CRL4Cdt2 E3 Ubiquitin Ligase and PCNA Cooperate to Degrade Thymine DNA Glycosylase in S-phase.

Thymine DNA Glycosylase (TDG) is an essential enzyme playing multiple roles in base excision repair, transcription regulation and DNA demethylation. TDG mediates the cytotoxicity of anti-cancer chemotherapeutic drug, 5-Fluorouracil (5-FU) by prolonging S-phase, generating DNA strand breaks and inducing DNA damage signalling. During S-phase of the cell cycle TDG is degraded via proteasomal pathway. Here we show that CRL4(Cdt2) E3 ubiquitin ligase promotes ubiquitination and proteasomal degradation of TDG in S-phase in a reaction that is dependent on the interaction of TDG with Proliferating Cell Nuclear Antigen (PCNA). siRNA mediated depletion of PCNA or components of CRL4(Cdt2), specifically Cullin4A/B, or substrate adaptor Cdt2, stabilizes TDG in human cells. Mutations in the PCNA-interacting-peptide (PIP) motif of TDG that disrupt the interaction of TDG with PCNA, or change critical basic residues essential for the action of the PIP-degron, prevent the ubiquitination and degradation of TDG. Thus physical interaction of TDG with PCNA through the PIP-degron is required for targeting TDG to CRL4(Cdt2) E3 ubiquitin ligase complex. Compared to forced expression of wild type TDG, CRL4(Cdt2)- resistant TDG (ΔPIP) slows cell proliferation and slightly increases the toxicity of 5-FU. Thus CRL4(Cdt2) dependent degradation of TDG occurs in S phase because of the requirement for TDG to interact with chromatin-loaded PCNA and this degradation is important for preventing toxicity from excess TDG.
J. Biol. Chem. Jun. 24, 2014; 0(0); [PUBMED:24962565]
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