Maintenance of genome integrity is critical for cell proliferation, organism survival and fertility. The DNA damage response (DDR) is an important aspect of genome maintenance that coordinates a range of cellular processes including DNA replication, cell cycle regulation and DNA repair. The phosphatidylinositol 3’ kinase (PI3K)-related kinase ATR, has emerged as a central regulator of the DDR by phosphorylating a diverse range of targets in response to DNA damage to coordinate DNA repair pathway choice with the cell cycle and other DNA metabolic activities. Recently, ATR has emerged as an attractive target for cancer treatment and ATR inhibitors are currently in clinical trials. Importantly, the ATR-activating scaffold protein TOPBP1 has also been implicated in regulating DNA repair pathway choice as well as coordinating distinct branches of ATR signaling. In this thesis, I will present a range of evidence that extends our understanding of the mechanism of the ATR-TOPBP1 signaling axis in somatic DNA repair.In addition to the roles of TOPBP1 and ATR in the somatic DNA damage response, TOPBP1 and ATR are essential for male meiotic progression by promoting DNA repair by homologous recombination, chromosome synapsis, and transcriptional silencing of unsynapsed chromosomes including the X and Y, which can only partially synapse by a small region known as the PAR (pseudoautosomal region). First, I present a characterization of ATR signaling events in mouse meiosis by using a dual approach to inhibit ATR followed by mass spectrometry. Specifically, we used a RAD1 conditional knockout model, which prevents TOPBP1-mediated ATR activation in meiocytes, and mice treated with ATR inhibitors. In this way, we can take advantage of both tissue-specific and acute ATR inhibition to generate a set of high-confidence ATR-dependent phosphorylation events. Second, in chapter 3, I utilize this database of ATR and RAD1-dependent events to understand the status of ATR activation in a new separation-of-function TOPBP1 model mouse with eight charge reversal mutations in BRCT5 of TOPBP1. These Topbp1KE/KE mice are grossly indistinguishable from wild type littermates and have no checkpoint or cell sensitivity phenotypes, yet males are completely sterile. By comparing the set of ATR and RAD1-dependent phosphosites to a testes phosphoproteome from the Topbp1KE/KE mice, we find that between 80-90% of ATR signaling remains unchanged, indicating that a small, but critical set of ATR targets are lost in the Topbp1KE/KE testes. Furthermore, in chapter 3, I present the characterization of the Topbp1KE/KE phenotype and describe several hypothesis and future directions to identify the critical ATR targets or changes in TOPBP1 protein interactions that contribute to the total loss of male fertility in these mice. Next, in chapter 4, I present my efforts towards understanding the role of the ATR signaling axis in the DNA damage response in cancer cell lines. Specifically, how ATR promotes the repair of DNA lesions by the error-free pathway homologous recombination (HR) by maintaining the expression of a set of HR factors. I established a protocol of low-dose, long-term treatment of ATR inhibitors that results in a depletion of HR factors which subsequently results in sensitivity to DNA damage induced by PARP inhibitors. I establish that this sensitivity is due to increased DNA-PK dependent mutagenic repair through the non-homologous end joining pathway (NHEJ) and both sensitivity and chromosomal aberrations can be rescued by DNA-PKcs inhibition. Finally, in Chapter 5, I present evidence that TOPBP1 regulates DNA repair pathway choice. Specifically, TOPBP1 coordinates the actions of the scaffold proteins 53BP1 and BRCA1 in regulating the decision between non-homologous end joining (NHEJ) or homologous recombination (HR). I show that by modulating the stability of the TOPBP1-53BP1 or TOPBP1-BRCA1 complexes by expression of protein fusion constructs that artificially strengthen these interactions or by mutating TOPBP1 BRCT domains to disrupt the interaction of TOPBP1 to phosphorylated 53BP1 or BRCA1, in mouse models. Furthermore, I present evidence that it is not possible to complement a loss of ATR activity after knockdown of endogenous TOPBP1 with expression of ectopic TOPBP1 in several cell culture systems. This result implies the existence of an unknown mechanism of TOPBP1 regulation that only occurs after expression from the endogenous locus. Overall, this thesis represents a body of work that extends our understanding of the role of the ATR-TOPBP1 signaling axis in spermatogenesis and in somatic DNA repair pathway choice.