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.