Direct Role for Proliferating Cell Nuclear Antigen in Substrate Recognition by the E3 Ubiquitin Ligase CRL4Cdt2

Abstract

Background: CRL4Cdt2 requires that a substrate bind to proliferating cell nuclear antigen (PCNA) on DNA prior to ligase recruitment, but the precise role of PCNA is unclear. Results: A specific PCNA residue is required for destruction of CRL4 Cdt2 substrates. Conclusion: CRL4Cdt2 recognizes a composite surface composed of PCNA and substrate residues. Significance: This is the first ubiquitin ligase whose substrate recognition requires creation of a bipartite substrate surface.

DDB2 association with PCNA is required for its degradation after UV-induced DNA damage

“…These results are in accordance with the ubiquitination process mediated by CUL4A-DDB1 Ctd2 that occurs via a PCNA-dependent mechanism. 31,32 Thus, p21 may be also targeted for degradation by a CRL4 complex formed by CUL4A-DDB1 DDB2 in a PCNA-dependent manner. The co-localization of DDB2 with PCNA, as well as the direct interaction between these proteins, shown not only after DNA damage but also in non-irradiated cells, suggested a specific biological role for this association.…”

“…32 However, the DDB2-PCNA interaction may have an alternative explanation, attributing to PCNA the role of substrate recognition factor for the E3 ligase CRL4 complex, 31 in a way similar to that occurring for p21 and Cdt1 degradation. 32 In support of this hypothesis, our results have shown that DDB2 mutated in the PIP-box (unable to bind PCNA, Fig.…”

Thymine DNA Glycosylase Is a CRL4Cdt2 Substrate

“…Cdt2 activity also requires residues on PCNA that probably make direct contact with Cdt2 (22,24). Because canonical PCNA-binding proteins such as replicative DNA polymerases lack the TD motif and/or Bϩ4 residues, they are not destroyed.…”

A Novel Function of CRL4Cdt2

“…it possesses the TD motif within the PIP-box, as well as a basic residue at the Bϩ4 position. The alignment also shows the Bϩ4 residue is generally one of a cluster of basic residues, all of which may have interactions with PCNA (52,53). Both p12 and Set8 have similarities in the N-terminal part of the PIP-box where they lack the conserved glutamine ( Fig.…”

“…Similar types of mutants have been used to establish the importance of the TD sequence and Bϩ4 basic residue in defining the degron determinants in Cdt1 and p21 (32,51,52). Mutation of all three basic residues was performed as it has been observed that all three are required for recruitment of CRL4 Cdt2 (52,53). The effects of UV on the degradation of the p12 TD/AA (Fig.…”