Abstract
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 the anti-cancer chemotherapeutic drug 5-fluorouracil (5-FU) by prolonging S phase, generating DNA strand breaks, and inducing DNA damage signaling. During S phase of the cell cycle, TDG is degraded via the 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 the CRL4(Cdt2) E3 ubiquitin ligase complex. Compared with 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.
Highlights
TDG is degraded in S phase via the proteasomal pathway
We demonstrate that the CRL4Cdt2 E3 ubiquitin ligase complex ubiquitinates and degrades TDG in a PCNAand PCNA-interacting peptide (PIP) degron-dependent manner
We confirmed by immunofluorescence that the amount of TDG protein is selectively diminished in S phase cells that can be distinguished by the appearance of foci of proliferating cell nuclear antigen (PCNA) in the nucleus (Fig. 1B, top)
Summary
TDG is degraded in S phase via the proteasomal pathway. Results: CRL4Cdt ubiquitinates and targets TDG associated with PCNA for degradation. We show that CRL4Cdt 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). The CRL4Cdt ubiquitin ligase uses Cdt as the DCAF and has been implicated in the polyubiquitination and degradation of several substrates involved in S phase regulation, in a reaction that is dependent on PCNA [17, 18]. The target substrates of the CRL4Cdt E3 ubiquitin ligase complex are known to have a PIP degron comprising a consensus PCNA-interacting protein motif (PIP motif) with additional sequence requirements [17, 18, 27, 28]. Because the association of PCNA with chromatin increases in S phase, the requirement that the substrate interact with PCNA before recognition by CRL4Cdt makes this an S phase-specific pathway of protein degradation. An excess of wild type TDG slows down cell proliferation, as does CRL4Cdt2-resistant TDG(⌬PIP), indicating that the active degradation of TDG by this pathway is critical for keeping cellular levels of the protein below toxic levels
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
