Academic literature on the topic 'CRL4Cdt2'

In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for each mitotic cell division. A key regulatory mechanism by which DNA re-replication is suppressed is the S phase-dependent proteolysis of Cdt1, an essential replication protein for licensing DNA replication origins by loading the Mcm2-7 replication helicase for DNA duplication in S phase. Cdt1 degradation is mediated by CRL4Cdt2 ubiquitin E3 ligase, which further requires Cdt1 binding to proliferating cell nuclear antigen (PCNA) through a PIP box domain in Cdt1 during DNA synthesis. Recent studies found that Cdt2, the specific subunit of CRL4Cdt2 ubiquitin E3 ligase that targets Cdt1 for degradation, also contains an evolutionarily conserved PIP box-like domain that mediates the interaction with PCNA. These findings suggest that the initiation and elongation of DNA replication or DNA damage-induced repair synthesis provide a novel mechanism by which Cdt1 and CRL4Cdt2 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4Cdt2. The proximity of PCNA-bound Cdt1 to CRL4Cdt2 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4Cdt2 ubiquitin E3 ligase may also regulate the degradation of other PIP box-containing proteins, such as CDK inhibitor p21 and histone methylase Set8, to regulate DNA replication licensing, cell cycle progression, DNA repair, and genome stability by directly interacting with PCNA during DNA replication and repair synthesis.

DNA synthesis–coupled proteolysis of the prereplicative complex component Cdt1 by the CRL4Cdt2 E3 ubiquitin ligase is thought to help prevent rereplication of the genome during S phase. To directly test whether CRL4Cdt2-triggered destruction of Cdt1 is required for normal cell cycle progression in vivo, we expressed a mutant version of Drosophila Cdt1 (Dup), which lacks the PCNA-binding PIP box (DupΔPIP) and which cannot be regulated by CRL4Cdt2. DupΔPIP is inappropriately stabilized during S phase and causes developmental defects when ectopically expressed. DupΔPIP restores DNA synthesis to dup null mutant embryonic epidermal cells, but S phase is abnormal, and these cells do not progress into mitosis. In contrast, DupΔPIP accumulation during S phase did not adversely affect progression through follicle cell endocycles in the ovary. In this tissue the combination of DupΔPIP expression and a 50% reduction in Geminin gene dose resulted in egg chamber degeneration. We could not detect Dup hyperaccumulation using mutations in the CRL4Cdt2 components Cul4 and Ddb1, likely because these cause pleiotropic effects that block cell proliferation. These data indicate that PIP box–mediated destruction of Dup is necessary for the cell division cycle and suggest that Geminin inhibition can restrain DupΔPIP activity in some endocycling cell types.

The CRL4Cdt2 ubiquitin ligase complex is an essential regulator of cell-cycle progression and genome stability, ubiquitinating substrates such as p21, Set8, and Cdt1, via a display of substrate degrons on proliferating cell nuclear antigens (PCNAs). Here, we examine the hierarchy of the ligase and substrate recruitment kinetics onto PCNA at sites of DNA replication. We demonstrate that the C-terminal end of Cdt2 bears a PCNA interaction protein motif (PIP box, Cdt2PIP), which is necessary and sufficient for the binding of Cdt2 to PCNA. Cdt2PIP binds PCNA directly with high affinity, two orders of magnitude tighter than the PIP box of Cdt1. X-ray crystallographic structures of PCNA bound to Cdt2PIP and Cdt1PIP show that the peptides occupy all three binding sites of the trimeric PCNA ring. Mutating Cdt2PIP weakens the interaction with PCNA, rendering CRL4Cdt2 less effective in Cdt1 ubiquitination and leading to defects in Cdt1 degradation. The molecular mechanism we present suggests a new paradigm for bringing substrates to the CRL4-type ligase, where the substrate receptor and substrates bind to a common multivalent docking platform to enable subsequent ubiquitination.

La protéine PCNA est un facteur d'échafaudage polyvalent pour plus de cinquante protéines impliquées dans le métabolisme d'ADN, notamment dans la réplication et la réparation. Comment les échanges entre les partenaires de PCNA sont régulés est actuellement mal compris. Parmi ses partenaires, CDT1, p21 et PR-Set7/Set8 possèdent un motif d'interaction avec PCNA particulier, nommé « PIP degron », qui favorise leur protéolyse d'une manière dépendante de l'E3 ubiquitine ligase CRL4Cdt2. Après irradiation aux UV-C, le facteur d'initiation de la réplication CDT1 est rapidement détruit d'une manière dépendante de son PIP degron, mais le rôle de cette dégradation est inconnu. Dans cette étude, j'ai analysé la fonction du PIP degron de CDT1 et fourni des évidences expérimentales qui montrent que l'inhibition de la dégradation de Cdt1 par CRL4Cdt2 dans les cellules de mammifères compromet la relocalisation de l'ADN polymérase translesionnelle eta en foyers nucléaires induits par les irradiations UV-C. En élargissant cette étude à d'autres partenaires de PCNA, nous avons constaté que seuls les protéines qui contiennent un PIP degron, et pas un PIP box canonique comme celui de FEN1 et p15 (PAF), interfèrent avec la formation de foyers de pol eta. La mutagenèse du PIP degron de CDT1 a révélé qu'un résidu de thréonine conservé parmi les PIP degrons est essentiel pour l'inhibition de la formation des foyers de pol eta. Les résultats obtenus suggèrent que l'élimination de protéines contenant des PIP degrons par la voie CRL4Cdt2 régule le recrutement de pol eta au niveau des sites de dommages induits par les UV-C<br>The sliding clamp PCNA is a versatile scaffold for more than fifty proteins involved in DNA metabolism such as replication and repair. How the switch between PCNA partners is regulated is currently not fully understood. Among its partners, Cdt1, p21 and PR-Set7/Set8 contain a specialized PCNA-binding motif named « PIP degron » that promotes their proteolysis in a fashion dependent on the E3 ubiquitin ligase CRL4Cdt2. Upon UV-irradiation, the replication initiation factor Cdt1 is rapidly destroyed in a PIP degron-dependent manner but the role of this degradation is unknown. Here we have analyzed the function of Cdt1 PIP degron and we provide evidence that interference with CRL4Cdt2-mediated destruction of Cdt1 in mammalian cells compromises PCNA-dependent relocalisation of the DNA translesion polymerase eta into UV-induced nuclear foci. By extending this analysis to other PCNA partners, we found that only PIP degrons, as compared to canonical PCNA-binding motifs of Fen1 and p15(PAF), interfere with pol eta focus formation. Mutagenesis of Cdt1 PIP degron revealed that a threonine residue conserved in PIP degrons is critical for inhibition of pol eta focus formation. Our results suggest that removal of high-affinity PIP degron-containing proteins from PCNA by CRL4Cdt2 pathway regulates pol eta recruitment to sites of UV-damage

PCNA est une protéine essentielle qui intervient dans de nombreux mécanismes cellulaires et qui possède de nombreuses modifications post-traductionnelles (MPTs) dont les fonctions de certaines, restent encore inconnues. Afin d’étudier la fonction de ces MPTs, nous avons développé un nouvel outil génétique permettant in cellulo, de substituer la protéine endogène PCNA par une version mutée de la protéine appelée version de complémentation. La technique consiste à cotransfecter des cellules en culture avec deux types de plasmides. Un premier plasmide permet l’invalidation du gène de PCNA endogène dans les cellules transfectées par le système CRISPR-Cas9. Le deuxième plasmide dit de complémentation permet l’expression d’une forme mutée de PCNA. Sur l’ensemble d’une banque de mutants testés, deux mutants de PCNA se sont avérés être létaux (D122A et E124A). Nous avons démontré que ces deux sites sont impliqués dans l’initiation d’une voie de dégradation ubiquitine dépendante CRL4Cdt2 essentielle pour la mise en place de la protéolyse d’un cocktail de protéines (cdt1, p21, set8) durant la phase S. Nous avons démontré que les cellules mutantes pour PCNA (D122A et E124A) accumulent la protéine p21. Ce défaut de dégradation de p21 provoque alors des évènements de re-réplication menant à terme à la mort des cellules mutantes<br>PCNA is an essential protein that is involved in many cellular mechanisms and has many post-translational modifications (PTMs). The functions of some PTMs, still remain unknown. In order to study the function of these PTMs, we have developed a new genetic tool allowing, in cellulo, the substitution of endogenous PCNA protein with a mutated version of the protein named complementation version. The technique involves cotransfection of the cells in culture with two types of plasmids. A first plasmid allows invalidation of the endogenous PCNA gene in transfected cells by the CRISPR-Cas9 system. The second plasmid, named complementation plasmid allows the expression of a mutated form of PCNA. In the whole bank of tested mutants, two PCNA mutants were found to be lethal (D122A and E124A). We have demonstrated that these two sites are involved in the initiation of an ubiquitin-dependent protein degradation CRL4Cdt2 pathway essential for the proteolysis of a protein cocktail (cdt1, p21, set8) during the S phase. We demonstrated that PCNA mutant cells (D122A and E124A) accumulate p21 protein. This lack of degradation of p21 then causes re-replication events leading ultimately to the mutant cells death