Meiosis is a highly regulated cellular process for generating haploid gametes. During meiosis, programmed double-strand breaks (DSB) allow homologous chromosomes to synapse, crossover, and segregate accurately. Chromosomal errors during meiosis are known to result in infertility or congenital disabilities. Consequently, DNA damage response (DDR) mechanisms are crucial during meiosis. A key DDR component is the heterotrimeric RAD9-RAD1-HUS1 (9-1-1) complex. In somatic cells, the 9-1-1 complex 1) directly recruits DNA repair proteins to damage sites and 2) activates DDR signaling via interactions between the phosphorylated C-terminal tail of RAD9 and TOPBP1, resulting in ATR activation, which in turn is crucial for DNA repair, cell cycle regulation, and overall genome maintenance. During meiosis, ATR is known to promote homologous recombination and is a critical player in promoting meiotic silencing at unsynapsed regions. However, the underlying mechanism of the 9-1-1 complex in regulating ATR signaling during meiosis remains poorly understood. Adding to the complexity, additional alternative 9-1-1 complexes involving the paralogs RAD9B and HUS1B form in spermatocytes. To test how all 9-1-1 complexes promote meiosis, we previously generated testis-specific Rad1 conditional knock-out (CKO) mice and observed severe asynapsis, compromised DSB repair, impaired meiotic silencing, and ATR signaling defects. Since Rad1 deletion disrupts clamp formation, the Rad1 CKO model does not differentiate between the signaling-dependent and independent roles of the 9-1-1 complexes in meiosis. To specifically understand the biological functions of 9-1-1 mediated ATR activation, we developed separation-of-function mutants with serine-to-alanine (SA) mutations in the C-terminal tail of RAD9A and RAD9B that disrupt RAD9-TOPBP1 interactions. These mouse mutants were viable, whereas null mutations in Rad9a or Rad9b cause embryonic lethality. Rad9aSA/SA and Rad9bSA/SA single mutants were inter-crossed to generate Rad9aSA/SA/9bSA/SA double mutants in which 9-1-1/ATR signaling is predicted to be fully disabled. In Aim 1, I will determine the effects of 9-1-1-dependent ATR signaling disruption on fertility and gametogenesis, including its impact on the repair of programmed DSBs, pairing of homologous chromosomes, and meiotic silencing during prophase I. In addition, I will compare the meiotic effects of ATR signaling disruption in Rad9 single or double mutants to determine overlapping or differential functions between the canonical and alternative 9-1-1 complexes. In Aim 2, I will analyze how RAD9-TOPBP1 interactions influence the phosphorylation of known and novel ATR substrates during meiosis. New 911- and ATR- dependent targets will be identified by systematic analysis of whole testes phosphoproteomes from Rad9aSA/SA/9bSA/SA, Rad1 CKO and ATR inhibitor-treated (ATRi) mice. Due to the importance of ATR signaling in meiosis, it is imperative to shed light on how the meiotic 9-1-1/ATR signaling network enables high-fidelity gamete production, with important implications for human fertility and congenital disabilities.