Regulation of MutL-gamma function in mediating crossing over in mammalian meiosis

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During prophase I of meiosis, homologous recombination (HR) is initiated by the formation of hundreds of DNA double strand breaks (DSBs), leading to the formation of crossovers (CO) that are essential for maintaining chromosome interactions until the first meiotic division. In mouse, less than 10% of DSBs become COs, but the placement and timing of these events must be orchestrated with exact fidelity before the cell cycle can commence. Critical to this is the sequential loading of two heterodimers required for CO formation: MutSg (MSH4/MSH5) loads early onto ~150 DSBs, followed by MutLg (MLH1/MLH3) which is recruited to ~23 of these MutSg-licensed sites. The PI’s lab has been a central player in elucidating the roles of MutSg and MutLg in designating a CO fate in mammals, and we recently determined that Cyclin N-terminal Domain- containing-1 (CNTD1) is essential for paring down MutSg sites and loading MutLg to generate precise CO numbers. Unexpectedly, we find that mammalian CNTD1 does not act as a cyclin, and instead executes its functions by recruiting two major complexes. The first is Replication Factor C (RFC) which loads two clamps onto DNA (PCNA and 9-1-1). In somatic cells, PCNA activates MLH1, while 9-1-1 is an essential checkpoint regulator in mouse meiosis. The second complex is the SKP1-Cullin 1-Fbox complex (SCF), a ubiquitin E3 ligase whose major target is WEE1, which inactivates CDK1 and CDK2 through phosphorylation. Phosphorylation of CDK1 prevents metaphase I progression by inactivating Maturation Promoting Factor. We hypothesize that the CNTD1 acts as a surveillance mechanism to monitor CO designation and maturation through its interactions with RFC, and then to promote cell cycle progression through its interactions with SCF, but only once CO formation is assured. Three aims are proposed: (1) to elucidate the mechanism by which CNTD1 regulates the choice and usage of RFC clamp loaders and the PCNA/9-1-1 clamps to orchestrate class I CO designation and maturation. We will elucidate the role of RFC, PCNA, and other clamp/clamp loader combinations in promoting CO and checkpoint control downstream of CNTD1. (2) To explore the CNTD1-dependent role of the SCF in class I CO regulation and cell cycle progression. Studies in this aim will determine the components of the meiotic SCF, how CNTD1 regulates SCF through its interactions in the cytoplasm, and how CNTD1/SCF regulates cell cycle progression. (3) To understand the spatial and functional requirements for CNTD1 in mammalian meiosis. In silico analysis suggests variable use of the cyclin domain of CNTD1 across eukaryotes, suggesting that CNTD1 can function in two distinct ways to facilitate prophase I progression (as a cyclin, recruiting CDKs, or as a regulator of RFC and SCF). We will explore this dual activity by driving expression of either form in the mouse. Additional studies will investigate the distribution and movement of CNTD1 between cellular compartments during prophase I. These genetic approaches will elucidate how the mammalian CNTD1 protein functions to orchestrate CO designation during prophase.