Infertility and birth defects often arise due to improper genetic quality control during meiosis. To ensure the production of gametes without genetic defects, the genome of meiocytes is monitored by a set of evolutionarily conserved kinases, known as checkpoint kinases. These kinases sense damage to DNA or problems in chromosome pairing and, upon activation, can block meiotic progression and induce cell death. But the action of checkpoint kinases is not solely utilized as a quality control mechanism. Programmed double strand break (DSB) formation is an abundant and essential event for normal meiotic progression, and checkpoint kinases are required to coordinate key events in recombination repair, crossover regulation and transcriptional silencing. How meiotic checkpoint signaling is regulated is not understood, especially in mammals. In particular, little is known about how checkpoint kinases can act both as essential regulators of normal meiotic progression as well as effectors of quality control mechanisms that lead to cell death. This knowledge gap poses a major barrier for understanding the determinants of genetic quality control and how mis-regulation of checkpoint signaling may aberrantly block meiotic progression and promote infertility. The same pathways also mediate fundamental DNA repair and checkpoint functions in mitotic cells, are sometimes deregulated in cancers, and are being targeted clinically as an emerging strategy for cancer treatment. This proposal applies innovative approaches to overcome long-standing barriers for the study of meiotic checkpoint signaling in mammals. The proposed studies focus on the essential checkpoint kinase ATR, which is important for DSB repair and transcriptional silencing of unsynapsed chromatin during meiosis and is regulated in part by the scaffolding protein TOPBP1 and other upstream regulators such as the RAD9A-RAD1-HUS1 complex. Cutting edge approaches for genome editing in the mouse will be used to generate rationally designed separation-of- function mouse mutants with the goal of revealing novel checkpoint regulatory mechanisms operative in meiosis. To guide and complement genetic and functional experiments, mass spectrometry analysis of testis extracts will be used for quantitative and unbiased characterization of checkpoint signaling in spermatocytes. Collectively, these studies are expected to reveal how meiotic ATR signaling is coordinated to achieve structure-specific signaling outputs in response to unrepaired DSBs or chromosome asynapsis, without inducing cell death. Beyond providing fundamental insights into the actions of genome maintenance pathways that function in virtually all cells, the results from this work will carry important implications related to the molecular origins of infertility and birth defects as well as the impact of ATR inhibition in clinical settings.
Failure of proper genetic quality control during meiosis, the process that generates haploid gametes (sperm and oocytes) for reproduction, frequently results in infertility or birth defects. This proposal utilizes innovative mouse models to resolve how the cellular DNA damage response ensures genetic quality control during meiosis and enables the efficient and accurate production of gametes. The findings from the proposed studies will provide fundamental knowledge about DNA repair and DNA damage signaling mechanisms that have key roles in organismal development, cancer, and reproduction.
