Akiyo HAYASHI

Loading PCNA onto chromatin is a pivotal step in DNA replication, cell cycle progression, and genome integrity. Conversely, unloading PCNA from chromatin is equally crucial for maintaining genome stability. Cells deficient in the PCNA unloader ATAD5-RFC exhibit elevated levels of chromatin-bound PCNA during S phase, but still show dissociation of PCNA from chromatin in mitosis. In this study, we found that depletion of TRAIP, an E3 ubiquitin ligase, results in the retention of PCNA on chromatin during mitosis. Although TRAIP-depleted cells with chromatin-bound PCNA during mitosis progressed into the subsequent G1 phase, they displayed reduced levels of Cdt1, a key replication licensing factor, and impaired S phase entry. In addition, TRAIP-depleted cells exhibited delayed S phase progression. These results suggest that TRAIP functions independently of ATAD5-RFC in removing PCNA from chromatin. Furthermore, TRAIP appears to be essential for precise pre-replication complexes (pre-RCs) formation necessary for faithful initiation of DNA replication and S phase progression.

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.