Cdt1 degradation to prevent DNA re-replication: conserved and non-conserved pathways

Abstract

In eukaryotes, DNA replication is strictly regulated so that it occurs only once per cell cycle. The mechanisms that prevent excessive DNA replication are focused on preventing replication origins from being reused within the same cell cycle. This regulation involves the temporal separation of the formation of the pre-replicative complex (pre-RC) from the initiation of DNA replication. The replication licensing factors Cdt1 and Cdc6 recruit the presumptive replicative helicase, the Mcm2-7 complex, to replication origins in late M or G1 phase to form pre-RCs. In fission yeast and metazoa, the Cdt1 licensing factor is degraded at the start of S phase by ubiquitin-mediated proteolysis to prevent the reassembly of pre-RCs. In humans, two E3 complexes, CUL4-DDB1CDT2 and SCFSkp2, are redundantly required for Cdt1 degradation. The two E3 complexes use distinct mechanisms to target Cdt1 ubiquitination. Current data suggests that CUL4-DDB1CDT2-mediated degradation of Cdt1 is S-phase specific, while SCFSkp2-mediated Cdt1 degradation occurs throughout the cell cycle. The degradation of Cdt1 by the CUL4-DDB1CDT2 E3 complex is an evolutionarily ancient pathway that is active in fungi and metazoa. In contrast, SCFSkp2-mediated Cdt1 degradation appears to have arisen relatively recently. A role for Skp2 in Cdt1 degradation has only been demonstrated in humans, and the pathway is not conserved in yeast, invertebrates, or even among other vertebrates.

Figure 1

Two distinct molecular pathways for Cdt1 degradation. (A and B) CUL4-DDB1^CDT2 and SCF^Skp2 CRL ubiquitin ligase complexes have similar modular structures: a cullin; a common RING H2-finger protein Rbx1; an adaptor protein, DDB1 or Skp1; and an SRS, CDT2 or Skp2. In the CUL4-DDB1^CDT2 complex, CDT2 binds to DDB1 through a WD-repeat region with a specific signature, termed a 'WDXR' or 'DXR' domain (marked in figure). In the SCF^Skp2 complex, Skp2 binds to Skp1 through an F-box motif (not marked). (A) CUL4-DDB1^CDT2 targets Cdt1 for degradation after Cdt1 binds to PCNA on chromatin. CDT2 is proposed to directly bind Cdt1 after Cdt1 binds to PCNA, although the CDT2-Cdt1 interaction has not yet been formally demonstrated. (B) SCF^Skp2 targets Cdt1 for degradation after CDK/Cyclin complexes phosphorylate Cdt1. See text for details.

Figure 2

The genesis of CUL4-DDB1^CDT2 and SCF^Skp2 E3 components. CUL4-DDB^CDT2 and SCF^Skp2 complex components were examined in representative organisms of diverse phyla (Table 2). A phylogenetic tree of the taxa analyzed, from eubacteria to mammals, is presented. Note that distances between branches are not to scale. Species and major classifications are color-coordinated, and the temporal locations of the presumed origins of E3 component genes are in red. CUL1-like and CUL4-like cullins, as well as their adaptor proteins DDB1 and Skp1, respectively, appear to have arisen early in eukaryotes, as they are absent from archaea and bacteria but are found in the eukaryotes examined. CDT2, the SRS for a CUL4-DDB1 E3 complex, appears to have arisen prior to the genesis of green plants. Skp2, the SRS for a CUL1 E3 complex, appears to have arisen after the genesis of fungi but prior to the genesis of metazoa. The branching order is based on a phylogenetic analysis using rRNA [76]. Note that other phylogenies, based on protein sequences, reverse the order of plants and slime molds [77]. Combining our genomic data with this alternative branching of phyla (not shown) would imply that CDT2 was created prior to plants in the main eukaryotic lineage but then lost within the slime mold lineage.