Meiosis is a specialized cell division that occurs in germ cells during the development of eggs and sperm, and must be completed with high fidelity to ensure the accurate transmission of genetic information to offspring. Several key chromosomal events occur during prophase I of meiosis, including genetic recombination and the pairing and close association (synapsis) of homologous chromosomes. In recent work, we showed that a ring-shaped DNA damage response protein complex called the RAD9A-RAD1-HUS1 (9-1-1) complex is essential for several aspects of these chromosomal processes in mammals, and that male mice with testis-specific targeted disruption of Hus1 show spermatogenesis defects and are unable to produce viable offspring. 9-1-1 had previously been determined to function as a scaffolding factor in mitotic cells to promote DNA repair and activate cell cycle checkpoint signaling, and was typically thought to function as an obligate heterotrimeric complex. Importantly, we determined that the RAD1 component of this complex can function separately from RAD9A and HUS1 during meiosis. We propose that the RAD1 functions that are independent of the canonical 9-1-1 complex involve the formation of two novel clamp complexes with the paralogs RAD9B and HUS1B, which are expressed predominantly in testis but whose functions are unknown. The highly focused, self-contained experiments proposed here will test the hypothesis that three different clamp complexes, 9-1-1, RAD9B-RAD1-HUS1 (9B-1-1), and RAD9B-RAD1-HUS1B (9B-1-1B), perform distinct but critical functions in maintaining chromosome integrity during mammalian meiosis.
In Aim 1, we will test the consequences of disabling the common subunit of all three putative 9-1-1 complexes in the male germ line by assessing fertility and chromosome integrity in mice in which Rad1 is conditionally deleted specifically in meiotic cells.
In Aim 2, we will address the molecular functions of the individual complexes by assessing physical interactions and chromosomal localization of proteins that we have identified as candidate binding partners and effectors of the checkpoint clamps, based on their co-evolution with 9-1-1 subunits in mammals. These studies have direct translational relevance to human fertility and developmental disorders, and build logically upon our expertise in using mouse models to study DNA repair and DNA damage signaling. Successful completion of the proposed experiments has great potential to significantly advance our understanding of the underlying molecular mechanisms driving chromosomal instability and aneuploidy, and may provide novel targets for the prevention and treatment of infertility.
While the fascinating chromosomal events of meiosis are what make us genetically unique, problems arising during these processes can also lead to subfertility, infertility, or intellectual and developmental disabilities, including aneuploidies sch as Down syndrome (Trisomy 21). The RAD9A-RAD1-HUS1 (9-1-1) complex is a ring-shaped protein complex that is crucial for maintaining meiotic chromosome integrity, and our recent findings established separable roles for the mammalian 9-1-1 subunits, particularly RAD1, during meiosis. By combining a mouse genetic approach with evolutionary computational analyses and physical interaction studies to define the meiotic functions of two novel RAD1-containing, 9-1-1-like complexes, we will provide a substantial step forward in our understanding of mammalian meiotic chromosome biology and facilitate future fertility-preserving medical advances.
