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A mechanism for sequential replication‐coupled destruction of CRL4‐Cdt2 substrates (616.1)
Author(s) -
Coleman Kate,
Varma Dileep,
Brantley Kristen,
Shibata Etsuko,
Dutta Anindya,
Cook Jean
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.616.1
Subject(s) - degron , dna replication factor cdt1 , proliferating cell nuclear antigen , biology , microbiology and biotechnology , dna replication , cell cycle , ubiquitin , dna repair , ubiquitin ligase , dna mismatch repair , cancer research , dna , genetics , cell , gene , eukaryotic dna replication
The CRL4Cdt2 ubiquitin E3 ligase targets a cohort of key cell cycle regulators during both S phase and DNA repair synthesis. These substrates include the p21 CDK inhibitor, the Cdt1 origin licensing protein, and the Set8 lysine methyltransferase. The intracellular concentrations of each of these proteins has profound consequences for cell proliferation and genome stability. CRL4‐Cdt2 only targets substrates if they are bound to DNA‐loaded PCNA, coupling substrate destruction to DNA synthesis. PCNA loading does not trigger simultaneous destruction of all CRL4‐Cdt2 substrates however; Cdt1 degradation begins nearly instantaneously with PCNA loading whereas p21 degradation is significantly delayed for example. As a consequence, the substrates of CRL4‐Cdt2 are destroyed in a stereotypical order, and we have observed this order in multiple cell lines during both DNA repair synthesis and in early S phase. To identify the molecular determinants of delayed vs. immediate destruction, we generated a series of fusion proteins between Cdt1 and p21. We demonstrate that fusing just the Cdt1 PCNA‐interacting motif, a.k.a. “PIP degron,” to the p21 N‐terminus is sufficient to confer rapid rather than delayed destruction. It is not the N‐terminal location, the presence of a second PIP box, or E2 utilization, but rather the degron sequence itself that defines the order of destruction. Surprisingly, rapid vs. delayed destruction is not due to degron features previously shown to be critical for either PCNA or CRL4‐Cdt2 interaction. We generated cell lines that destroy p21 according to the Cdt1 pattern (rapid) rather than the normal p21 pattern (delayed), and we will show the effects of this conversion on DNA replication parameters during S phase and on DNA damage checkpoint responses. This study sheds light on how the substrates of a single ubiquitin E3 ligase are targeted sequentially rather than simultaneously. Grant Funding Source : Supported by NIH GM083024 to J.G.C. and NIH F31 CA165891 to K.C.