No abstract

Crossover recombination is essential for accurate chromosome segregation during meiosis. The MutSγ-complex, Msh4-Msh5, facilitates crossing over by binding and stabilizing nascent recombination intermediates. We show that these activities are governed by regulated proteolysis. MutSγ is initially inactive for crossing over due to an N-terminal degron on Msh4 that renders it unstable by directly targeting proteasomal degradation. Activation of MutSγ requires the Dbf4dependent kinase, Cdc7 (DDK), which directly phosphorylates and thereby neutralizes the Msh4 degron. Genetic requirements for Msh4 phosphorylation indicate that DDK targets MutSγ only after it has bound to nascent JMs in the context of synapsing chromosomes. Overexpression studies confirm that the steady-state level of Msh4, not phosphorylation perse, is the critical determinant for crossing over. At the DNA level, Msh4 phosphorylation enables the formation and crossover-biased resolution of double-Holliday Junction intermediates. Our study establishes regulated protein degradation as a fundamental mechanism underlying meiotic crossing over.

No abstract

The cardiovascular disease of atherosclerosis is characterised by aged vascular smooth muscle cells and compromised cell survival. Analysis of human and murine plaques highlights markers of DNA damage such as p53, Ataxia telangiectasia mutated (ATM), and defects in mitochondrial oxidative metabolism as significant observations. The antiageing protein Klotho could prolong VSMC survival in the atherosclerotic plaque and delay the consequences of plaque rupture by improving VSMC phenotype to delay heart attacks and stroke. Comparing wild-type VSMCs from an ApoE model of atherosclerosis with a flox’d Pink1 knockout of inducible mitochondrial dysfunction we show WT Pink1 is essential for normal cell viability, while Klotho mediates energetic switching which may preserve cell survival. Methods: Wild-type ApoE VSMCs were screened to identify potential drug candidates that could improve longevity without inducing cytotoxicity. The central regulator of cell metabolism AMP Kinase was used as a readout of energy homeostasis. Functional energetic switching between oxidative and glycolytic metabolism was assessed using XF24 technology. Live cell imaging was then used as a functional readout for the WT drug response, compared with Pink1 (phosphatase-and-tensin-homolog (PTEN)-induced kinase-1) knockout cells. Results: Candidate drugs were assessed to induce pACC, pAMPK, and pLKB1 before selecting Klotho for its improved ability to perform energetic switching. Klotho mediated an inverse dose-dependent effect and was able to switch between oxidative and glycolytic metabolism. Klotho mediated improved glycolytic energetics in wild-type cells which were not present in Pink1 knockout cells that model mitochondrial dysfunction. Klotho improved WT cell survival and migration, increasing proliferation and decreasing necrosis independent of effects on apoptosis. Conclusions: Klotho plays an important role in VSMC energetics which requires Pink1 to mediate energetic switching between oxidative and glycolytic metabolism. Klotho improved VSMC phenotype and, if targeted to the plaque early in the disease, could be a useful strategy to delay the effects of plaque ageing and improve VSMC survival.

The MutSγ complex, Msh4-Msh5, binds DNA joint-molecule (JM) intermediates during homologous recombination to promote crossing over and accurate chromosome segregation at the first division of meiosis. MutSγ facilitates the formation and biased resolution of crossoverspecific JM intermediates called double Holliday junctions. Here we show that these activities are governed by regulated proteasomal degradation. MutSγ is initially inactive for crossing over due to an N-terminal degron on Msh4 that renders it unstable. Activation of MutSγ requires the Dbf4-dependent kinase, Cdc7 (DDK), which directly phosphorylates and thereby neutralizes the Msh4 degron. Phosphorylated Msh4 is chromatin bound and requires DNA strand exchange and chromosome synapsis, implying that DDK specifically targets MutSγ that has already bound nascent JMs. Our study establishes regulated protein degradation as a fundamental mechanism underlying meiotic crossover control. the differentiation of crossover sites manifests as the selective retention and accumulation of specific recombination factors. One such factor is MutSγ, a heterodimer of Msh4 and Msh5, two homologs of the DNA mismatch-recognition factor MutS (Manhart and Alani, 2016;Snowden et al., 2004). Msh4 and Msh5 are members the ZMM proteins (Zip1, Zip2, Zip3, Zip4, Msh4, Msh5, Mer3, and Spo16), a diverse set of activities that facilitate crossover-specific events of recombination, and couple these events to chromosome synapsis (Fung et al., 2004;Hunter, 2015;Lynn et al., 2007;Shinohara et al., 2008). As seen in a variety of species, initial numbers of MutSγ immunostaining foci greatly outnumber final crossover numbers (De Muyt et al., 2014; de Vries et al., 1999;Edelmann et al., 1999;Higgins et al., 2008;Kneitz et al., 2000;Yokoo et al., 2012;Zhang et al., 2014). As prophase I progresses, MutSγ is lost from most recombination sites but retained at sites that will go on to mature into crossovers. This patterning process is dependent on the Zip3/RNF212/ZHP-3/HEI10 family of RING E3 ligases (Agarwal and Roeder, Wang et al., 2012;Yokoo et al., 2012;Zhang et al., 2018). Additional evidence implicates the SUMO-modification and ubiquitin-proteasome systems in meiotic crossover control (Ahuja et al., 2017; Rao et al., 2017) and suggests a model in which factors such as MutSγ are selectively stabilized at crossover sites by protecting them from proteolysis. Implicit in this model is the notion that MutSγ may be intrinsically unstable.Here, we show that regulated proteolysis plays a direct and essential role in meiotic crossing over. Msh4 is identified as an intrinsically unstable protein that is targeted for proteasomal degradation by an N-terminal degron thereby rendering MutSγ inactive for crossing over. Activation of MutSγ occurs by neutralizing the Msh4 degron via Cdc7-catalyzed phosphorylation. Thus, a key meiotic pro-crossover factor is activated by attenuating its proteolysis.