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

June 24th, 2014 by Shibata, E., Dar, A., Dutta, A.

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 CRL4Cdt2 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 CRL4Cdt2, 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 CRL4Cdt2 E3 ubiquitin ligase complex. Compared to forced expression of wild type TDG, CRL4Cdt2- resistant TDG (ΔPIP) slows cell proliferation and slightly increases the toxicity of 5-FU. Thus CRL4Cdt2 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.