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J Biol Chem. 2014 Aug 15;289(33):23056-64. doi: 10.1074/jbc.M114.574210. Epub 2014 Jun 24.

CRL4Cdt2 E3 ubiquitin ligase and proliferating cell nuclear antigen (PCNA) cooperate to degrade thymine DNA glycosylase in S phase.

Author information

1
From the Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908.
2
From the Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908 ad8q@virginia.edu.

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.

KEYWORDS:

DNA Damage Response; E3 Ubiquitin Ligase; Proliferating Cell Nuclear Antigen (PCNA); Protein Degradation; Thymine-DNA Glycosylase (TDG); Ubiquitin

PMID:
24962565
PMCID:
PMC4132804
DOI:
10.1074/jbc.M114.574210
[Indexed for MEDLINE]
Free PMC Article
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