During sexual reproduction, the chromosome complement is precisely halved during meiosis to produce haploid gametes. The chromosome repair process called homologous recombination plays essential roles in meiosis. Recombination promotes chromosome pairing and mediates crossing over to create connections between homologous chromosomes that facilitate their accurate segregation. Defects in meiotic recombination are linked to infertility, pregnancy miscarriage, and congenital disease in humans. Although meiotic recombination has been intensively studied, the intermediate steps that include de novo DNA synthesis remain poorly understood. Recombination-associated DNA synthesis (RADS) is intrinsically difficult to characterize in vivo because DNA replication factors are essential for cell viability, and cell cultures lack the synchrony required to cleanly separate DNA synthesis during chromosome replication from that associated with recombination. RADS is predicted to be mechanistically distinct from the DNA synthesis associated with chromosome replication because it is primed by recombination intermediates and involves limited synthesis of single strands of DNA. This study will characterize the nature, function and mechanism of RADS during meiosis.
These aims will be realized using a novel assay system in the budding yeast Saccharomyces cerevisiae that exploits a combination of chemical and real-time genetic tools to allow essential replication factors to be inactivated at will in meiotic yeast cultures synchronized at precisely the point when recombination initiates. This will enable mutational analysis of RADS without perturbing the preceding chromosome replication.
The first aim will characterize the role of RADS in meiotic prophase. The effects of generally inhibiting RADS on the efficiency of chromosome pairing and the DNA events of homologous recombination will be determined.
The second aim will characterize the nature and mechanism of RADS by exploiting conditional alleles of essential replication factors created using the auxin-inducible degron system. Using this system, the components of the replication machinery will be systematically tested to delineate the factors required for meiotic RADS. Implicated factors will then be characterized in detail to elucidate the nature and mechanism of RADS. Together, these experiments will address a key gap in our understanding of chromosome metabolism and move towards a mechanistic understanding of the essential process of RADS. Given the conserved nature of meiotic recombination and the eukaryotic replication machinery, the results will be broadly relevant for understanding human meiosis. The sponsor/co-sponsor team, located in a world-class chromosome biology center, will implement a comprehensive training plan to furnish the applicant with training and skills in the following key areas: confidence and critical thinking through deep knowledge; advanced experimental and analytical skills; presentation and public speaking skills; writing and grantsmanship skills; and nurturing a network of colleagues and collaborators.
Defects in homologous recombination repair during meiosis are associated with infertility, pregnancy miscarriage, and congenital disorders. The proposed research seeks to fill a critical gap in knowledge by characterizing the intermediate DNA synthesis steps of meiotic recombination. Understanding these intermediate steps will improve our understanding of this fundamental process and how it goes awry in human disease. !
| Owens, Shannon; Tang, Shangming; Hunter, Neil (2018) Monitoring Recombination During Meiosis in Budding Yeast. Methods Enzymol 601:275-307 |
