon the DNA damage response has been well studied. However, most studies of ATR function have relied on a few well-established substrates, and we still lack a comprehensive and unbiased understanding of ATR action in vivo. I employed a combined genetic-phosphoproteomic approach to monitor Mec1 (S. cerevisiae) substrates in a systematic, unbiased and quantitative manner. Unexpectedly, I found from this analysis that Mec1 is highly active during normal DNA replication, at levels comparable to or higher than Mec1’s activation state induced by replication stress. This “replication-correlated” mode of Mec1 action requires the Dna2 or the Ddc1 lagging-strand replication factors and is distinguishable from Mec1’s action in activating the downstream kinase Rad53. I also found evidence supporting that in humans, ATR can also function in a replication-correlated manner. Overall, we proposed that Mec1/ATR performs key functions during ongoing DNA synthesis to prevent genomic instability that are largely distinct from its canonical checkpoint role during replication stress. To study further the ATR kinase, I discovered a new mechanism for how ATR is essential for proper genome maintenance. I found that long-term chronic inhibition of ATR signaling severely impairs the ability of cells to utilize homologous recombination (HR)-mediated DNA repair. Proteomic analysis revealed that chronic ATR inhibition reduces the abundance of key HR factors, suggesting that spontaneous ATR signaling promotes enhanced HR capacity by controlling the abundance of the HR machinery. This key role of ATR is mediated largely via CHK1-dependent transcription control and may also involve protein stabilization of specific HR factors. Interestingly, cancer cells seem to exhibit a stronger dependency on ATR signaling for maintaining the levels of HR factors, and I propose that elevated constitutive ATR signaling caused by augmented replication stress in cancer cells drives the enhanced HR capacity observed in certain tumor types. Overall, these findings show that constitutive ATR signaling plays a major role in shaping the capacity and abundance of the HR machinery, and provides rationale for combination chemotherapy using ATR inhibitors.
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Genetics; DNA replication; Biochemistry; ATR/ATM; DNA Repair; Mass Spec; DNA damage
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Smolka, Marcus B.
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Schimenti, John C.
Koren, Amnon
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Biochemistry, Molecular and Cell Biology
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Ph. D., Biochemistry, Molecular and Cell Biology
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Doctor of Philosophy
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Government Document
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Attribution-NonCommercial 4.0 International
dissertation or thesis
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