Mechanism of CRL4(Cdt2), a PCNA-dependent E3 ubiquitin ligase

Abstract

Eukaryotic cell cycle transitions are driven by E3 ubiquitin ligases that catalyze the ubiquitylation and destruction of specific protein targets. For example, the anaphase-promoting complex/cyclosome (APC/C) promotes the exit from mitosis via destruction of securin and mitotic cyclins, whereas CRL1(Skp2) allows entry into S phase by targeting the destruction of the cyclin-dependent kinase (CDK) inhibitor p27. Recently, an E3 ubiquitin ligase called CRL4(Cdt2) has been characterized, which couples proteolysis to DNA synthesis via an unusual mechanism that involves display of substrate degrons on the DNA polymerase processivity factor PCNA. Through its destruction of Cdt1, p21, and Set8, CRL4(Cdt2) has emerged as a master regulator that prevents rereplication in S phase. In addition, it also targets other factors such as E2F and DNA polymerase η. In this review, we discuss our current understanding of the molecular mechanism of substrate recognition by CRL4(Cdt2) and how this E3 ligase helps to maintain genome integrity.

Figure 1.

Models of CRL1^Fbw7 and CRL4^Ddb2 structures. (A) A ribbon diagram model depicting the CRL1^Fbw7 ubiquitin ligase and its substrate, Cyclin E, was generated using PyMOL (http://www.pymol.org). Cul1 N terminus (slate), Cul1 C terminus (slate), and Rbx1 (purple) are from Zheng et al. (2002) (Protein Data Bank [PDB] accession no. 1LDK). Skp1 (light blue), Fbw7 (dark blue), and the substrate, Cyclin E C-terminal degron (red), are from Hao et al. (2007) (PDB 2OVQ). (B) A ribbon diagram model depicting CRL4^Ddb2 bound to abasic DNA was generated using PyMOL. Cul4a (slate) and Rbx1 (purple) are from Angers et al. (2006) (PDB 2HYE) and aligned to the Cul1 N terminus (PDB 1LDK). Ddb1 (light blue), zebrafish Ddb2 (dark blue), and 16 base pairs of dsDNA containing an Abasic site (red) are from Scrima et al. (2008) (PDB 3EI2) and aligned to BPB (β propeller B) of Ddb1 (PDB 2HYE).

Figure 2.

CRL4^Cdt2 degrons. (A) PIP box and PIP degron consenuses. Canonical PIP box residues are shown in violet, and PIP degron-specific residues are shown in blue. The TD motif promotes high-affinity binding to PCNA, whereas the basic residue four amino acids downstream from the PIP box is required for docking of CRL4^Cdt2 onto the PCNA–PIP degron complex. (B) The Cdt1 PIP degron sequence from various species. In all cases, the degron is at the extreme N terminus. Same color scheme as in A. (C) C-terminal and internal PIP degrons of various other substrates. Confirmed substrates are in bold. Asterisk denotes the C terminus.

Figure 3.

The role of CRL4^Cdt2 in preventing rereplication. Origins of replication are primed for initiation in the G1 phase of the cell cycle via the ordered loading of ORC, Cdc6, Cdt1, and the MCM2–7 helicase (licensing), a process that requires histone H4 methylation on Lys 20 by Set8. In S phase, the MCM2–7 helicase is activated by Cdk2 and many additional factors, whereupon origins are unwound and two replisomes are assembled. Each replisome includes the MCM2–7 helicase, leading (pol ɛ) and lagging (pol δ) DNA polymerases, and the processivity factor PCNA. As replication proceeds, MCM2–7 travels away from the origin. Reinitiation is inhibited because once cells are in S phase, new MCM2–7 recruitment is not allowed, largely due to CRL4^Cdt2, whose activity is coupled to chromatin-bound PCNA (green arrow). Thus, CRL4^Cdt2 marks the licensing factors Cdt1 and Set8 for destruction (red arrows). In addition, it destabilizes p21, an inhibitor of Cdk2. Cdk2 activity in S phase promotes replication initiation but also phosphorylates Cdc6, leading to its export to the cytoplasm, where it is unavailable for licensing.

Figure 4.

Model for docking of CRL4^Cdt2 onto the PCNA–PIP degron complex. (A) Crystal structure of PCNA with the PIP degron of p21 (residues X to Y) (Gulbis et al. 1996). The p21 peptide is show in cyan. (B) Close-up of the PCNA–p21 complex, including the PIP degron sequence (same color coding as in Fig. 2A) and a putative contact between the B+4 residue and Cdt2 (dashed line).

Figure 5.

Model for assembly of the PCNA–PIP degron–CRL4^Cdt2 complex on DNA. (A) RFC loads PCNA onto a primer–template junction during DNA replication or repair-associated gap filling. (B) A PIP degron-containing protein docks onto the PCNA–DNA complex. (C) CRL4^Cdt2 recognizes the PIP–degron–PCNA complex. (D) CRL4^Cdt2 promotes ubiquitin transfer to the substrate. The three double arrows depict possible interactions between PCNA, the PIP degron, and CRL4^Cdt2 in the absence of DNA (see the text for details).