One fundamental goal of any organism is the preservation and faithful propagation of genetic information. However, DNA replication is an inherently stressful process. Cells constantly confront DNA damage, caused by a range of endogenous and exogenous insults. Failure to repair DNA damage has severe consequences and can lead to genomic instability. In eukaryotic cells, the phosphatidylinositol 3′ kinase (PI3K)‐related kinases (PIKKs), including ATR, ATM, and DNA-PKcs in mammals and Mec1 and Tel1 in budding yeast, are key sensors of DNA damage. Once activated, these kinases will initiate a signaling cascade, often referred as the DNA damage checkpoint, that promotes a range of cellular responses, including the arrest of the cell cycle, inhibition of origin firing, protection of stalled replication forks, induction of transcriptional responses, initiation of apoptosis, and control of dNTP levels. Besides the checkpoint function, the DNA damage signaling kinases have also been shown to phosphorylate dozens of DNA repair proteins. While the function of these kinases in the canonical DNA damage checkpoint has been well characterized, the precise mechanisms by which they control DNA repair remain incompletely understood, representing a significant knowledge gap in the field.My thesis work focuses on understanding how the Mec1/ATR kinase regulates DNA repair processes to maintain genome integrity. Using Saccharomyces cerevisiae as a model system, I led two main projects. In the first project, I proposed that phosphorylation of the helicase Sgs1 by Mec1 is important for genome integrity. Using a traditional gross chromosomal rearrangement (GCR) assay, I showed that Sgs1 phosphorylation by Mec1 is crucial for GCR suppression. I further demonstrated that Mec1 phosphorylates Sgs1 to promote rejection of homeologous recombination, therefore preventing rearrangements and genomic instability. In the second project, I curated a list of high-confidence Mec1 substrates by merging phosphoproteomic datasets from previously published studies and found that Mec1 phosphorylates Rad18, a key regulator of DNA damage tolerance pathways, at its SAP domain. Phospho-mimetic mutations of Rad18 impairs its DNA binding ability, leading to sensitivity of genotoxins. Furthermore, I showed that Rad18 phosphorylation by Mec1 is necessary for constraining the usage of translesion synthesis (TLS) and mutagenic homologous recombination (HR), which contributes to genome integrity. In addition, I found that ATR also regulates RAD18-mediated DNA repair via phosphorylation in mammalian cells. In summary, my thesis work highlights two novel phosphorylation-based regulatory mechanisms that control the quality of DNA repair. This expands our understanding of the role of Mec1 in coordinating DNA repair processes. My work also opens important questions to be addressed in the future. Given the conservation of this kinase in eukaryotes, my work sheds light on the function of ATR in regulating DNA repair in mammalian cells. Since ATRi therapy is currently undergoing clinical trials against cancer, my work could provide new insights into improving treatment efficacy and reducing side effects.