Summary/Abstract Homologous recombination is a chromosome repair process that plays essential roles in meiosis, the specialized cell division that produces gametes. Defects in meiotic recombination are a leading cause of infertility, pregnancy loss and congenital disease in humans. A major gap in our understanding of meiotic recombination is the mechanism of the recombination-associated DNA synthesis (RADS) that is essential to restore chromosome integrity. This knowledge gap persists because of inherent challenges to studying meiotic RADS in vivo, in particular the essential nature of DNA replication factors, the need to study RADS in isolation from chromosomal replication, and the requirement for special assays to measure RADS. These hurdles have now been overcome using an innovative combination of chemical, real-time and molecular genetics tools in budding yeast that enable acute inactivation of essential replication factors specifically during recombination, and measurement of de novo DNA synthesis. This system utilizes an ATP-analog sensitive allele of the Cdc7 kinase (cdc7-as3) to synchronize cells after S-phase, but before recombination is initiated. Real-time inactivation of essential replication factors is achieved using the auxin-inducible degron (AID) system, which has been rewired and optimized for use in meiotic cells. To monitor RADS, newly synthesized DNA is labeled, isolated and quantified using 5-ethynyl-2′-deoxyuridine (Edu) incorporation, biotin-azide click chemistry, streptavidin purification and quantitative PCR (qPCR). Exploiting these tools, the long-term objectives of this project are to understand the nature, function, mechanism and regulation of RADS. These objectives will be pursued through three aims. Aim 1 will determine the role of RADS for both the DNA events of meiotic recombination and the chromosomal events of meiotic prophase using the comprehensive battery of molecular, genetic and cytological assays uniquely available in budding yeast. Aim 2 will test models of RADS by delineating the replication factors involved and systematically analyzing their roles. Complementary studies in mouse will analyze the localization and dynamics of replication factors at sites of recombination. Aim 3 will identify and characterize factors involved in the recruitment of replication factors to recombination sites and the regulation of RADS. Immunofluorescence cytology will be used to monitor chromosomal dynamics of replication factors and determine the genetic requirements for their localization. The timing and extent of RADS will be analyzed in strains mutant for factors predicted to modulate RADS, including meiosis-specific recombination proteins, DNA helicases and topoisomerases. The results of these aims will provide unprecedented insights into the mechanism and regulation of RADS, filling a major gap in our understanding of meiotic recombination. These findings will be germane to und...