PCNA Is a Cofactor for Cdt1 Degradation by CUL4/DDB1-mediated N-terminal Ubiquitination

Takeshi Senga,Umasundari Sivaprasad,Wenge Zhu,Jong Hoon Park,Emily E Arias,Johannes C Walter,Anindya Dutta

doi:10.1074/jbc.m512705200

Abstract

Cdt1, a protein essential in G1 for licensing of origins for DNA replication, is inhibited in S-phase, both by binding to geminin and degradation by proteasomes. Cdt1 is also degraded after DNA damage to stop licensing of new origins until after DNA repair. Phosphorylation of Cdt1 by cyclin-dependent kinases promotes its binding to SCF-Skp2 E3 ubiquitin ligase, but the Cdk2/Skp2-mediated pathway is not essential for the degradation of Cdt1. Here we show that the N terminus of Cdt1 contains a second degradation signal that is active after DNA damage and in S-phase and is dependent on the interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) through a PCNA binding motif. The degradation involves N-terminal ubiquitination and requires Cul4 and Ddb1 proteins, components of an E3 ubiquitin ligase implicated in protein degradation after DNA damage. Therefore PCNA, the matchmaker for many proteins involved in DNA and chromatin metabolism, also serves to promote the targeted degradation of associated proteins in S-phase or after DNA damage.

Highlights

Cdt1 is targeted for degradation by ubiquitin ligases after DNA damage (14 –16)
In this report we identify the signal for the Skp2-independent degradation of Cdt1 and demonstrate that this signal operates through interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) and uses the Cul4/Ddb1 ubiquitin ligases
One concern after PCNA siRNA is that inhibition of DNA replication might activate checkpoint pathways similar to what we have reported after ORC2 depletion [28], and the cells might arrest in G1 with low Cdk2 kinase activity

Summary

Introduction

Cdt1 is targeted for degradation by ubiquitin ligases after DNA damage (14 –16). When determining the N-terminal limit of the degradation signal, deletion of the first 5 amino acids of full-length Cdt1 (⌬5) was sufficient to stabilize the protein after UV irradiation (Fig. 1D).

Results

Conclusion