Akiyo HAYASHI

The CRL4^Cdt2 ubiquitin ligase complex is an essential regulator of cell-cycle progression and genome stability, ubiquitinating substrates such as p21, Set8, and Cdt1, via a display of substrate degrons on proliferating cell nuclear antigens (PCNAs). Here, we examine the hierarchy of the ligase and substrate recruitment kinetics onto PCNA at sites of DNA replication. We demonstrate that the C-terminal end of Cdt2 bears a PCNA interaction protein motif (PIP box, Cdt2^PIP), which is necessary and sufficient for the binding of Cdt2 to PCNA. Cdt2^PIP binds PCNA directly with high affinity, two orders of magnitude tighter than the PIP box of Cdt1. X-ray crystallographic structures of PCNA bound to Cdt2^PIP and Cdt1^PIP show that the peptides occupy all three binding sites of the trimeric PCNA ring. Mutating Cdt2^PIP weakens the interaction with PCNA, rendering CRL4^Cdt2 less effective in Cdt1 ubiquitination and leading to defects in Cdt1 degradation. The molecular mechanism we present suggests a new paradigm for bringing substrates to the CRL4-type ligase, where the substrate receptor and substrates bind to a common multivalent docking platform to enable subsequent ubiquitination.

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.