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

在减数分裂前期，同源重组（HR）是由数百个DNA双链断裂（dsb）的形成引发的，导致交叉（CO）的形成，这对于维持染色体相互作用至关重要，直到第一次减数分裂。在小鼠中，只有不到10%的dsb成为COs，但在细胞周期开始之前，这些事件的位置和时间必须精确地协调。关键是CO形成所需的两种异源二聚体的顺序加载：MutSg （MSH4/MSH5）最早加载到约150个dsb上，然后是MutLg (MLH1/MLH3)，它被招募到约23个这些MutSg许可的位点上。PI的实验室在阐明MutSg和MutLg在哺乳动物中指定CO命运中的作用方面一直发挥着核心作用，并且我们最近确定Cyclin n-末端结构域-1 （CNTD1）对于减少MutSg位点和加载MutLg以产生精确的CO数量至关重要。出乎意料的是，我们发现哺乳动物CNTD1并不作为周期蛋白，而是通过招募两个主要复合物来执行其功能。第一个是复制因子C (RFC)，它将两个夹子装载到DNA上（PCNA和9-1-1）。在体细胞中，PCNA激活MLH1，而9-1-1是小鼠减数分裂中必不可少的检查点调节因子。第二个复合体是SKP1-Cullin 1-Fbox复合体（SCF），这是一种泛素E3连接酶，其主要靶点是WEE1，通过磷酸化使CDK1和CDK2失活。CDK1磷酸化通过失活成熟促进因子阻止I中期进展。我们假设CNTD1作为一种监视机制，通过其与RFC的相互作用来监测CO的指定和成熟，然后通过其与SCF的相互作用来促进细胞周期进程，但前提是CO的形成是确定的。本文提出了三个目标：(1)阐明CNTD1调控RFC钳加载器和PCNA/9-1-1钳的选择和使用的机制，以协调I类CO的指定和成熟。我们将阐明RFC、PCNA和其他箝位/箝位加载器组合在促进CNTD1下游CO和检查点控制中的作用。(2)探讨SCF在I类CO调控和细胞周期进程中的cntd1依赖性作用。这方面的研究将确定减数分裂SCF的组成，CNTD1如何通过其在细胞质中的相互作用调节SCF，以及CNTD1/SCF如何调节细胞周期进程。(3)了解CNTD1在哺乳动物减数分裂中的空间和功能需求。计算机分析表明CNTD1的细胞周期蛋白结构域在真核生物中有不同的用途，这表明CNTD1可以以两种不同的方式促进I前期的进展（作为细胞周期蛋白，募集CDKs，或作为RFC和SCF的调节剂）。我们将通过在鼠标中驱动任意一种形式的表达来探索这种双重活动。进一步的研究将调查前期1细胞间CNTD1的分布和运动。这些遗传方法将阐明哺乳动物CNTD1蛋白如何在前期协调CO的指定。

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.