Summary F-box proteins are the substrate binding subunits of SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complexes. Using affinity purifications and mass spectrometry, we identified RRM2 (the ribonucleotide reductase family member 2) as a new interactor of the F-box protein Cyclin F. Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides (dNTPs), which are necessary for both replicative and repair DNA synthesis. We found that, during G2, following CDK-mediated phosphorylation of Thr33, RRM2 is degraded via SCFCyclin F to maintain balanced dNTP pools and genome stability. After DNA damage, Cyclin F is downregulated in an ATR-dependent manner to allow accumulation of RRM2. Defective elimination of Cyclin F delays DNA repair and sensitizes cells to DNA damage, a phenotype that is reverted by expressing a non-degradable RRM2 mutant. In summary, we have identified a novel biochemical pathway that controls the abundance of dNTPs and ensures efficient DNA repair in response to genotoxic stress.
Generally, F-box proteins are the substrate recognition subunits of SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complexes, which mediate the timely proteolysis of important eukaryotic regulatory proteins1,2. Mammalian genomes encode roughly 70 F-box proteins, but only a handful have established functions3,4. The F-box protein family obtained its name from Cyclin F (also called Fbxo1), in which the F-box motif (the ~40 amino acid domain required for binding to Skp1) was first described5. Cyclin F, which is encoded by an essential gene, also contains a cyclin box domain, but in contrast to most cyclins, it does not bind or activate any cyclin-dependent kinases (CDKs)5–7. However, like other cyclins, Cyclin F oscillates during the cell cycle, with protein levels peaking in G2. Despite its essential nature and status as the founding member of the F-box protein family, Cyclin F remains an orphan protein, whose functions are unknown. Starting from an unbiased screen, we identified CP110, a protein essential for centrosome duplication, as an interactor and substrate of Cyclin F. Utilizing a mode of substrate binding distinct from other F-box protein-substrate pairs, CP110 and Cyclin F physically associate on the centrioles during the G2 phase of the cell cycle, and CP110 is ubiquitylated via the SCFCyclin F ubiquitin ligase complex, leading to its degradation. siRNA-mediated depletion of Cyclin F in G2 induces centrosomal and mitotic abnormalities, such as multipolar spindles and asymmetric, bipolar spindles with lagging chromosomes. These phenotypes were reverted by co-silencing CP110 and were recapitulated by expressing a stable mutant of CP110 that is unable to bind Cyclin F. Finally, expression of a stable CP110 mutant in cultured cells also promotes the formation of micronuclei, a hallmark of chromosome instability. We propose that SCFCyclin F–mediated degradation of CP110 is required for the fidelity of mitosis and genome integrity.
Self-renewing naïve mouse embryonic stem cells (mESCs) contain few mitochondria, which increase in number and volume at the onset of differentiation. KBP (encoded by Kif1bp) is an interactor of the mitochondrial-associated kinesin Kif1Bα. We found that TDH, responsible for mitochondrial production of acetylCoA in mESCs, and the acetyl-transferase GCN5L1 cooperate to acetylate Lys501 in KBP, allowing its recognition by and degradation via Fbxo15, an F-box protein transcriptionally controlled by the pluripotency core factors and repressed upon differentiation. Defects in KBP degradation in mESCs result in unscheduled increase in mitochondrial biogenesis, enhanced respiration and ROS production, and inhibition of cell proliferation. Silencing of Kif1Bα reverts the aberrant increase in mitochondria induced by KBP stabilization. Notably, upon differentiation, Kif1bp−/− mESCs display impaired expansion of the mitochondrial mass and form smaller embryoid bodies. Thus, KBP proteolysis limits the accumulation of mitochondria in mESCs to preserve their optimal fitness, whereas KBP accumulation promotes mitochondrial biogenesis in differentiating cells.
ABSTRACT. Sugarcane culture is an important source of income for the Brazilian economy. The aim of this study was to identify somaclonal variation in sugarcane varieties RB943365 and RB92579 arising from micropropagation using inter-simple sequence repeat (ISSR) DNA markers. The evaluated plants were generated from the in vitro propagation of shoot tips grown in MS medium supplemented with vitamins, myoinositol, glycine, and sucrose, without the use of growth regulators. Fifteen consecutive subcultures with intervals of 14 days were carried out, and DNA was extracted from young leaves obtained from each of the subcultures. The DNA was amplified with ISSR markers and separated by electrophoresis on 2% agarose gels. No evidence of polymorphism was observed in subcultures of the varieties analyzed, suggesting the absence of somaclonal variants. In this way, the ISSR marker was efficient at analyzing somaclonal variation, and in vitro propagation of sugarcane can be considered efficient for 15 consecutive subcultures of the varieties analyzed.
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