DTL/CDT2 is essential for both CDT1 regulation and the early G2/M checkpoint

Abstract

Checkpoint genes maintain genomic stability by arresting cells after DNA damage. Many of these genes also control cell cycle events in unperturbed cells. By conducting a screen for checkpoint genes in zebrafish, we found that dtl/cdt2 is an essential component of the early, radiation-induced G2/M checkpoint. We subsequently found that dtl/cdt2 is required for normal cell cycle control, primarily to prevent rereplication. Both the checkpoint and replication roles are conserved in human DTL. Our data indicate that the rereplication reflects a requirement for DTL in regulating CDT1, a protein required for prereplication complex formation. CDT1 is degraded in S phase to prevent rereplication, and following DNA damage to prevent origin firing. We show that DTL associates with the CUL4-DDB1 E3 ubiquitin ligase and is required for CDT1 down-regulation in unperturbed cells and following DNA damage. The cell cycle defects of Dtl-deficient zebrafish are suppressed by reducing Cdt1 levels. In contrast, the early G2/M checkpoint defect appears to be Cdt1-independent. Thus, DTL promotes genomic stability through two distinct mechanisms. First, it is an essential component of the CUL4-DDB1 complex that controls CDT1 levels, thereby preventing rereplication. Second, it is required for the early G2/M checkpoint.

PCNA-dependent regulation of p21 ubiquitylation and degradation via the CRL4Cdt2 ubiquitin ligase complex

“…The degradation of p21 in response to UV is critical for optimal DNA repair by alleviating the repression of PCNA and promoting translesion DNA synthesis (Bendjennat et al 2003;Chen et al 2004;Lee et al 2006). Thus, our findings suggest that CRL4 Cdt2 E3 ubiquitin ligase is critical for the cell's response to DNA damage, because it not only suppresses replication licensing by targeting Cdt1 for degradation Sansam et al 2006;Senga et al 2006), but also promotes DNA repair by promoting the degradation of p21 (Fig. 8).…”

“…The ability to degrade Cdt1 under these conditions is critical for preventing rereplication and the induction of irradiation-induced early G2/M checkpoint (Sansam et al 2006). Cdt1 interacts with PCNA via a conserved PCNAinteracting motif (PIP Box) in the N-terminal end of Cdt1, and this interaction is required for Cdt1 ubiquitylation via the CRL4 Cdt2 E3 ligase Higa et al 2006;Senga et al 2006).…”

“…Following UV irradiation, as well as in S phase of the cell cycle, the CRL4 Cdt2 E3 ligase (composed of the Cul4A/B, DDB1 [damage-specific DNA-binding protein-1], and the DCAF subunit Cdt2) promotes the ubiquitylation and degradation of the replication factor Cdt1 in cooperation with Proliferating Cell Nuclear Antigen (PCNA) (Higa et al 2003Hu et al 2004; Arias and Walter 2006;Hu and Xiong 2006;Jin et al 2006;Ralph et al 2006;Sansam et al 2006;Senga et al 2006). The ability to degrade Cdt1 under these conditions is critical for preventing rereplication and the induction of irradiation-induced early G2/M checkpoint (Sansam et al 2006).…”

Thymine DNA Glycosylase Is a CRL4Cdt2 Substrate

“…Fewer than 10 CRL4 Cdt2 substrates are known, but all have a role in DNA replication or cell cycle regulation (2). The best-characterized substrate is the replication licensing factor Cdt1 (3)(4)(5)(6), which is required to recruit the MCM2-7 helicase to origins of replication in the G 1 phase of the cell cycle. After cells enter S phase,…”

Regulation of cell cycle progression by forkhead transcription factor FOXO3 through its binding partner DNA replication factor Cdt1

“…Phosphorylation of Cdt1 by Cdk2 promotes its binding to SCF-Skp2 E3 ubiquitin ligase (8)(9)(10), which results in its degradation in S phase. In addition to the Skp2 pathway, PCNA/ DDB1/Cul4-dependent signaling was found to degrade Cdt1 during S phase via the interaction of Cdt1 with PCNA (11)(12)(13)(14)(15). Recently, APC/C Cdh1 was proposed as a third ubiquitin ligase regulating Cdt1 degradation (16).…”