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

Cell Division BioMed Central Review Open Access Cdt1 degradation to prevent DNA re-replication: conserved and non-conserved pathways Youngjo Kim and Edward T Kipreos* Address: Department of Cellular Biology, University of Georgia, Athens, GA 30602-2607 USA Email: Youngjo Kim - ; Edward T Kipreos* - * Corresponding author Published: 12 June 2007 Cell Division 2007, 2:18 doi:10.1186/1747-1028-2-18 Received: 26 May 2007 Accepted: 12 June 2007 This article is available from: http://www.celldiv.com/content/2/1/18 © 2007 Kim and Kipreos; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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 Mcm27 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. Cdt1 degradation and the control of DNA replication To maintain genome integrity, DNA replication must be strictly regulated to occur only once per cell cycle. Replication is, therefore, tightly regulated to prevent the re-initiation of DNA replication within the same S phase. A failure to restrict DNA replication results in 're-replication', in which the genome is over-replicated within the same cell cycle via origin re-firing. In eukaryotes, the extent of DNA replication is controlled by temporally restricting the assembly of the pre-replicative complex (pre-RC) through a process termed 'replication licensing' (reviewed in [1,2]). Pre-RCs form on replication origins through the sequential binding of DNA replication proteins during late mitosis or G1 phase. The six-member origin recognition complex (ORC) binds replication origins on newlysynthesized chromatin. During late mitosis or G1 phase, the replication licensing factors Cdt1 and Cdc6 are recruited to the origin. Cdt1 and Cdc6 together load the presumptive replicative helicase, the Mcm2-7 complex, onto the origin to complete pre-RC formation. During S phase, pre-RCs are activated by phosphorylation via CDK and DDK (Dbf4-dependent kinase) activity. This phosphorylation allows the recruitment of essential replication Page 1 of 9 (page number not for citation purposes) Cell Division 2007, 2:18 factors, including Cdc45, Mcm10, RPA, proliferating cell nuclear antigen (PCNA), and DNA polymerases α and δ. Cdt1 and Cdc6 are essential loading factors for the Mcm27 complex, and they are negatively regulated during S phase to ensure that the Mcm2-7 complex cannot re-bind to origins that have already fired. In budding yeast, Cdt1 is exported from the nucleus during S phase [3]. In contrast, fission yeast and metazoan Cdt1 homologs are degraded during S phase [1,2,4]. The other replication licensing factor, Cdc6, is regulated by degradation during S phase in budding and fission yeast, while in metazoa, Cdc6 is exported from the nucleus [1,2,4,5]. In most eukaryotic species examined, redundant regulation prevents reassembly of pre-RCs in S phase. The exact regulation varies between eukaryotes, and can include controls of each of the pre-RC components: Cdt1, Cdc6, ORC subunits, and the Mcm2-7 complex [1,2,4]. In all eukaryotic species examined, Cdt1 is a major focus of replication licensing regulation. In Drosophila and vertebrates, Cdt1 activity is redundantly regulated by its degradation and the binding of a Cdt1inhibitor called Geminin [2]. Loss of Geminin leads to rereplication in Drosophila and in certain human cell lines but not in others [6-11]. In human HeLa cells, Cdt1 is degraded prior to the expression of Geminin, suggesting that Geminin is a back-up system that functions after the majority of Cdt1 has been degraded [12]. HeLa cells do not undergo re-replication when Cdt1 degradation is blocked or when Geminin is inactivated; however, when both pathways are deregulated simultaneously, re-replication is observed, indicating that the two pathways redundantly restrain Cdt1 activity [13]. In both C. elegans and Xenopus embryos, loss of Geminin is not associated with re-replication [14-16]. In contrast, Cdt1 degradation is more critical for regulating DNA replication in these species. A failure to degrade CDT-1 in C. elegans is associated with re-replication [17]. Likewise, the expression of a non-degradable Cdt1 (but not wild-type Cdt1) induces re-replication in Xenopus egg extract [18]. Cdt1 is degraded by the ubiquitin-proteasome system. In this pathway, ubiquitin ligases (E3s) provide the specificity for the degradation because they bind specific substrates and then facilitate the transfer of ubiquitin from the ubiquitin conjugating enzyme (E2) to the substrate [19]. The covalent attachment of a tandem array of ubiquitins to the substrate (in the proper linkage) induces the degradation of the substrate by the 26S proteasome [20]. In humans, two distinct E3 complexes, CUL4-DDB1CDT2 and SCFSkp2, have been reported to target Cdt1 for ubiquitin-mediated degradation. Both of these E3s are members http://www.celldiv.com/content/2/1/18 of the cullin-RING ligase (CRL) class of ubiquitin ligases. The two E3 complexes utilize distinct mechanisms for targeting Cdt1 ubiquitination. In this review, we will focus on the regulation of Cdt1 degradation in different species and explore the conservation of pathway components and mechanisms across species and phyla. The CUL4-DDB1 complex targets Cdt1 for degradation Studies in C. elegans first suggested the involvement of CUL4 in Cdt1 degradation. The inactivation of the C. elegans cul-4 gene by RNAi caus (...truncated)