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

MutL-gamma 功能在介导哺乳动物减数分裂中交叉的调节

• 批准号: 10626923
• 负责人: Paula Elaine Cohen
• 金额: $ 43.6万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10626923
• 关键词: Automobile Driving; Binding; CDC2 gene; CDK2 gene; CUL1 gene; Cell Cycle; Cell Cycle Progression; Cell Nucleus; Chromosomes; Closure by clamp; Complex; Cullin Proteins; Cyclin B; Cyclins; Cytoplasm; DNA; DNA Double Strand Break; DNA Sequence; Distant; Ensure; Enzymes; Eukaryota; Event; Experimental Genetics; F-Box Proteins; Failure; Family; Family member; Frequencies; Genetic Crossing Over; Genome; Heart; Licensing; MLH1 gene; MSH4 gene; Mammals; Maturation-Promoting Factor; Mediating; Meiosis; Meiotic Prophase I; Metaphase; Mismatch Repair; Monitor; Movement; Mus; N-terminal; Pathway interactions; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Process; Prophase; Proteins; Regulation; Role; Sexual Reproduction; Site; Somatic Cell; Transcript; Variant; Zygonema; egg; experimental study; genetic approach; homologous recombination; in silico; member; novel; prevent; protein function; recruit; repaired; replication factor C; sperm cell; success; ubiquitin-protein ligase

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.

在减数分裂的前期I，同源重组（HR）由数百个DNA双链断裂（DSB）的形成启动，导致形成对于维持染色体相互作用至关重要的交换（CO），直到第一次减数分裂。在小鼠中，不到10%的DSB成为CO，但这些事件的位置和时间必须在细胞周期开始之前精确地协调。关键在于CO形成所需的两个异二聚体的顺序加载：MutSg（MSH 4/MSH 5）早期加载到约150个DSB上，然后是MutLg（MLH 1/MLH 3），其被募集到这些MutSg许可的位点中的约23个。PI的实验室一直是阐明MutSg和MutLg在哺乳动物中指定CO命运中的作用的核心参与者，我们最近确定了细胞周期蛋白N末端结构域-1（CNTD 1）对于削减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CNTD 1不作为细胞周期蛋白，而是通过招募两个主要的复合物来执行其功能。第一种是复制因子C（RFC），它将两个夹子（PCNA和9-1-1）加载到DNA上。在体细胞中，PCNA激活MLH 1，而9-1-1是小鼠减数分裂中的重要检查点调节剂。第二种复合物是SKP 1-Cullin 1-Fbox复合物（SCF），一种泛素E3连接酶，其主要靶标是WEE 1，通过磷酸化使CDK 1和CDK 2失活。CDK 1的磷酸化通过使成熟促进因子失活来阻止中期I进程。我们假设CNTD 1作为一种监测机制，通过与RFC的相互作用来监测CO的指定和成熟，然后通过与SCF的相互作用来促进细胞周期的进展，但只有在CO形成得到保证的情况下。提出了三个目标：（1）阐明CNTD 1调节RFC夹装载器和PCNA/9-1-1夹的选择和使用以协调I类CO命名和成熟的机制。我们将阐明RFC，PCNA，和其他钳/钳加载器组合在促进CO和CNTD 1下游的检查点控制的作用。(2)探讨CNTD 1依赖的SCF在I类CO调节和细胞周期进程中的作用。这一目标的研究将确定减数分裂SCF的组成部分，CNTD 1如何通过其在细胞质中的相互作用调节SCF，以及CNTD 1/SCF如何调节细胞周期进程。(3)了解哺乳动物减数分裂中CNTD 1的空间和功能需求。计算机模拟分析表明，CNTD 1的细胞周期蛋白结构域在真核生物中的可变用途，表明CNTD 1可以以两种不同的方式发挥作用，以促进前期I进展（作为细胞周期蛋白，招募CDK，或作为RFC和SCF的调节剂）。我们将通过驱动小鼠中任一形式的表达来探索这种双重活性。进一步的研究将调查在前期I期间CNTD 1在细胞区室之间的分布和运动。这些遗传学方法将阐明哺乳动物CNTD 1蛋白如何在前期协调CO指定的功能。

项目成果

MUS81 generates a subset of MLH1-MLH3-independent crossovers in mammalian meiosis.

• DOI: 10.1371/journal.pgen.1000186
• 发表时间: 2008-09-12
• 期刊: PLoS genetics
• 影响因子: 4.5
• 作者: Holloway JK;Booth J;Edelmann W;McGowan CH;Cohen PE
• 通讯作者: Cohen PE

Mammalian BTBD12 (SLX4) protects against genomic instability during mammalian spermatogenesis.

• DOI: 10.1371/journal.pgen.1002094
• 发表时间: 2011-06
• 期刊: PLoS genetics
• 影响因子: 4.5
• 作者: Holloway JK;Mohan S;Balmus G;Sun X;Modzelewski A;Borst PL;Freire R;Weiss RS;Cohen PE
• 通讯作者: Cohen PE

Conditional inactivation of the DNA damage response gene Hus1 in mouse testis reveals separable roles for components of the RAD9-RAD1-HUS1 complex in meiotic chromosome maintenance.

• DOI: 10.1371/journal.pgen.1003320
• 发表时间: 2013
• 期刊: PLoS genetics
• 影响因子: 4.5
• 作者: Lyndaker AM;Lim PX;Mleczko JM;Diggins CE;Holloway JK;Holmes RJ;Kan R;Schlafer DH;Freire R;Cohen PE;Weiss RS
• 通讯作者: Weiss RS

Mismatch repair proteins, meiosis, and mice: understanding the complexities of mammalian meiosis.

• DOI: 10.1016/j.yexcr.2004.03.020
• 发表时间: 2004-05
• 期刊: Experimental cell research
• 影响因子: 3.7
• 作者: A. Svetlanov;P. Cohen

Phosphoproteomics of ATR signaling in mouse testes.

• DOI: 10.7554/elife.68648
• 发表时间: 2022-02-08
• 期刊: eLife
• 影响因子: 7.7
• 作者: Sims JR;Faça VM;Pereira C;Ascenção C;Comstock W;Badar J;Arroyo-Martinez GA;Freire R;Cohen PE;Weiss RS;Smolka MB
• 通讯作者: Smolka MB