DNA damage response (DDR) pathways give cells the ability to sense and repair DNA lesions or initiate apoptosis when the damage is unrepairable. The RAD9A-HUS1-RAD1 (9A-1-1) complex is a heterotrimeric DNA clamp that plays an important role in checkpoint signaling and DNA repair. The 9A-1-1 complex interacts with TOPBP1 at the site of damaged DNA, which promotes CHK1 phosphorylation by stimulating the upstream kinase ATR. The 9A-1-1 complex also directly interacts with multiple components of several DNA repair pathways, such as base excision repair and homologous recombination. Loss of any subunit of the 9A-1-1 complex causes severe genomic instability, heightened sensitivity to genotoxic stress, and embryonic lethality. In order to study this complex in vivo I am using two genetic approaches to determine 1) how the 9A-1-1 complex along with alternative 9-1-1 complexes act during male mammalian meiosis and 2) what the physiological requirements are for 9A-1-1 complex-mediated checkpoint signaling in mitotic and meiotic cells. Our first approach utilized a genetic model to study the functions of 9A-1-1 and alternative 9-1-1 complexes throughout prophase I of meiosis. Previous studies revealed that conditional knockout of Hus1 in the testes causes germ cell loss and infertility due to unresolved meiotic DSBs. Surprisingly, RAD1 was able to localize to meiotic chromosome cores even in the absence of Hus1. This led us to hypothesize that RAD1 interacts with paralogs of HUS1 and RAD9A, termed HUS1B and RAD9B respectively to form alternative 9-1-1 complexes during meiosis. We created a testis-specific conditional knockout of Rad1 and found that Rad1 loss led to significantly increased asynapsis of homologous chromosomes, compromised DSB repair and impaired phosphorylation of known ATR targets such as CHK1. Phosphoproteomic analysis of testes from Rad1 CKO mice or ATR inhibitor-treated wild-type mice further implicated RAD1 and ATR signaling in the regulation of cohesin subunits, synaptonemal complex proteins and various DNA repair factors. To differentiate between the DNA repair and checkpoint signaling roles of the 9A-1-1 complex, a RAD9A separation-of-function mutant was created. Serine 385 of the C-terminal tail of RAD9A is the main phosphorylation site responsible for interacting with TOPBP1, ultimately leading to activation of ATR and its downstream substrate CHK1. By creating a phospho-dead mutant (RAD9A-S385A) we selectively disrupted checkpoint signaling without compromising other DNA repair functions of the 9A-1-1 complex. Although Rad9aSA/SA mice were viable, they were born at less than expected frequency and were smaller in size as compared to control littermates. Rad9aSA/SA mice had a 10-fold increase in micronucleated red blood cells, a marker of genomic instability, and were hypersensitive to the DNA interstrand crosslinking agent mitomycin C (MMC). Cultured Rad9aSA/SA cells demonstrated reduced genotoxin-induced CHK1 phosphorylation as compared to control cell lines, confirming that ATR signaling and CHK1 activation were disrupted as anticipated. Exposure to DNA damaging agents also triggered increased chromosomal aberrations, including radials, fusions and breaks, in Rad9aSA/SA cells relative to controls. Rad9aSA/SA cells also showed defects in replication fork protection, reflected by significantly increased MRE11-dependent nascent strand degradation. Together, these studies provide a deeper understanding of the multifaceted functions that the canonical 9A-1-1 complex and alternative clamps play in maintaining genomic stability both within the germline and in somatic cells.