林 晃世

CRL4Cdt2 ubiquitin ligase plays an important role maintaining genome integrity during the cell cycle. A recent report suggested that Cdk1 negatively regulates CRL4Cdt2 activity through phosphorylation of its receptor, Cdt2, but the involvement of phosphorylation remains unclear. To address this, we mutated all CDK consensus phosphorylation sites located in the C-terminal half region of Cdt2 (Cdt2-18A) and examined the effect on substrate degradation. We show that both cyclinA/Cdk2 and cyclinB/Cdk1 phosphorylated Cdt2 in vitro and that phosphorylation was reduced by the 18A mutation both in vitro and in vivo. The 18A mutation increased the affinity of Cdt2 to PCNA, and a high amount of Cdt2-18A was colocalized with PCNA foci during S phase in comparison with Cdt2-WT. Poly-ubiquitination activity to Cdt1 was concomitantly enhanced in cells expressing Cdt2-18A. Other CRL4Cdt2 substrates, Set8 and thymine DNA glycosylase, begin to accumulate around late S phase to G2 phase, but the accumulation was prevented in Cdt2-18A cells. Furthermore, mitotic degradation of Cdt1 after UV irradiation was induced in these cells. Our results suggest that CDK-mediated phosphorylation of Cdt2 inactivates its ubiquitin ligase activity by reducing its affinity to PCNA, an important strategy for regulating the levels of key proteins in the cell cycle.

Cdt1 is rapidly degraded by CRL4(Cdt2) E3 ubiquitin ligase after UV (UV) irradiation. Previous reports revealed that the nucleotide excision repair (NER) pathway is responsible for the rapid Cdt1-proteolysis. Here, we show that mismatch repair (MMR) proteins are also involved in the degradation of Cdt1 after UV irradiation in the G1 phase. First, compared with the rapid (within approximate to 15min) degradation of Cdt1 in normal fibroblasts, Cdt1 remained stable for approximate to 30min in NER-deficient XP-A cells, but was degraded within approximate to 60min. The delayed degradation was also dependent on PCNA and CRL4(Cdt2). The MMR proteins Msh2 and Msh6 were recruited to the UV-damaged sites of XP-A cells in the G1 phase. Depletion of these factors with small interfering RNAs prevented Cdt1 degradation in XP-A cells. Similar to the findings in XP-A cells, depletion of XPA delayed Cdt1 degradation in normal fibroblasts and U2OS cells, and co-depletion of Msh6 further prevented Cdt1 degradation. Furthermore, depletion of Msh6 alone delayed Cdt1 degradation in both cell types. When Cdt1 degradation was attenuated by high Cdt1 expression, repair synthesis at the damaged sites was inhibited. Our findings demonstrate that UV irradiation induces multiple repair pathways that activate CRL4(Cdt2) to degrade its target proteins in the G1 phase of the cell cycle, leading to efficient repair of DNA damage.

Numerous cell cycle-regulating proteins are controlled by protein degradation. Recent work shows that ubiquitination-dependent proteolysis plays an important role in once-per-cell cycle control of DNA replication. Cdt1 is a licensing factor essential for assembling the pre-replicative complex on replication origins. Cdt1 is present in G1 phase, but after S phase ubiquitin-mediated proteolysis maintains Cdt1 at low levels. This is important to prevent the re-replication of chromosomal DNA. The cell cycle-dependent degradation of Cdt1 can be monitored by dual staining of the cell nuclei with antibodies against Cdt1- and S/G2-phase marker proteins, such as cyclin A or geminin. © Springer Science+Business Media New York 2014.

PCNA is a DNA clamp, acting on chromatin as a platform for various proteins involved in many aspects of DNA replication-linked processes. Most of these proteins have the PCNA-interaction protein motif (PIP box) that associates with PCNA. Recent works show that PCNA plays an important role as a matchmaker, connecting PCNA-interacting proteins to the ubiquitin ligase CRL4&lt sup&gt Cdt2&lt /sup&gt for their degradation. Proteins degraded by CRL4&lt sup&gt Cdt2&lt /sup&gt include Cdt1, p21, and Set8 in mammalian cells. These CRL4&lt sup&gt Cdt2&lt /sup&gt substrates have a PIP degron that consists of the canonical PIP-box sequence and additional conserved amino acids required for ubiquitination. The degradation of these proteins is triggered when PCNA is loaded onto chromatin at the onset of S phase, and this process is important to prevent re-replication of DNA. These CRL4&lt sup&gt Cdt2&lt /sup&gt substrates are also degraded through the same mechanism in response to DNA damage. In this chapter, we describe several approaches to investigate how PIP degron-containing proteins are degraded in a PCNA-dependent manner. © Springer Science+Business Media New York 2014.

Cells respond to DNA damage by activating alternate signaling pathways that induce proliferation arrest or apoptosis. The correct balance between these two pathways is important for maintaining genomic integrity and preventing unnecessary cell death. The mechanism by which DNA-damaged cells escape from apoptosis during DNA repair is poorly understood. We show that the DNA replication-initiating kinase Cdc7 actively prevents unnecessary death in DNA-damaged cells. In response to mild DNA damage, Tob levels increase through both a transcriptional mechanism and protein stabilization, resulting in inhibition of pro-apoptotic signaling. Cells lacking Cdc7 expression undergo apoptosis after mild DNA damage, where Cul4-DDB1(Cdt2) induces Tob ubiquitination and subsequent degradation. Cdc7 phosphorylates and interacts with Tob to inhibit the Cul4-DDB1(Cdt2)-dependent Tob degradation. Thus, Cdc7 defines an essential pro-survival signaling pathway by contributing to stabilization of Tob, thereby the viability of DNA-damaged cells being maintained.

Recent work identified the E3 ubiquitin ligase CRL4(Cdt2) as mediating the timely degradation of Cdt1 during DNA replication and following DNA damage. In both cases, proliferating cell nuclear antigen (PCNA) loaded on chromatin mediates the CRL4(Cdt2)-dependent proteolysis of Cdt1. Here, we demonstrate that while replication factor C subunit 1 (RFC1)-RFC is required for Cdt1 degradation after UV irradiation during the nucleotide excision repair process, another RFC complex, Ctf18-RFC, which is known to be involved in the establishment of cohesion, has a key role in Cdt1 degradation in S phase. Cdt1 segments having only the degron, a specific sequence element in target protein for ubiquitination, for CRL4(Cdt2) were stabilized during S phase in Ctf18-depleted cells. Additionally, endogenous Cdt1 was stabilized when both Skp2 and Ctf18 were depleted. Since a substantial amount of PCNA was detected on chromatin in Ctf18-depleted cells, Ctf18 is required in addition to loaded PCNA for Cdt1 degradation in S phase. Our data suggest that Ctf18 is involved in recruiting CRL4(Cdt2) to PCNA foci during S phase. Ctf18-mediated Cdt1 proteolysis occurs independent of cohesion establishment, and depletion of Ctf18 potentiates rereplication. Our findings indicate that individual RFC complexes differentially control CRL4(Cdt2)-dependent proteolysis of Cdt1 during DNA replication and repair.